Medical Physiology [3 ed.] 9781455743773, 9780323427968, 2016005260

Table of contents :
Medical Physiology
Copyright Page
Contributors
Video Table of Contents
Preface to the Third Edition
The eBook
Acknowledgments
Preface to the First Edition
Target Audience
Content of the Textbook
Emphasis of the Textbook
Creating the Textbook
Special Features
Acknowledgments
Section I: Introduction
Chapter 1: Foundations of Physiology
What is physiology?
Physiological genomics is the link between the organ and the gene
Cells live in a highly protected milieu intérieur
Homeostatic mechanisms—operating through sophisticated feedback control mechanisms— are responsible for maintaining the constancy of the milieu intérieur
Medicine is the study of “physiology gone awry”
References
Section II: Physiology of Cells and Molecules
Chapter 2: Functional Organization of the Cell
Structure of Biological Membranes
The surface of the cell is defined by a membrane
The cell membrane is composed primarily of phospholipids
Phospholipids form complex structures in aqueous solution
The diffusion of individual lipids within a leaflet of a bilayer is determined by the chemical makeup of its constituents
Phospholipid bilayer membranes are impermeable to charged molecules
The plasma membrane is a bilayer
Membrane proteins can be integrally or peripherally associated with the plasma membrane
The membrane-spanning portions of transmembrane proteins are usually hydrophobic α helices
Some membrane proteins are mobile in the plane of the bilayer
Function of Membrane Proteins
Integral membrane proteins can serve as receptors
Integral membrane proteins can serve as adhesion molecules
Integral membrane proteins can carry out the transmembrane movement of water-soluble substances
Integral membrane proteins can also be enzymes
Integral membrane proteins can participate in intracellular signaling
Peripheral membrane proteins participate in intracellular signaling and can form a submembranous cytoskeleton
Cellular Organelles and the Cytoskeleton
The cell is composed of discrete organelles that subserve distinct functions
The nucleus stores, replicates, and reads the cell’s genetic information
Lysosomes digest material derived from the interior and exterior of the cell
The mitochondrion is the site of oxidative energy production
The cytoplasm is not amorphous but is organized by the cytoskeleton
Intermediate filaments provide cells with structural support
Microtubules provide structural support and provide the basis for several types of subcellular motility
Thin filaments (actin) and thick filaments (myosin) are present in almost every cell type
Synthesis and Recycling of Membrane Proteins
Secretory and membrane proteins are synthesized in association with the rough ER
Simultaneous protein synthesis and translocation through the rough ER membrane requires machinery for signal recognition and protein translocation
Proper insertion of membrane proteins requires start- and stop-transfer sequences
Newly synthesized secretory and membrane proteins undergo post-translational modification and folding in the lumen of the rough ER
Secretory and membrane proteins follow the secretory pathway through the cell
Carrier vesicles control the traffic between the organelles of the secretory pathway
Specialized protein complexes, such as clathrin and coatamers, mediate the formation and fusion of vesicles in the secretory pathway
Vesicle Formation in the Secretory Pathway
Vesicle Fusion in the Secretory Pathway
Newly synthesized secretory and membrane proteins are processed during their passage through the secretory pathway
Newly synthesized proteins are sorted in the trans-Golgi network
A mannose-6-phosphate recognition marker is required to target newly synthesized hydrolytic enzymes to lysosomes
Cells internalize extracellular material and plasma membrane through the process of endocytosis
Receptor-mediated endocytosis is responsible for internalizing specific proteins
Endocytosed proteins can be targeted to lysosomes or recycled to the cell surface
Certain molecules are internalized through an alternative process that involves caveolae
Specialized Cell Types
Epithelial cells form a barrier between the internal and external milieu
Tight Junctions
Adhering Junctions
Gap Junctions
Desmosomes
Epithelial cells are polarized
References
Books and Reviews
Journal Articles
Chapter 3: Signal Transduction
Mechanisms of Cellular Communication
Cells can communicate with one another via chemical signals
Soluble chemical signals interact with target cells via binding to surface or intracellular receptors
Cells can also communicate by direct interactions—juxtacrine signaling
Gap Junctions
Adhering and Tight Junctions
Membrane-Associated Ligands
Ligands in the Extracellular Matrix
Second-messenger systems amplify signals and integrate responses among cell types
Receptors That are Ion Channels
Ligand-gated ion channels transduce a chemical signal into an electrical signal
Receptors Coupled to G Proteins
General Properties of G Proteins
G proteins are heterotrimers that exist in many combinations of different α, β, and γ subunits
G-protein activation follows a cycle
Activated α subunits couple to a variety of downstream effectors, including enzymes and ion channels
βγ subunits can activate downstream effectors
Small GTP-binding proteins are involved in a vast number of cellular processes
G-Protein Second Messengers: Cyclic Nucleotides
cAMP usually exerts its effect by increasing the activity of protein kinase A
Protein phosphatases reverse the action of kinases
cGMP exerts its effect by stimulating a nonselective cation channel in the retina
G-Protein Second Messengers: Products of Phosphoinositide Breakdown
Many messengers bind to receptors that activate phosphoinositide breakdown
IP3 liberates Ca2+ from intracellular stores
Calcium activates calmodulin-dependent protein kinases
DAGs and Ca2+ activate protein kinase C
G-Protein Second Messengers: Arachidonic Acid Metabolites
Phospholipase A2 is the primary enzyme responsible for releasing AA
Cyclooxygenases, lipoxygenases, and epoxygenases mediate the formation of biologically active eicosanoids
Prostaglandins, prostacyclins, and thromboxanes (cyclooxygenase products) are vasoactive, regulate platelet action, and modulate ion transport N3-16
The leukotrienes (5-lipoxygenase products) play a major role in inflammatory responses
The HETEs and EETs (epoxygenase products) tend to enhance Ca2+ release from intracellular stores and to enhance cell proliferation
Degradation of the eicosanoids terminates their activity
Receptors That are Catalytic
The receptor guanylyl cyclase transduces the activity of atrial natriuretic peptide, whereas a soluble guanylyl cyclase transduces the activity of nitric oxide
Receptor (Membrane-Bound) Guanylyl Cyclase
Soluble Guanylyl Cyclase
Some catalytic receptors are serine/threonine kinases
RTKs produce phosphotyrosine motifs recognized by SH2 and phosphotyrosine-binding domains of downstream effectors
Creation of Phosphotyrosine Motifs
Recognition of pY Motifs by SH2 and Phosphotyrosine-Binding Domains
The MAPK Pathway
The Phosphatidylinositol-3-Kinase Pathway
Tyrosine kinase–associated receptors activate cytosolic tyrosine kinases such as Src and JAK
Receptor tyrosine phosphatases are required for lymphocyte activation
Nuclear Receptors
Steroid and thyroid hormones enter the cell and bind to members of the nuclear receptor superfamily in the cytoplasm or nucleus
Activated nuclear receptors bind to sequence elements in the regulatory region of responsive genes and either activate or repress DNA transcription
References
Books and Reviews
Journal Articles
Chapter 4: Regulation of Gene Expression
From Genes to Proteins
Gene expression differs among tissues and—in any tissue—may vary in response to external stimuli
Genetic information flows from DNA to proteins
The gene consists of a transcription unit
DNA is packaged into chromatin
Gene expression may be regulated at multiple steps
Transcription factors are proteins that regulate gene transcription
The Promoter and Regulatory Elements
The basal transcriptional machinery mediates gene transcription
The promoter determines the initiation site and direction of transcription
Positive and negative regulatory elements modulate gene transcription
Locus control regions and insulator elements influence transcription within multigene chromosomal domains
Transcription Factors
DNA-binding transcription factors recognize specific DNA sequences
Transcription factors that bind to DNA can be grouped into families based on tertiary structure
Zinc Finger
Basic Zipper
Basic Helix-Loop-Helix
Helix-Turn-Helix
Coactivators and corepressors are transcription factors that do not bind to DNA
Transcriptional activators stimulate transcription by three mechanisms
Recruitment of the Basal Transcriptional Machinery
Chromatin Remodeling
Stimulation of Pol II
Transcriptional activators act in combination
Transcriptional repressors act by competition, quenching, or active repression
The activity of transcription factors may be regulated by post-translational modifications
Phosphorylation
Site-Specific Proteolysis
Other Post-Translational Modifications
The expression of some transcription factors is tissue specific
Regulation of Inducible Gene Expression by Signal-Transduction Pathways
cAMP regulates transcription via the transcription factors CREB and CBP
Receptor tyrosine kinases regulate transcription via a Ras-dependent cascade of protein kinases
Tyrosine kinase–associated receptors can regulate transcription via JAK-STAT
Nuclear receptors are transcription factors
Modular Construction
Dimerization
Activation of Transcription
Repression of Transcription
Physiological stimuli can modulate transcription factors, which can coordinate complex cellular responses
Epigenetic Regulation of Gene Expression
Epigenetic regulation can result in long-term gene silencing
Alterations in chromatin structure may mediate epigenetic regulation, stimulating or inhibiting gene transcription
Histone methylation may stimulate or inhibit gene expression
DNA methylation is associated with gene inactivation
Post-Translational Regulation of Gene Expression
Alternative splicing generates diversity from single genes
Retained Intron
Alternative 3′ Splice Sites
Alternative 5′ Splice Sites
Cassette Exons
Mutually Exclusive Exons
Alternative 5′ Ends
Alternative 3′ Ends
Regulatory elements in the 3′ untranslated region control mRNA stability
MicroRNAs regulate mRNA abundance and translation
References
Books and Reviews
Journal Articles
Glossary
Chapter 5: Transport of Solutes and Water
The Intracellular and Extracellular Fluids
Total-body water is the sum of the ICF and ECF volumes
Plasma Volume
Interstitial Fluid
Transcellular Fluid
ICF is rich in K+, whereas ECF is rich in Na+ and Cl−
Volume Occupied by Plasma Proteins
Effect of Protein Charge
All body fluids have approximately the same osmolality, and each fluid has equal numbers of positive and negative charges
Osmolality
Electroneutrality
Solute Transport Across Cell Membranes
In passive, noncoupled transport across a permeable membrane, a solute moves down its electrochemical gradient
At equilibrium, the chemical and electrical potential energy differences across the membrane are equal but opposite
(Vm − EX) is the net electrochemical driving force acting on an ion
In simple diffusion, the flux of an uncharged substance through membrane lipid is directly proportional to its concentration difference
Some substances cross the membrane passively through intrinsic membrane proteins that can form pores, channels, or carriers
Water-filled pores can allow molecules, some as large as 45 kDa, to cross membranes passively
Gated channels, which alternately open and close, allow ions to cross the membrane passively
Na+ Channels
K+ Channels
Ca2+ Channels
Proton Channels
Anion Channels
Some carriers facilitate the passive diffusion of small solutes such as glucose
The physical structures of pores, channels, and carriers are quite similar
The Na-K pump, the most important primary active transporter in animal cells, uses the energy of ATP to extrude Na+ and take up K+
Besides the Na-K pump, other P-type ATPases include the H-K and Ca pumps
H-K Pump
Ca Pumps
Other Pumps
The F-type and the V-type ATPases transport H+
F-type or FoF1 ATPases
V-type H Pump
ATP-binding cassette transporters can act as pumps, channels, or regulators
ABCA Subfamily
MDR Subfamily
MRP/CFTR Subfamily
Cotransporters, one class of secondary active transporters, are generally driven by the energy of the inwardly directed Na+ gradient
Na/Glucose Cotransporter
Na+-Driven Cotransporters for Organic Solutes
Na/HCO3 Cotransporters
Na+-Driven Cotransporters for Other Inorganic Anions
Na/K/Cl Cotransporter
Na/Cl Cotransporter
K/Cl Cotransporter
H+-Driven Cotransporters
Exchangers, another class of secondary active transporters, exchange ions for one another
Na-Ca Exchanger
Na-H Exchanger
Na+-Driven Cl-HCO3 Exchanger
Cl-HCO3 Exchanger
Other Anion Exchangers
Regulation of Intracellular Ion Concentrations
The Na-K pump keeps [Na+] inside the cell low and [K+] high
The Ca pump and the Na-Ca exchanger keep intracellular [Ca2+] four orders of magnitude lower than extracellular [Ca2+]
Ca Pump (SERCA) in Organelle Membranes
Ca Pump (PMCA) on the Plasma Membrane
Na-Ca Exchanger (NCX) on the Plasma Membrane
In most cells, [Cl−] is modestly above equilibrium because Cl− uptake by the Cl-HCO3 exchanger and Na/K/Cl cotransporter balances passive Cl− efflux through channels
The Na-H exchanger and Na+-driven transporters keep the intracellular pH and [] above their equilibrium values
Water Transport and the Regulation of Cell Volume
Water transport is driven by osmotic and hydrostatic pressure differences across membranes
Because of the presence of impermeant, negatively charged proteins within the cell, Donnan forces will lead to cell swelling
The Na-K pump maintains cell volume by doing osmotic work that counteracts the passive Donnan forces
Cell volume changes trigger rapid changes in ion channels or transporters, returning volume toward normal
Response to Cell Shrinkage
Response to Cell Swelling
Cells respond to long-term hyperosmolality by accumulating new intracellular organic solutes
The gradient in tonicity—or effective osmolality—determines the osmotic flow of water across a cell membrane
Water Exchange Across Cell Membranes
Water Exchange Across the Capillary Wall
Adding isotonic saline, pure water, or pure NaCl to the ECF will increase ECF volume but will have divergent effects on ICF volume and ECF osmolality
Infusion of Isotonic Saline
Infusion of “Solute-Free” Water
Ingestion of Pure NaCl Salt
Whole-body Na+ content determines ECF volume, whereas whole-body water content determines osmolality
Transport of Solutes and Water Across Epithelia
The epithelial cell generally has different electrochemical gradients across its apical and basolateral membranes
Tight and leaky epithelia differ in the permeabilities of their tight junctions
Epithelial cells can absorb or secrete different solutes by inserting specific channels or transporters at either the apical or basolateral membrane
Na+ Absorption
K+ Secretion
Glucose Absorption
Cl− Secretion
Water transport across epithelia passively follows solute transport
Absorption of a Hyperosmotic Fluid
Absorption of an Isosmotic Fluid
Absorption of a Hypo-osmotic Fluid
Epithelia can regulate transport by controlling transport proteins, tight junctions, and the supply of the transported substances
Increased Synthesis (or Degradation) of Transport Proteins
Recruitment of Transport Proteins to the Cell Membrane
Post-translational Modification of Pre-existing Transport Proteins
Changes in the Paracellular Pathway
Luminal Supply of Transported Species and Flow Rate
References
Books and Reviews
Journal Articles
Chapter 6: Electrophysiology of the Cell Membrane
Ionic Basis of Membrane Potentials
Principles of electrostatics explain why aqueous pores formed by channel proteins are needed for ion diffusion across cell membranes
Membrane potentials can be measured with microelectrodes as well as dyes or fluorescent proteins that are voltage sensitive
Membrane potential is generated by ion gradients
For mammalian cells, Nernst potentials for ions typically range from −100 mV for K+ to +100 mV for Ca2+
Currents carried by ions across membranes depend on the concentration of ions on both sides of the membrane, the membrane potential, and the permeability of the membrane to each ion
Membrane potential depends on ionic concentration gradients and permeabilities
Electrical Model of a Cell Membrane
The cell membrane model includes various ionic conductances and electromotive forces in parallel with a capacitor
The separation of relatively few charges across the bilayer capacitance maintains the membrane potential
Ionic current is directly proportional to the electrochemical driving force (Ohm’s law)
Capacitative current is proportional to the rate of voltage change
A voltage clamp measures currents across cell membranes
The patch-clamp technique resolves unitary currents through single channel molecules
Single channel currents sum to produce macroscopic membrane currents
Single channels can fluctuate between open and closed states
Molecular Physiology of Ion Channels
Classes of ion channels can be distinguished on the basis of electrophysiology, pharmacological and physiological ligands, intracellular messengers, and sequence homology
Electrophysiology
Pharmacological Ligands
Physiological Ligands
Intracellular Messengers
Sequence Homology
Many channels are formed by a radially symmetric arrangement of subunits or domains around a central pore
Gap junction channels are made up of two connexons, each of which has six identical subunits called connexins
An evolutionary tree called a dendrogram illustrates the relatedness of ion channels
Hydrophobic domains of channel proteins can predict how these proteins weave through the membrane
Protein superfamilies, subfamilies, and subtypes are the structural bases of channel diversity
Connexins
K+ Channels
HCN, CNG, and TRP Channels
NAADP Receptor
Voltage-Gated Na+ Channels
Voltage-Gated Ca2+ Channels
CatSper Channels
Hv Channels
Ligand-Gated Channels
Other Ion Channels
References
Books and Reviews
Journal Articles
Chapter 7: Electrical Excitability and Action Potentials
Mechanisms of Nerve and Muscle Action Potentials
An action potential is a transient depolarization triggered by a depolarization beyond a threshold
In contrast to an action potential, a graded response is proportional to stimulus intensity and decays with distance along the axon
Excitation of a nerve or muscle depends on the product (strength × duration) of the stimulus and on the refractory period
The action potential arises from changes in membrane conductance to Na+ and K+
The Na+ and K+ currents that flow during the action potential are time and voltage dependent
Time Dependence of Na+ and K+ Currents
Voltage Dependence of Na+ and K+ Currents
Macroscopic Na+ and K+ currents result from the opening and closing of many channels
The Hodgkin-Huxley model predicts macroscopic currents and the shape of the action potential
Physiology of Voltage-Gated Channels and Their Relatives
A large superfamily of structurally related membrane proteins includes voltage-gated and related channels
Na+ channels generate the rapid initial depolarization of the action potential
Na+ channels are blocked by neurotoxins and local anesthetics
Ca2+ channels contribute to action potentials in some cells and also function in electrical and chemical coupling mechanisms
Ca2+ channels are characterized as L-, T-, P/Q-, N-, and R-type channels on the basis of kinetic properties and inhibitor sensitivity
K+ channels determine resting potential and regulate the frequency and termination of action potentials
The Kv (or Shaker-related) family of K+ channels mediates both the delayed outward-rectifier current and the transient A-type current
Two families of KCa K+ channels mediate Ca2+-activated K+ currents
The Kir K+ channels mediate inward-rectifier K+ currents, and K2P channels may sense stress
Propagation of Action Potentials
The propagation of electrical signals in the nervous system involves local current loops
Myelin improves the efficiency with which axons conduct action potentials
The cable properties of the membrane and cytoplasm determine the velocity of signal propagation
References
Books and Reviews
Journal Articles
Chapter 8: Synaptic Transmission and the Neuromuscular Junction
Mechanisms of Synaptic Transmission
Electrical continuity between cells is established by electrical or chemical synapses
Electrical synapses directly link the cytoplasm of adjacent cells
Chemical synapses use neurotransmitters to provide electrical continuity between adjacent cells
Neurotransmitters can activate ionotropic or metabotropic receptors
Synaptic Transmission at the Neuromuscular Junction
Neuromuscular junctions are specialized synapses between motor neurons and skeletal muscle
ACh activates nicotinic AChRs to produce an excitatory end-plate current
The nicotinic AChR is a member of the pentameric Cys-loop receptor family of ligand-gated ion channels
Activation of AChR channels requires binding of two ACh molecules
Miniature EPPs reveal the quantal nature of transmitter release from the presynaptic terminals
Direct sensing of extracellular transmitter also shows quantal release of transmitter
Synaptic vesicles package, store, and deliver neurotransmitters
Neurotransmitter release occurs by exocytosis of synaptic vesicles
Re-uptake or cleavage of the neurotransmitter terminates its action
Toxins and Drugs Affecting Synaptic Transmission
Guanidinium neurotoxins such as tetrodotoxin prevent depolarization of the nerve terminal, whereas dendrotoxins inhibit repolarization
ω-Conotoxin blocks Ca2+ channels that mediate Ca2+ influx into nerve terminals, inhibiting synaptic transmission
Bacterial toxins such as tetanus and botulinum toxins cleave proteins involved in exocytosis, preventing fusion of synaptic vesicles
Both agonists and antagonists of the nicotinic AChR can prevent synaptic transmission
Inhibitors of AChE prolong and magnify the EPP
References
Books and Reviews
Journal Articles
Chapter 9: Cellular Physiology of Skeletal, Cardiac, and Smooth Muscle
Skeletal Muscle
Contraction of skeletal muscle is initiated by motor neurons that innervate motor units
Action potentials propagate from the sarcolemma to the interior of muscle fibers along the transverse tubule network
Depolarization of the T-tubule membrane results in Ca2+ release from the SR at the triad
Striations of skeletal muscle fibers correspond to ordered arrays of thick and thin filaments within myofibrils
Thin and thick filaments are supramolecular assemblies of protein subunits
Thin Filaments
Thick Filaments
During the cross-bridge cycle, contractile proteins convert the energy of ATP hydrolysis into mechanical energy
An increase in [Ca2+]i triggers contraction by removing the inhibition of cross-bridge cycling
Termination of contraction requires re-uptake of Ca2+ into the SR
Muscle contractions produce force under isometric conditions and force with shortening under isotonic conditions
Muscle length influences tension development by determining the degree of overlap between actin and myosin filaments
At higher loads, the velocity of shortening is lower because more cross-bridges are simultaneously active
In a single skeletal muscle fiber, the force developed may be increased by summing multiple twitches in time
In a whole skeletal muscle, the force developed may be increased by summing the contractions of multiple fibers
Cardiac Muscle
Action potentials propagate between adjacent cardiac myocytes through gap junctions
Cardiac contraction requires Ca2+ entry through L-type Ca2+ channels
Cross-bridge cycling and termination of cardiac muscle contraction are similar to the events in skeletal muscle
In cardiac muscle, increasing the entry of Ca2+ enhances the contractile force
Smooth Muscle
Smooth muscles may contract in response to synaptic transmission or electrical coupling
Action potentials of smooth muscles may be brief or prolonged
Some smooth-muscle cells spontaneously generate either pacemaker currents or slow waves
Some smooth muscles contract without action potentials
In smooth muscle, both entry of extracellular Ca2+ and intracellular Ca2+ spark activate contraction
Ca2+ Entry via Voltage-Gated Channels
Ca2+ Release from the SR
Ca2+ Entry through Store-Operated Ca2+ Channels (SOCs)
Ca2+-dependent phosphorylation of the myosin regulatory light chain activates cross-bridge cycling in smooth muscle
Termination of smooth-muscle contraction requires dephosphorylation of myosin light chain
Smooth-muscle contraction may also occur independently of increases in [Ca2+]i
In smooth muscle, increases in both [Ca2+]i and the Ca2+ sensitivity of the contractile apparatus enhance contractile force
Smooth muscle maintains high force at low energy consumption
Diversity among Muscles
Skeletal muscle is composed of slow-twitch and fast-twitch fibers
The properties of cardiac cells vary with location in the heart
The properties of smooth-muscle cells differ markedly among tissues and may adapt with time
Smooth-muscle cells express a wide variety of neurotransmitter and hormone receptors
References
Books and Reviews
Journal Articles
Section III: The Nervous System
Chapter 10: Organization of the Nervous System
The nervous system can be divided into central, peripheral, and autonomic nervous systems
Each area of the nervous system has unique nerve cells and a different function
Cells of the Nervous System
The neuron doctrine first asserted that the nervous system is composed of many individual signaling units—the neurons
Nerve cells have four specialized regions: cell body, dendrites, axon, and presynaptic terminals
Cell Body
Dendrites
Axon
Presynaptic Terminals
The cytoskeleton helps compartmentalize the neuron and also provides the tracks along which material travels between different parts of the neuron
Fast Axoplasmic Transport
Fast Retrograde Transport
Slow Axoplasmic Transport
Neurons can be classified on the basis of their axonal projection, their dendritic geometry, and the number of processes emanating from the cell body
Axonal Projection
Dendritic Geometry
Number of Processes
Glial cells provide a physiological environment for neurons
Development of Neurons and Glial Cells
Neurons differentiate from the neuroectoderm
Neurons and glial cells originate from cells in the proliferating germinal matrix near the ventricles
Neurons migrate to their correct anatomical position in the brain with the help of adhesion molecules
Neurons do not regenerate
Neurons
Axons
Glia
Subdivisions of the Nervous System
The CNS consists of the telencephalon, cerebellum, diencephalon, midbrain, pons, medulla, and spinal cord
Telencephalon
Cerebellum
Diencephalon
Brainstem (Midbrain, Pons, and Medulla)
Spinal Cord
The PNS comprises the cranial and spinal nerves, their associated sensory ganglia, and various sensory receptors
The ANS innervates effectors that are not under voluntary control
References
Books and Reviews
Journal Articles
Chapter 11: The Neuronal Microenvironment
Extracellular fluid in the brain provides a highly regulated environment for central nervous system neurons
The brain is physically and metabolically fragile
Cerebrospinal Fluid
CSF fills the ventricles and subarachnoid space
The brain floats in CSF, which acts as a shock absorber
The choroid plexuses secrete CSF into the ventricles, and the arachnoid granulations absorb it
The epithelial cells of the choroid plexus secrete the CSF
Brain Extracellular Space
Neurons, glia, and capillaries are packed tightly together in the CNS
The CSF communicates freely with the BECF, which stabilizes the composition of the neuronal microenvironment
The ion fluxes that accompany neural activity cause large changes in extracellular ion concentration
The Blood-Brain Barrier
The blood-brain barrier prevents some blood constituents from entering the brain extracellular space
Continuous tight junctions link brain capillary endothelial cells
Uncharged and lipid-soluble molecules more readily pass through the blood-brain barrier
Transport by capillary endothelial cells contributes to the blood-brain barrier
Glial Cells
Glial cells constitute half the volume of the brain and outnumber neurons
Astrocytes supply fuel to neurons in the form of lactic acid
Astrocytes are predominantly permeable to K+ and also help regulate [K+]o
Gap junctions couple astrocytes to one another, allowing diffusion of small solutes
Astrocytes synthesize neurotransmitters, take them up from the extracellular space, and have neurotransmitter receptors
Astrocytes secrete trophic factors that promote neuronal survival and synaptogenesis
Astrocytic endfeet modulate cerebral blood flow
Oligodendrocytes and Schwann cells make and sustain myelin
Oligodendrocytes are involved in pH regulation and iron metabolism in the brain
Microglial cells are the macrophages of the CNS
References
Books and Reviews
Journal Articles
Chapter 12: Physiology of Neurons
Neurons receive, combine, transform, store, and send information
Neural information flows from dendrite to soma to axon to synapse
Signal Conduction in Dendrites
Dendrites attenuate synaptic potentials
Dendritic membranes have voltage-gated ion channels
Control of Spiking Patterns in the Soma
Neurons can transform a simple input into a variety of output patterns
Intrinsic firing patterns are determined by a variety of ion currents with relatively slow kinetics
Axonal Conduction
Axons are specialized for rapid, reliable, and efficient transmission of electrical signals
Action potentials are usually initiated at the initial segment
Conduction velocity of a myelinated axon increases linearly with diameter
Demyelinated axons conduct action potentials slowly, unreliably, or not at all
References
Books and Reviews
Journal Articles
Chapter 13: Synaptic Transmission in the Nervous System
Neuronal Synapses
The molecular mechanisms of neuronal synapses are similar but not identical to those of the neuromuscular junction
Presynaptic terminals may contact neurons at the dendrite, soma, or axon and may contain both clear vesicles and dense-core granules
The postsynaptic membrane contains transmitter receptors and numerous proteins clustered in the postsynaptic density
Some transmitters are used by diffusely distributed systems of neurons to modulate the general excitability of the brain
Electrical synapses serve specialized functions in the mammalian nervous system
Neurotransmitter Systems of the Brain
Most of the brain’s transmitters are common biochemicals
Synaptic transmitters can stimulate, inhibit, or modulate the postsynaptic neuron
Excitatory Synapses
Inhibitory Synapses
Modulatory Synapses
G proteins may affect ion channels directly, or indirectly through second messengers
Signaling cascades allow amplification, regulation, and a long duration of transmitter responses
Neurotransmitters may have both convergent and divergent effects
Fast Amino Acid–Mediated Synapses in the CNS
Most EPSPs in the brain are mediated by two types of glutamate-gated channels
Most IPSPs in the brain are mediated by the GABAA receptor, which is activated by several classes of drugs
The ionotropic receptors for ACh, serotonin, GABA, and glycine belong to the superfamily of ligand-gated/pentameric channels
Most neuronal synapses release a very small number of transmitter quanta with each action potential
When multiple transmitters colocalize to the same synapse, the exocytosis of large vesicles requires high-frequency stimulation
Plasticity of Central Synapses
Use-dependent changes in synaptic strength underlie many forms of learning
Short-term synaptic plasticity usually reflects presynaptic changes
Long-term potentiation in the hippocampus may last for days or weeks
Long-term depression exists in multiple forms
Long-term depression in the cerebellum may be important for motor learning
References
Books and Reviews
Journal Articles
Chapter 14: The Autonomic Nervous System
Organization of the Visceral Control System
The ANS has sympathetic, parasympathetic, and enteric divisions
Sympathetic preganglionic neurons originate from spinal segments T1 to L3 and synapse with postganglionic neurons in paravertebral or prevertebral ganglia
Preganglionic Neurons
Paravertebral Ganglia
Prevertebral Ganglia
Postganglionic Neurons
Cranial Nerves III, VII, and IX
Cranial Nerve X
Sacral Nerves
The visceral control system also has an important afferent limb
The enteric division is a self-contained nervous system of the GI tract and receives sympathetic and parasympathetic input
Synaptic Physiology of the Autonomic Nervous System
The sympathetic and parasympathetic divisions have opposite effects on most visceral targets
All preganglionic neurons—both sympathetic and parasympathetic—release acetylcholine and stimulate N2 nicotinic receptors on postganglionic neurons
All postganglionic parasympathetic neurons release ACh and stimulate muscarinic receptors on visceral targets
Most postganglionic sympathetic neurons release norepinephrine onto visceral targets
Postganglionic sympathetic and parasympathetic neurons often have muscarinic as well as nicotinic receptors
Nonclassic transmitters can be released at each level of the ANS
Two of the most unusual nonclassic neurotransmitters, ATP and nitric oxide, were first identified in the ANS
ATP
Nitric Oxide
Central Nervous System Control of the Viscera
Sympathetic output can be massive and nonspecific, as in the fight-or-flight response, or selective for specific target organs
Parasympathetic neurons participate in many simple involuntary reflexes
A variety of brainstem nuclei provide basic control of the ANS
The forebrain can modulate autonomic output, and reciprocally, visceral sensory input integrated in the brainstem can influence or even overwhelm the forebrain
CNS control centers oversee visceral feedback loops and orchestrate a feed-forward response to meet anticipated needs
The ANS has multiple levels of reflex loops
References
Books and Reviews
Journal Articles
Chapter 15: Sensory Transduction
Sensory receptors convert environmental energy into neural signals
Sensory transduction uses adaptations of common molecular signaling mechanisms
Sensory transduction requires detection and amplification, usually followed by a local receptor potential
Chemoreception
Chemoreceptors are ubiquitous, diverse, and evolutionarily ancient
Taste receptors are modified epithelial cells, whereas olfactory receptors are neurons
Taste Receptor Cells
Olfactory Receptor Cells
Complex flavors are derived from a few basic types of taste receptors, with contributions from sensory receptors of smell, temperature, texture, and pain
Taste transduction involves many types of molecular signaling systems
Salty
Sour
Sweet
Bitter
Amino Acids
Olfactory transduction involves specific receptors, G protein–coupled signaling, and a cyclic nucleotide–gated ion channel
Visual Transduction
The optical components of the eye collect light and focus it onto the retina
The retina is a small, displaced part of the CNS
There are three primary types of photoreceptors: rods, cones, and intrinsically photosensitive ganglion cells
Rods and cones hyperpolarize in response to light
Rhodopsin is a G protein–coupled “receptor” for light
The eye uses a variety of mechanisms to adapt to a wide range of light levels
Color vision depends on the different spectral sensitivities of the three types of cones
The ipRGCs have unique properties and functions
Vestibular and Auditory Transduction: Hair Cells
Bending the stereovilli of hair cells along one axis causes cation channels to open or to close
The otolithic organs (saccule and utricle) detect the orientation and linear acceleration of the head
The semicircular canals detect the angular acceleration of the head
The outer and middle ears collect and condition air pressure waves for transduction within the inner ear
Outer Ear
Middle Ear
The cochlea is a spiral of three parallel, fluid-filled tubes
Inner hair cells transduce sound, whereas the active movements of outer hair cells amplify the signal
The frequency sensitivity of auditory hair cells depends on their position along the basilar membrane of the cochlea
Somatic Sensory Receptors, Proprioception, and Pain
A variety of sensory endings in the skin transduce mechanical, thermal, and chemical stimuli
Mechanoreceptors in the skin provide sensitivity to specific stimuli such as vibration and steady pressure
Separate thermoreceptors detect warmth and cold
Nociceptors are specialized sensory endings that transduce painful stimuli
Muscle spindles sense changes in the length of skeletal muscle fibers, whereas Golgi tendon organs gauge the muscle’s force
References
Books and Reviews
Journal Articles
Chapter 16: Circuits of the Central Nervous System
Elements of Neural Circuits
Neural circuits process sensory information, generate motor output, and create spontaneous activity
Nervous systems have several levels of organization
Most local circuits have three elements: input axons, interneurons, and projection (output) neurons
Simple, Stereotyped Responses: Spinal Reflex Circuits
Passive stretching of a skeletal muscle causes a reflexive contraction of that same muscle and relaxation of the antagonist muscles
Force applied to the Golgi tendon organ regulates muscle contractile strength
Noxious stimuli can evoke complex reflexive movements
Spinal reflexes are strongly influenced by control centers within the brain
Rhythmic Activity: Central Pattern Generators
Central pattern generators in the spinal cord can create a complex motor program even without sensory feedback
Pacemaker cells and synaptic interconnections both contribute to central pattern generation
Central pattern generators in the spinal cord take advantage of sensory feedback, interconnections among spinal segments, and interactions with brainstem control centers
Spatial Representations: Sensory and Motor Maps in the Brain
The nervous system contains maps of sensory and motor information
The cerebral cortex has multiple visuotopic maps
Maps of somatic sensory information magnify some parts of the body more than others
The cerebral cortex has a motor map that is adjacent to and well aligned with the somatosensory map
Sensory and motor maps are fuzzy and plastic
Temporal Representations: Time-Measuring Circuits
To localize sound, the brain compares the timing and intensity of input to the ears
The brain measures interaural timing by a combination of neural delay lines and coincidence detectors
References
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Journal Articles
Section IV: The Cardiovascular System
Chapter 17: Organization of the Cardiovascular System
Elements of the Cardiovascular System
The circulation is an evolutionary consequence of body size
The heart is a dual pump that drives the blood in two serial circuits: the systemic and the pulmonary circulations
Hemodynamics
Blood flow is driven by a constant pressure head across variable resistances
Blood pressure is always measured as a pressure difference between two points
Total blood flow, or cardiac output, is the product (heart rate) × (stroke volume)
Flow in an idealized vessel increases with the fourth power of radius (Poiseuille equation)
Viscous resistance to flow is proportional to the viscosity of blood but does not depend on properties of the blood vessel walls
The viscosity of blood is a measure of the internal slipperiness between layers of fluid
How Blood Flows
Blood flow is laminar
Pressure and flow oscillate with each heartbeat between maximum systolic and minimum diastolic values
Origins of Pressure in the Circulation
Gravity causes a hydrostatic pressure difference when there is a difference in height
Low compliance of a vessel causes the transmural pressure to increase when the vessel blood volume is increased
The viscous resistance of blood causes an axial pressure difference when there is flow
The inertia of the blood and vessels causes pressure to decrease when the velocity of blood flow increases
How to Measure Blood Pressure, Blood Flow, and Cardiac Volumes
Blood pressure can be measured directly by puncturing the vessel
Blood pressure can be measured indirectly by use of a sphygmomanometer
Blood flow can be measured directly by electromagnetic and ultrasound flowmeters
Invasive Methods
Noninvasive Methods
Cardiac output can be measured indirectly by the Fick method, which is based on the conservation of mass
Cardiac output can be measured indirectly by dilution methods
Regional blood flow can be measured indirectly by “clearance” methods
Ventricular dimensions, ventricular volumes, and volume changes can be measured by angiography and echocardiography
References
Books and Reviews
Journal Articles
Chapter 18: Blood
Blood Composition
Whole blood is a suspension of cellular elements in plasma
Bone marrow is the source of most blood cells
RBCs are mainly composed of hemoglobin
Leukocytes defend against infections
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
Platelets are nucleus-free fragments
Blood Viscosity
Whole blood has an anomalous viscosity
Blood viscosity increases with the hematocrit and the fibrinogen plasma concentration
Fibrinogen
Hematocrit
Vessel Radius
Velocity of Flow
Temperature
Hemostasis and Fibrinolysis
Platelets can plug holes in small vessels
Adhesion
Activation
Aggregation
A controlled cascade of proteolysis creates a blood clot
Intrinsic Pathway (Surface Contact Activation)
Extrinsic Pathway (Tissue Factor Activation)
Common Pathway
Coagulation as a Connected Diagram
Anticoagulants keep the clotting network in check
Paracrine Factors
Anticoagulant Factors
Fibrinolysis breaks up clots
References
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Journal Articles
Chapter 19: Arteries and Veins
Arterial Distribution and Venous Collection Systems
Physical properties of vessels closely follow the level of branching in the circuit
Most of the blood volume resides in the systemic veins
The intravascular pressures along the systemic circuit are higher than those along the pulmonary circuit
Under normal conditions, the steepest pressure drop in the systemic circulation occurs in arterioles, the site of greatest vascular resistance
Local intravascular pressure depends on the distribution of vascular resistance
Elastic Properties of Blood Vessels
Blood vessels are elastic tubes
Because of the elastic properties of vessels, the pressure-flow relationship of passive vascular beds is nonlinear
Contraction of smooth muscle halts blood flow when driving pressure falls below the critical closing pressure
Elastic and collagen fibers determine the distensibility and compliance of vessels
Differences in compliance cause arteries to act as resistors and veins to act as capacitors
Laplace’s law describes how tension in the vessel wall increases with transmural pressure
The vascular wall is adapted to withstand wall tension, not transmural pressure
Elastin and collagen separately contribute to the wall tension of vessels
Aging reduces the distensibility of arteries
Active tension from smooth-muscle activity adds to the elastic tension of vessels
Elastic tension helps stabilize vessels under vasomotor control
References
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Journal Articles
Chapter 20: The Microcirculation
The microcirculation serves both nutritional and non-nutritional roles
The microcirculation extends from the arterioles to the venules
Capillary Exchange of Solutes
The exchange of O2 and CO2 across capillaries depends on the diffusional properties of the surrounding tissue
The O2 extraction ratio of a whole organ depends primarily on blood flow and metabolic demand
According to Fick’s law, the diffusion of small water-soluble solutes across a capillary wall depends on both the permeability and the concentration gradient
The whole-organ extraction ratio for small hydrophilic solutes provides an estimate of the solute permeability of capillaries
Small polar molecules have a relatively low permeability because they can traverse the capillary wall only by diffusing through water-filled pores (small-pore effect)
The exchange of macromolecules across capillaries can occur by transcytosis (large-pore effect)
Capillary Exchange of Water
Fluid transfer across capillaries is convective and depends on net hydrostatic and osmotic forces (i.e., Starling forces)
Capillary blood pressure (Pc) falls from ~35 mm Hg at the arteriolar end to ~15 mm Hg at the venular end
Arteriolar (Pa) and Venular (Pv) Pressure
Location
Time
Gravity
Interstitial fluid pressure (Pif) is slightly negative, except in encapsulated organs
Capillary colloid osmotic pressure (πc), which reflects the presence of plasma proteins, is ~25 mm Hg
Interstitial fluid colloid osmotic pressure (πif) varies between 0 and 10 mm Hg among different organs
The Starling principle predicts ultrafiltration at the arteriolar end and absorption at the venular end of most capillary beds
For continuous capillaries, the endothelial barrier for fluid exchange is more complex than considered by Starling
Lymphatics
Lymphatics return excess interstitial fluid to the blood
Flow in Initial Lymphatics
Flow in Collecting Lymphatics
Transport of Proteins and Cells
The circulation of extracellular fluids involves three convective loops: blood, interstitial fluid, and lymph
Regulation of the Microcirculation
The active contraction of vascular smooth muscle regulates precapillary resistance, which controls capillary blood flow
Contraction of Vascular Smooth Muscle
Relaxation of Vascular Smooth Muscle
Tissue metabolites regulate local blood flow in specific vascular beds, independently of the systemic regulation
The endothelium of capillary beds is the source of several vasoactive compounds, including nitric oxide, endothelium-derived hyperpolarizing factor, and endothelin
Nitric Oxide
Endothelium-Derived Hyperpolarizing Factor
Prostacyclin (Prostaglandin I2)
Endothelins
Thromboxane A2
Other Endothelial Factors
Autoregulation stabilizes blood flow despite large fluctuations in systemic arterial pressure
Blood vessels proliferate in response to growth factors by a process known as angiogenesis
Promoters of Vessel Growth
Inhibitors of Vessel Growth
References
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Journal Articles
Chapter 21: Cardiac Electrophysiology and the Electrocardiogram
Electrophysiology of Cardiac Cells
The cardiac action potential starts in specialized muscle cells of the sinoatrial node and then propagates in an orderly fashion throughout the heart
The cardiac action potential conducts from cell to cell via gap junctions
Cardiac action potentials have as many as five distinctive phases
The Na+ current is the largest current in the heart
The Ca2+ current in the heart passes primarily through L-type Ca2+ channels
The repolarizing K+ current turns on slowly
Early Outward K+ Current (A-type Current)
G Protein–Activated K+ Current
KATP Current
The If current is mediated by a nonselective cation channel
Different cardiac tissues uniquely combine ionic currents to produce distinctive action potentials
The SA node is the primary pacemaker of the heart
The Concept of Pacemaker Activity
SA Node
AV Node
Purkinje Fibers
Atrial and ventricular myocytes fire action potentials but do not have pacemaker activity
Atrial Muscle
Ventricular Muscle
Acetylcholine and catecholamines modulate pacemaker activity, conduction velocity, and contractility
Acetylcholine
Catecholamines
The Electrocardiogram
An ECG generally includes five waves
A pair of ECG electrodes defines a lead
The Limb Leads
The Precordial Leads
A simple two-cell model can explain how a simple ECG can arise
Cardiac Arrhythmias
Conduction abnormalities are a major cause of arrhythmias
Partial (or Incomplete) Conduction Block
Complete Conduction Block
Re-Entry
Accessory Conduction Pathways
Fibrillation
Altered automaticity can originate from the sinus node or from an ectopic locus
Depolarization-Dependent Triggered Activity
Long QT Syndrome
Ca2+ overload and metabolic changes can also cause arrhythmias
Ca2+ Overload
Metabolism-Dependent Conduction Changes
Electromechanical Dissociation
References
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Chapter 22: The Heart as a Pump
The Cardiac Cycle
The closing and opening of the cardiac valves define four phases of the cardiac cycle
Changes in ventricular volume, pressure, and flow accompany the four phases of the cardiac cycle N22-1
Diastasis Period (Middle of Phase 1)
Atrial Contraction (End of Phase 1)
Isovolumetric Contraction (Phase 2)
Ejection or Outflow (Phase 3)
Isovolumetric Relaxation (Phase 4)
Rapid Ventricular Filling Period (Beginning of Phase 1)
The ECG, phonocardiogram, and echocardiogram all follow the cyclic pattern of the cardiac cycle
Aortic Blood Flow
Jugular Venous Pulse
Electrocardiogram
Phonocardiogram and Heart Sounds
Echocardiogram
The cardiac cycle causes flow waves in the aorta and peripheral vessels
Aortic Arch
Thoracic-Abdominal Aorta and Large Arteries
The cardiac cycle also causes pressure waves in the aorta and peripheral vessels
Terminal Arteries and Arterioles
Capillaries
Distortion of pressure waves is the result of their propagation along the arterial tree
Effect of Frequency on Wave Velocity and Damping
Effect of Wall Stiffness on Wave Velocity
Pressure waves in veins do not originate from arterial waves
Effect of the Cardiac Cycle
Effect of the Respiratory Cycle
Effect of Skeletal Muscle Contraction (“Muscle Pump”)
Cardiac Dynamics
The right ventricle contracts like a bellows, whereas the left ventricle contracts like a hand squeezing a tube of toothpaste
The right atrium contracts before the left, but the left ventricle contracts before the right
Atrial Contraction
Initiation of Ventricular Contraction
Ventricular Ejection
Ventricular Relaxation
Measurements of ventricular volumes, pressures, and flows allow clinicians to judge cardiac performance
Definitions of Cardiac Volumes
Measurements of Cardiac Volumes
Measurement of Ventricular Pressures
Measurement of Flows
The pressure-volume loop of a ventricle illustrates the ejection work of the ventricle
Segment AB
Segment BC
Segment CD
Segment DE
Segment EF
Segment FA
The “pumping work” done by the heart accounts for a small fraction of the total energy the heart consumes
From Contractile Filaments to a Regulated Pump
The entry of Ca2+ from the outside triggers Ca2+-induced Ca2+ release from the sarcoplasmic reticulum
A global rise in [Ca2+]i initiates contraction of cardiac myocytes
Phosphorylation of phospholamban and of troponin I speeds cardiac muscle relaxation
Extrusion of Ca2+ into the ECF
Reuptake of Ca2+ by the SR
Uptake of Ca2+ by Mitochondria
Dissociation of Ca2+ from Troponin C
The overlap of thick and thin filaments cannot explain the unusual shape of the cardiac length-tension diagram
Starling’s law states that a greater fiber length (i.e., greater ventricular volume) causes the heart to deliver more mechanical energy
The velocity of cardiac muscle shortening falls when the contraction occurs against a greater opposing force (or pressure) or at a shorter muscle length (or lower volume)
Increases in heart rate enhance myocardial tension
Contractility is an intrinsic measure of cardiac performance
Effect of Changes in Contractility
Effect of Changes in Preload (i.e., Initial Sarcomere Length)
Effect of Changes in Afterload
Positive inotropic agents increase myocardial contractility by raising [Ca2+]i
Positive Inotropic Agents
Negative Inotropic Agents
References
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Journal Articles
Chapter 23: Regulation of Arterial Pressure and Cardiac Output
Short-Term Regulation of Arterial Pressure
Systemic mean arterial blood pressure is the principal variable that the cardiovascular system controls
Neural reflexes mediate the short-term regulation of mean arterial blood pressure
High-pressure baroreceptors at the carotid sinus and aortic arch are stretch receptors that sense changes in arterial pressure
Increased arterial pressure raises the firing rate of afferent baroreceptor nerves
The medulla coordinates afferent baroreceptor signals
The efferent pathways of the baroreceptor response include both sympathetic and parasympathetic divisions of the autonomic nervous system
Sympathetic Efferents
Parasympathetic Efferents
The principal effectors in the neural control of arterial pressure are the heart, the arteries, the veins, and the adrenal medulla
Sympathetic Input to the Heart (Cardiac Nerves)
Parasympathetic Input to the Heart (Vagus Nerve)
Sympathetic Input to Blood Vessels (Vasoconstrictor Response)
Parasympathetic Input to Blood Vessels (Vasodilator Response)
Sympathetic Input to Blood Vessels in Skeletal Muscle (Vasodilator Response)
Adrenal Medulla
The unique combination of agonists and receptors determines the end response in cardiac and vascular effector cells
Adrenergic Receptors in the Heart
Cholinergic Receptors in the Heart
Adrenergic Receptors in Blood Vessels
Cholinergic Receptors in or near Blood Vessels
Nonadrenergic, Noncholinergic Receptors in Blood Vessels
The medullary cardiovascular center tonically maintains blood pressure and is under the control of higher brain centers
Secondary neural regulation of arterial blood pressure depends on chemoreceptors
Carotid Bodies
Aortic Bodies
Afferent Fiber Input to the Medulla
Physiological Role of the Peripheral Chemoreceptors in Cardiovascular Control
Central Chemoreceptors
Regulation of Cardiac Output
Mechanisms intrinsic to the heart modulate both heart rate and stroke volume
Intrinsic Control of Heart Rate
Intrinsic Control of Stroke Volume
Mechanisms extrinsic to the heart also modulate heart rate and stroke volume
Baroreceptor Regulation
Chemoreceptor Regulation
Low-pressure baroreceptors in the atria respond to increased “fullness” of the vascular system, triggering tachycardia, renal vasodilation, and diuresis
Atrial Receptors
Ventricular Receptors
Cardiac output is roughly proportional to effective circulating blood volume
Matching of Venous Return and Cardiac Output
Increases in cardiac output cause right atrial pressure to fall
Changes in blood volume shift the vascular function curve to different RAPs, whereas changes in arteriolar tone alter the slope of the curve
Because vascular function and cardiac function depend on each other, cardiac output and venous return match at exactly one value of RAP
Intermediate- and Long-Term Control of the Circulation
Endocrine and paracrine vasoactive compounds control the circulatory system on an intermediate- to long-term basis
Biogenic Amines
Peptides
Prostaglandins
Nitric Oxide
Pathways for the renal control of ECF volume are the primary long-term regulators of mean arterial pressure
References
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Journal Articles
Chapter 24: Special Circulations
The blood flow to individual organs must vary to meet the needs of the particular organ, as well as of the whole body
Neural, myogenic, metabolic, and endothelial mechanisms control regional blood flow
Neural Mechanisms
Myogenic Mechanisms
Metabolic Mechanisms
Endothelial Mechanisms
The Brain
Anastomoses at the circle of Willis and among the branches of distributing arteries protect the blood supply to the brain, which is ~15% of resting cardiac output
Arteries
Veins
Capillaries
Lymphatics
Vascular Volume
Neural, metabolic, and myogenic mechanisms control blood flow to the brain
Neural Control
Metabolic Control
Myogenic Control
The neurovascular unit matches blood flow to local brain activity
Autoregulation maintains a fairly constant cerebral blood flow across a broad range of perfusion pressures
The Heart
The coronary circulation receives 5% of the resting cardiac output from the left heart and mostly returns it to the right heart
Extravascular compression impairs coronary blood flow during systole
Myocardial blood flow parallels myocardial metabolism
Although sympathetic stimulation directly constricts coronary vessels, accompanying metabolic effects predominate, producing an overall vasodilation
Collateral vessel growth can provide blood flow to ischemic regions
Vasodilator drugs may compromise myocardial flow through “coronary steal”
The Skeletal Muscle
A microvascular unit is the capillary bed supplied by a single terminal arteriole
Metabolites released by active muscle trigger vasodilation and an increase in blood flow
Sympathetic innervation increases the intrinsic tone of resistance vessels
Rhythmic contraction promotes blood flow through the “muscle pump”
The Splanchnic Organs
The vascular supply to the gut is highly interconnected
Blood flow to the gastrointestinal tract increases up to eight-fold after a meal (postprandial hyperemia)
Sympathetic activity directly constricts splanchnic blood vessels, whereas parasympathetic activity indirectly dilates them
Changes in the splanchnic circulation regulate total peripheral resistance and the distribution of blood volume
Exercise and hemorrhage can substantially reduce splanchnic blood flow
The liver receives its blood flow from both the systemic and the portal circulation
The Skin
The skin is the largest organ of the body
Specialized arteriovenous anastomoses in apical skin help control heat loss
Apical Skin
Nonapical Skin
Mechanical stimuli elicit local vascular responses in the skin
White Reaction
“Triple Response”
References
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Chapter 25: Integrated Control of the Cardiovascular System
Interaction among the Different Cardiovascular Control Systems
The control of the cardiovascular system involves “linear,” “branched,” and “connected” interactions
Regulation of the entire cardiovascular system depends on the integrated action of multiple subsystem controls as well as noncardiovascular controls
Response to Erect Posture
Because of gravity, standing up (orthostasis) tends to shift blood from the head and heart to veins in the legs
The ANS mediates an “orthostatic response” that raises heart rate and peripheral vascular resistance and thus tends to restore mean arterial pressure
Nonuniform Initial Distribution of Blood
Nonuniform Distensibility of the Vessels
Muscle Pumps
Autonomic Reflexes
Postural Hypotension
Temperature Effects
Responses to Acute Emotional Stress
The fight-or-flight reaction is a sympathetic response that is centrally controlled in the cortex and hypothalamus
The common faint reflects mainly a parasympathetic response caused by sudden emotional stress
Response to Exercise
Early physiologists suggested that muscle contraction leads to mechanical and chemical changes that trigger an increase in cardiac output
Mechanical Response: Increased Venous Return
Chemical Response: Local Vasodilation in Active Muscle
Central command organizes an integrated cardiovascular response to exercise
Muscle and baroreceptor reflexes, metabolites, venous return, histamine, epinephrine, and increased temperature reinforce the response to exercise
Response to Hemorrhage
After hemorrhage, cardiovascular reflexes restore mean arterial pressure
Tachycardia and Increased Contractility
Arteriolar Constriction
Venous Constriction
Circulating Vasoactive Agonists
After hemorrhage, transcapillary refill, fluid conservation, and thirst restore the blood volume
Transcapillary Refill
Renal Conservation of Salt and Water
Thirst
Positive-feedback mechanisms cause irreversible hemorrhagic shock
Failure of the Vasoconstrictor Response
Failure of the Capillary Refill
Failure of the Heart
CNS Depression
References
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Section V: The Respiratory System
Chapter 26: Organization of the Respiratory System
Comparative Physiology of Respiration
External respiration is the exchange of O2 and CO2 between the atmosphere and the mitochondria
Diffusion is the major mechanism of external respiration for small aquatic organisms
Convection enhances diffusion by producing steeper gradients across the diffusion barrier
Surface area amplification enhances diffusion
Respiratory pigments such as hemoglobin increase the carrying capacity of the blood for both O2 and CO2
Pathophysiology recapitulates phylogeny … in reverse
Organization of the Respiratory System in Humans
Humans optimize each aspect of external respiration—ventilation, circulation, area amplification, gas carriage, local control, and central control
Conducting airways deliver fresh air to the alveolar spaces
Alveolar air spaces are the site of gas exchange
The lungs play important nonrespiratory roles, including filtering the blood, serving as a reservoir for the left ventricle, and performing several biochemical conversions
Olfaction
Processing of Inhaled Air Before It Reaches the Alveoli
Left Ventricular Reservoir
Filtering Small Emboli from the Blood
Biochemical Reactions
Lung Volumes and Capacities
The spirometer measures changes in lung volume
The volume of distribution of helium or nitrogen in the lung is an estimate of the RV
Helium-Dilution Technique
N2-Washout Method
The plethysmograph, together with Boyle’s law, is a tool for estimation of RV
References
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Chapter 27: Mechanics of Ventilation
Static Properties of the Lung
The balance between the outward elastic recoil of the chest wall and the inward elastic recoil of the lungs generates a subatmospheric intrapleural pressure
Contraction of the diaphragm and selected intercostal muscles increases the volume of the thorax, producing an inspiration
Relaxation of the muscles of inspiration produces a quiet expiration
An increase of the static compliance makes it easier to inflate the lungs
Surface tension at the air-water interface of the airways accounts for most of the elastic recoil of the lungs
Pulmonary surfactant is a mixture of lipids—mainly dipalmitoylphosphatidylcholine—and apoproteins
Pulmonary surfactant reduces surface tension and increases compliance
Dynamic Properties of the Lung
Airflow is proportional to the difference between alveolar and atmospheric pressure, but inversely proportional to airway resistance
In the lung, airflow is transitional in most of the tracheobronchial tree
The smallest airways contribute only slightly to total airway resistance in healthy lungs
Vagal tone, histamine, and reduced lung volume all increase airway resistance
Intrapleural pressure has a static component (−PTP) that determines lung volume and a dynamic component (Pa) that determines airflow
Transpulmonary Pressure
Alveolar Pressure
During inspiration, a sustained negative shift in PIP causes Pa to become transiently more negative
Dynamic compliance falls as respiratory frequency rises
Transmural pressure differences cause airways to dilate during inspiration and to compress during expiration
Static Conditions
Inspiration
Expiration
Because of airway collapse, expiratory flow rates become independent of effort at low lung volumes
References
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Chapter 28: Acid-Base Physiology
pH and Buffers
pH values vary enormously among different intracellular and extracellular compartments
Buffers minimize the size of the pH changes produced by adding acid or alkali to a solution
According to the Henderson-Hasselbalch equation, pH depends on the ratio [CO2]/[]
has a far higher buffering power in an open than in a closed system
Acid-Base Chemistry When Is the Only Buffer
In the absence of other buffers, doubling causes pH to fall by 0.3 but causes almost no change in []
In the absence of other buffers, doubling [] causes pH to rise by 0.3
Acid-Base Chemistry in the Presence of and Buffers—The Davenport Diagram
The Davenport diagram is a graphical tool for interpreting acid-base disturbances in blood
The Buffer
Buffers
Solving the Problem
The amount of formed or consumed during “respiratory” acid-base disturbances increases with
Adding or removing an acid or base—at a constant —produces a “metabolic” acid-base disturbance
During metabolic disturbances, makes a greater contribution to total buffering when pH and are high and when is low
A metabolic change can compensate for a respiratory disturbance
A respiratory change can compensate for a metabolic disturbance
Position on a Davenport diagram defines the nature of an acid-base disturbance
pH Regulation of Intracellular Fluid
Ion transporters at the plasma membrane closely regulate the pH inside of cells
Indirect interactions between K+ and H+ make it appear as if cells have a K-H exchanger
Changes in intracellular pH are often a sign of changes in extracellular pH, and vice versa
References
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Chapter 29: Transport of Oxygen and Carbon Dioxide in the Blood
Carriage of O2
The amount of O2 dissolved in blood is far too small to meet the metabolic demands of the body
Hemoglobin consists of two α and two β subunits, each of which has an iron-containing “heme” and a polypeptide “globin”
The Hb-O2 dissociation curve has a sigmoidal shape because of cooperativity among the four subunits of the Hb molecule
Increases in temperature, [CO2], and [H+], all of which are characteristic of metabolically active tissues, cause Hb to dump O2
Temperature
Acid
Carbon Dioxide
2,3-Diphosphoglycerate reduces the affinity of adult, but not of fetal, Hb
Carriage of CO2
Blood carries “total CO2” mainly as
CO2 transport depends critically on carbonic anhydrase, the Cl-HCO3 exchanger, and Hb
The high in the lungs causes the blood to dump CO2
The O2-CO2 diagram describes the interaction of and in the blood
References
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Chapter 30: Gas Exchange in the Lungs
Diffusion of Gases
Gas flow across a barrier is proportional to diffusing capacity and concentration gradient (Fick’s law)
The total flux of a gas between alveolar air and blood is the summation of multiple diffusion events along each pulmonary capillary during the respiratory cycle
The flow of O2, CO, and CO2 between alveolar air and blood depends on the interaction of these gases with red blood cells
Diffusion and Perfusion Limitations on Gas Transport
The diffusing capacity normally limits the uptake of CO from alveolar air to blood
Perfusion normally limits the uptake of N2O from alveolar air to blood
In principle, CO transport could become perfusion limited and N2O transport could become diffusion limited under special conditions
The uptake of CO provides an estimate of DL
For both O2 and CO2, transport is normally perfusion limited
Uptake of O2
Escape of CO2
Pathological changes that reduce DL do not necessarily produce hypoxia
References
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Chapter 31: Ventilation and Perfusion of the Lungs
Ventilation
About 30% of total ventilation in a respiratory cycle is wasted ventilating anatomical dead space (i.e., conducting airways)
The Fowler single-breath N2-washout technique estimates anatomical dead space
The Bohr expired-[CO2] approach estimates physiological dead space
Alveolar ventilation is the ratio of CO2 production rate to CO2 mole fraction in alveolar air
Alveolar and arterial are inversely proportional to alveolar ventilation
Alveolar and arterial rise with increased alveolar ventilation
Because of the action of gravity on the lung, regional ventilation in an upright subject is normally greater at the base than the apex
Restrictive and obstructive pulmonary diseases can exacerbate the nonuniformity of ventilation
Restrictive Pulmonary Disease
Obstructive Pulmonary Disease
Perfusion of the Lung
The pulmonary circulation has low pressure and resistance but high compliance
Overall pulmonary vascular resistance is minimal at FRC
Alveolar Vessels
Extra-Alveolar Vessels
Increases in pulmonary arterial pressure reduce pulmonary vascular resistance by recruiting and distending pulmonary capillaries
Recruitment
Distention
Hypoxia is a strong vasoconstrictor, opposite to its effect in the systemic circulation
Oxygen
Carbon Dioxide and Low pH
Autonomic Nervous System
Hormones and Other Humoral Agents
Because of gravity, regional perfusion in an upright subject is far greater near the base than the apex of the lung
Zone 1: Pa > PPA > PPV
Zone 2: PPA > Pa > PPV
Zone 3: PPA > PPV > Pa
Zone 4: PPA > PPV > Pa
Matching Ventilation and Perfusion
The greater the ventilation-perfusion ratio, the higher the and the lower the in the alveolar air
Because of the action of gravity, the regional ratio in an upright subject is greater at the apex of the lung than at the base
The ventilation of unperfused alveoli (local = ∞) triggers compensatory bronchoconstriction and a fall in surfactant production
Alveolar Dead-Space Ventilation
Redirection of Blood Flow
Regulation of Local Ventilation
The perfusion of unventilated alveoli (local = 0) triggers a compensatory hypoxic vasoconstriction
Shunt
Redirection of Airflow
Asthma
Normal Anatomical Shunts
Pathological Shunts
Regulation of Local Perfusion
Even if whole-lung and are normal, exaggerated local mismatches produce hypoxia and respiratory acidosis
Normal Lungs
Alveolar Dead-Space Ventilation Affecting One Lung
Shunt Affecting One Lung
Mixed Mismatches
References
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Chapter 32: Control of Ventilation
Overview of the Respiratory Control System
Automatic centers in the brainstem activate the respiratory muscles rhythmically and subconsciously
Peripheral and central chemoreceptors—which sense , , and pH—drive the CPG
Other receptors as well as higher brain centers also modulate ventilation
Neurons That Control Ventilation
The neurons that generate the respiratory rhythm are located in the medulla
The pons modulates—but is not essential for—respiratory output
The dorsal and ventral respiratory groups contain many neurons that fire in phase with respiratory motor output
The dorsal respiratory group processes sensory input and contains primarily inspiratory neurons
The ventral respiratory group is primarily motor and contains both inspiratory and expiratory neurons
Generation of the Respiratory Rhythm
Different RRNs fire at different times during inspiration and expiration
The firing patterns of RRNs depend on the ion channels in their membranes and the synaptic inputs they receive
Intrinsic Membrane Properties
Synaptic Input
Pacemaker properties and synaptic interactions may both contribute to the generation of the respiratory rhythm
Pacemaker Activity
Synaptic Interactions
The respiratory CPG for eupnea could reside in a single site or in multiple sites, or could emerge from a complex network
Restricted-Site Model
Distributed Oscillator Models
Emergent Property Model
Chemical Control of Ventilation
Peripheral Chemoreceptors
Peripheral chemoreceptors (carotid and aortic bodies) respond to hypoxia, hypercapnia, and acidosis
Sensitivity to Decreased Arterial
Sensitivity to Increased Arterial
Sensitivity to Decreased Arterial pH
The glomus cell is the chemosensor in the carotid and aortic bodies
Hypoxia, hypercapnia, and acidosis inhibit K+ channels, raise glomus cell [Ca2+]i, and release neurotransmitters
Hypoxia N32-17
Hypercapnia
Extracellular Acidosis
Central Chemoreceptors
The blood-brain barrier separates the central chemoreceptors in the medulla from arterial blood
Central chemoreceptors are located in the ventrolateral medulla and other brainstem regions
Some neurons of the medullary raphé and VLM are unusually pH sensitive
Integrated Responses to Hypoxia, Hypercapnia, and Acidosis
Hypoxia accentuates the acute response to respiratory acidosis
Respiratory Acidosis
Metabolic Acidosis
Respiratory acidosis accentuates the acute response to hypoxia
Modulation of Ventilatory Control
Stretch and chemical/irritant receptors in the airways and lung parenchyma provide feedback about lung volume and the presence of irritants
Slowly Adapting Pulmonary Stretch Receptors
Rapidly Adapting Pulmonary Stretch (Irritant) Receptors
C-Fiber Receptors
Higher brain centers coordinate ventilation with other behaviors and can override the brainstem’s control of breathing
Coordination with Voluntary Behaviors That Use Respiratory Muscles
Coordination with Complex Nonventilatory Behaviors
Modification by Affective States
Balancing Conflicting Demands of Gas Exchange and Other Behaviors
References
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Section VI: The Urinary System
Chapter 33: Organization of the Urinary System
Functional Anatomy of the Kidney
The kidneys are paired, retroperitoneal organs with vascular and epithelial elements
The kidneys have a very high blood flow and glomerular capillaries flanked by afferent and efferent arterioles
The functional unit of the kidney is the nephron
The renal corpuscle has three components: vascular elements, the mesangium, and Bowman’s capsule and space
The tubule components of the nephron include the proximal tubule, loop of Henle, distal tubule, and collecting duct
The tightness of tubule epithelia increases from the proximal to the medullary collecting tubule
Main Elements of Renal Function
The nephron forms an ultrafiltrate of the blood plasma and then selectively reabsorbs the tubule fluid or secretes solutes into it
The JGA is a region where each thick ascending limb contacts its glomerulus
Sympathetic nerve fibers to the kidney regulate renal blood flow, glomerular filtration, and tubule reabsorption
The kidneys, as endocrine organs, produce renin, 1,25-dihydroxyvitamin D, erythropoietin, prostaglandins, and bradykinin
Measuring Renal Clearance and Transport
The clearance of a solute is the virtual volume of plasma that would be totally cleared of a solute in a given time
A solute’s urinary excretion is the algebraic sum of its filtered load, reabsorption by tubules, and secretion by tubules
Microscopic techniques make it possible to measure single-nephron rates of filtration, absorption, and secretion
Single-Nephron GFR
Handling of Water by Tubule Segments in a Single Nephron
Handling of Solutes by Tubule Segments in a Single Nephron
The Ureters and Bladder
The ureters propel urine from the renal pelvis to the bladder by peristaltic waves conducted along a syncytium of smooth-muscle cells
Sympathetic, parasympathetic, and somatic fibers innervate the urinary bladder and its sphincters
Bladder filling activates stretch receptors, initiating the micturition reflex, a spinal reflex under control of higher central nervous system centers
References
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Chapter 34: Glomerular Filtration and Renal Blood Flow
Glomerular Filtration
A high glomerular filtration rate is essential for maintaining stable and optimal extracellular levels of solutes and water
The clearance of inulin is a measure of GFR
The clearance of creatinine is a useful clinical index of GFR
Molecular size and electrical charge determine the filterability of solutes across the glomerular filtration barrier
Hydrostatic pressure in glomerular capillaries favors glomerular ultrafiltration, whereas oncotic pressure in capillaries and hydrostatic pressure in Bowman’s space oppose it
Renal Blood Flow
Increased glomerular plasma flow leads to an increase in GFR
Afferent and efferent arteriolar resistances control both glomerular plasma flow and GFR
Peritubular capillaries provide tubules with nutrients and retrieve reabsorbed fluid
Blood flow in the renal cortex exceeds that in the renal medulla
The clearance of para-aminohippurate is a measure of RPF
Control of Renal Blood Flow and Glomerular Filtration
Autoregulation keeps RBF and GFR relatively constant
Myogenic Response
Tubuloglomerular Feedback
Volume expansion and a high-protein diet increase GFR by reducing TGF
Four factors that modulate RBF and GFR play key roles in regulating effective circulating volume
Renin-Angiotensin-Aldosterone Axis
Sympathetic Nerves
Arginine Vasopressin
Atrial Natriuretic Peptide
Other vasoactive agents modulate RBF and GFR
Epinephrine
Dopamine
Endothelins
Prostaglandins
Leukotrienes
Nitric Oxide
References
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Chapter 35: Transport of Sodium and Chloride
Na+ and Cl− Transport by Different Segments of The Nephron
Na+ and Cl− reabsorption decreases from proximal tubules to Henle’s loops to classic distal tubules to collecting tubules and ducts
The tubule reabsorbs Na+ via both the transcellular and the paracellular pathways
Transcellular Na+ Reabsorption
Paracellular Na+ Reabsorption
Na+ and Cl−, and Water Transport at the Cellular and Molecular Level
Na+ reabsorption involves apical transporters or ENaCs and a basolateral Na-K pump
Proximal Tubule
Thin Limbs of Henle’s Loop
Thick Ascending Limb
Distal Convoluted Tubule
Initial and Cortical Collecting Tubules
Medullary Collecting Duct
Cl− reabsorption involves both paracellular and transcellular pathways
Proximal Tubule
Thick Ascending Limb
Distal Convoluted Tubule
Collecting Ducts
Water reabsorption is passive and secondary to solute transport
Proximal Tubule
Loop of Henle and Distal Nephron
The kidney’s high O2 consumption reflects a high level of active Na+ transport
Regulation of Na+ and Cl− Transport
Glomerulotubular balance stabilizes fractional Na+ reabsorption by the proximal tubule in the face of changes in the filtered Na+ load
The proximal tubule achieves GT balance by both peritubular and luminal mechanisms
Peritubular Factors in the Proximal Tubule
Luminal Factors in the Proximal Tubule
ECF volume contraction or expansion upsets GT balance
The distal nephron also increases Na+ reabsorption in response to an increased Na+ load
Four parallel pathways that regulate effective circulating volume all modulate Na+ reabsorption
Renin-Angiotensin-Aldosterone Axis
Sympathetic Division of the Autonomic Nervous System
Arginine Vasopressin (Antidiuretic Hormone)
Atrial Natriuretic Peptide
Dopamine, elevated plasma [Ca2+], an endogenous steroid, prostaglandins, and bradykinin all decrease Na+ reabsorption
Dopamine
Elevated Plasma [Ca2+]
Endogenous Na-K Pump Inhibitor
Prostaglandins
Bradykinin
References
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Journal Articles
Chapter 36: Transport of Urea, Glucose, Phosphate, Calcium, Magnesium, and Organic Solutes
Urea
The kidney filters, reabsorbs, and secretes urea
Urea excretion rises with increasing urinary flow
Glucose
The proximal tubule reabsorbs glucose via apical, electrogenic Na/glucose cotransport and basolateral facilitated diffusion
Glucose excretion in the urine occurs only when the plasma concentration exceeds a threshold
Other Organic Solutes
The proximal tubule reabsorbs amino acids using a wide variety of apical and basolateral transporters
An H+-driven cotransporter takes up oligopeptides across the apical membrane, whereas endocytosis takes up proteins and other large organic molecules
Oligopeptides
Proteins
Two separate apical Na+-driven cotransporters reabsorb monocarboxylates and dicarboxylates/tricarboxylates
The proximal tubule secretes PAH and a variety of other organic anions
PAH secretion is an example of a Tm-limited mechanism
The proximal tubule both reabsorbs and secretes urate
Reabsorption
Secretion
The late proximal tubule secretes several organic cations
Nonionic diffusion of neutral weak acids and bases across tubules explains why their excretion is pH dependent
Phosphate
The proximal tubule reabsorbs phosphate via apical Na/phosphate cotransporters
Phosphate excretion in the urine already occurs at physiological plasma concentrations
PTH inhibits apical Na/phosphate uptake, promoting phosphate excretion
Fibroblast growth factor 23 and other phosphatonins also inhibit apical Na/phosphate uptake, promoting phosphate excretion
Calcium
Binding to plasma proteins and formation of Ca2+-anion complexes influence the filtration and reabsorption of Ca2+
The proximal tubule reabsorbs two thirds of filtered Ca2+, with more distal segments reabsorbing nearly all of the remainder
Proximal Tubule
Thick Ascending Limb
Distal Convoluted Tubule
Transcellular Ca2+ movement is a two-step process, involving passive Ca2+ entry through apical channels and basolateral extrusion by electrogenic Na/Ca exchange and a Ca pump
PTH and vitamin D stimulate—whereas high plasma Ca2+ inhibits—Ca2+ reabsorption
Parathyroid Hormone
Vitamin D
Plasma Ca2+ Levels
Diuretics
Magnesium
Most Mg2+ reabsorption takes place along the TAL
Mg2+ reabsorption increases with depletion of Mg2+ or Ca2+, or with elevated PTH levels
Mg2+ Depletion
Hypermagnesemia and Hypercalcemia
Hormones
Diuretics
References
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Chapter 37: Transport of Potassium
Potassium Balance and the Overall Renal Handling of Potassium
Changes in K+ concentrations can have major effects on cell and organ function
K+ homeostasis involves external K+ balance between environment and body, and internal K+ balance between intracellular and extracellular compartments
External K+ Balance
Internal K+ Balance
Ingested K+ moves transiently into cells for storage before excretion by the kidney
The kidney excretes K+ by a combination of filtration, reabsorption, and secretion
Potassium Transport by Different Segments of the Nephron
The proximal tubule reabsorbs most of the filtered K+, whereas the distal nephron reabsorbs or secretes K+, depending on K+ intake
Low Dietary K+
Normal or High Dietary K+
Medullary trapping of K+ helps to maximize K+ excretion when K+ intake is high
Potassium Transport at the Cellular and Molecular Levels
Passive K+ reabsorption along the proximal tubule follows Na+ and fluid movements
K+ reabsorption along the TAL occurs predominantly via a transcellular route that exploits secondary active Na/K/Cl cotransport
K+ secretion by principal and intercalated cells of the ICT and CCT involves active K+ uptake across the basolateral membrane
K+ reabsorption by intercalated cells involves apical uptake via an H-K pump
K+ reabsorption along the MCD is both passive and active
Regulation of Renal Potassium Excretion
Increased luminal flow increases K+ secretion
An increased lumen-negative transepithelial potential increases K+ secretion
Low luminal [Cl−] enhances K+ secretion
Aldosterone increases K+ secretion
Mineralocorticoids
Glucocorticoids
High K+ intake promotes renal K+ secretion
Dietary K+ Loading
Dietary K+ Deprivation
Acidosis decreases K+ secretion
Epinephrine reduces and AVP enhances K+ excretion
Opposing factors stabilize K+ secretion
Attenuating Effects
Additive Effects
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Chapter 38: Urine Concentration and Dilution
Water Balance and the Overall Renal Handling of Water
The kidney can generate a urine as dilute as 40 mOsm (one seventh of plasma osmolality) or as concentrated as 1200 mOsm (four times plasma osmolality)
Free-water clearance () is positive if the kidney produces urine that is less concentrated than plasma and negative if the kidney produces urine that is more concentrated than plasma
Isosmotic Urine
Dilute Urine
Concentrated Urine
Water Transport by Different Segments of the Nephron
The kidney concentrates urine by driving water via osmosis from the tubule lumen into a hyperosmotic interstitium
Tubule fluid is isosmotic in the proximal tubule, becomes dilute in the loop of Henle, and then either remains dilute or becomes concentrated by the end of the collecting duct
Generation of a Hyperosmotic Medulla and Urine
The renal medulla is hyperosmotic to blood plasma during both antidiuresis (low urine flow) and water diuresis
NaCl transport generates only a ~200-mOsm gradient across any portion of the ascending limb, but countercurrent exchange can multiply this single effect to produce a 900-mOsm gradient between cortex and papilla
The single effect is the result of passive NaCl reabsorption in the thin ascending limb and active NaCl reabsorption in the TAL
The IMCD reabsorbs urea, producing high levels of urea in the interstitium of the inner medulla
Urea Handling
Urea Recycling
The vasa recta’s countercurrent exchange and relatively low blood flow minimize washout of medullary hyperosmolality
The MCD produces a concentrated urine by osmosis, driven by the osmotic gradient between the medullary interstitium and the lumen
Regulation by Arginine Vasopressin
AVP increases water permeability in all nephron segments beyond the DCT
AVP, via cAMP, causes vesicles containing AQP2 to fuse with apical membranes of principal cells of collecting tubules and ducts
AVP increases NaCl reabsorption in the outer medulla and urea reabsorption in the IMCD, enhancing urinary concentrating ability
References
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Chapter 39: Transport of Acids and Bases
Acid-Base Balance and the Overall Renal Handling of Acid
Whereas the lungs excrete the large amount of CO2 formed by metabolism, the kidneys are crucial for excreting nonvolatile acids
To maintain acid-base balance, the kidney must not only reabsorb virtually all filtered but also secrete generated nonvolatile acids
Secreted H+ titrates to CO2 ( reabsorption) and also titrates filtered buffers and endogenously produced NH3
Titration of Filtered (“ Reabsorption”)
Titration of Filtered Buffers (Titratable-Acid Formation)
Titration of Filtered and Secreted NH3 (Ammonium Excretion)
Acid-Base Transport by Different Segments of the Nephron
The nephron reclaims virtually all the filtered in the proximal tubule (~80%), thick ascending limb (~10%), and distal nephron (~10%)
The nephron generates new , mostly in the proximal tubule
Formation of Titratable Acid
Excretion
Acid-Base Transport at the Cellular and Molecular Levels
H+ moves across the apical membrane from tubule cell to lumen by Na-H exchange, electrogenic H pumping, and K-H pumping
Na-H Exchanger
Electrogenic H Pump
H-K Exchange Pump
CAs in the lumen and cytosol stimulate H+ secretion by accelerating the interconversion of CO2 and
Apical CA (CA IV)
Cytoplasmic CA (CA II)
Basolateral CA (CA IV and CA XII)
Inhibition of CA
efflux across the basolateral membrane takes place by electrogenic Na/HCO3 cotransport and Cl-HCO3 exchange
Electrogenic Na/HCO3 Cotransport
Cl-HCO3 Exchange
is synthesized by proximal tubules, partly reabsorbed in the loop of Henle, and secreted passively into papillary collecting ducts
Regulation of Renal Acid Secretion
Respiratory acidosis stimulates renal H+ secretion
Metabolic acidosis stimulates both proximal H+ secretion and NH3 production
Metabolic alkalosis reduces proximal H+ secretion and, in the CCT, may even provoke secretion
A rise in GFR increases delivery to the tubules, enhancing reabsorption (glomerulotubular balance for )
Extracellular volume contraction—via ANG II, aldosterone, and sympathetic activity—stimulates renal H+ secretion
Hypokalemia increases renal H+ secretion
Both glucocorticoids and mineralocorticoids stimulate acid secretion
Diuretics can change H+ secretion, depending on how they affect transepithelial voltage, ECF volume, and plasma [K+]
References
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Chapter 40: Integration of Salt and Water Balance
Sodium Balance
Water Balance
Control of Extracellular Fluid Volume
In the steady state, Na+ intake via the gastrointestinal tract equals Na+ output from renal and extrarenal pathways
The kidneys increase Na+ excretion in response to an increase in ECF volume, not to an increase in extracellular Na+ concentration
It is not the ECF volume as a whole, but the effective circulating volume, that regulates Na+ excretion
Decreases in effective circulating volume trigger four parallel effector pathways to decrease renal Na+ excretion
Increased activity of the renin-angiotensin-aldosterone axis is the first of four parallel pathways that correct a low effective circulating volume
Increased sympathetic nerve activity, increased AVP, and decreased ANP are the other three parallel pathways that correct a low effective circulating volume
Renal Sympathetic Nerve Activity
Arginine Vasopressin (Antidiuretic Hormone)
Atrial Natriuretic Peptide
High arterial pressure raises Na+ excretion by hemodynamic mechanisms, independent of changes in effective circulating volume
Large and Acute Decrease in Arterial Blood Pressure
Large Increase in Arterial Pressure
Control of Water Content (Extracellular Osmolality)
Increased plasma osmolality stimulates hypothalamic osmoreceptors that trigger the release of AVP, inhibiting water excretion
Hypothalamic neurons synthesize AVP and transport it along their axons to the posterior pituitary, where they store it in nerve terminals prior to release
Increased osmolality stimulates a second group of osmoreceptors that trigger thirst, which promotes water intake
Several nonosmotic stimuli also enhance AVP secretion
Reduced Effective Circulating Volume
Volume Expansion
Pregnancy
Other Factors
Decreased effective circulating volume and low arterial pressure also trigger thirst
Defense of the effective circulating volume usually has priority over defense of osmolality
References
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Section VII: The Gastrointestinal System
Chapter 41: Organization of the Gastrointestinal System
Overview of Digestive Processes
The gastrointestinal tract is a tube that is specialized along its length for the sequential processing of food
Assimilation of dietary food substances requires digestion as well as absorption
Digestion requires enzymes secreted in the mouth, stomach, pancreas, and small intestine
Ingestion of food initiates multiple endocrine, neural, and paracrine responses
In addition to its function in nutrition, the GI tract plays important roles in excretion, fluid and electrolyte balance, and immunity
Regulation of Gastrointestinal Function
The ENS is a “minibrain” with sensory neurons, interneurons, and motor neurons
ACh, peptides, and bioactive amines are the ENS neurotransmitters that regulate epithelial and motor function
The brain-gut axis is a bidirectional system that controls GI function via the ANS, GI hormones, and the immune system
Gastrointestinal Motility
Tonic and rhythmic contractions of smooth muscle are responsible for churning, peristalsis, and reservoir action
Segments of the GI tract have both longitudinal and circular arrays of muscles and are separated by sphincters that consist of specialized circular muscles
Location of a sphincter determines its function
Upper Esophageal Sphincter
Lower Esophageal Sphincter
Pyloric Sphincter
Ileocecal Sphincter
Internal and External Anal Sphincters
Motility of the small intestine achieves both churning and propulsive movement, and its temporal pattern differs in the fed and fasted states
Motility of the large intestine achieves both propulsive movement and a reservoir function
References
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Chapter 42: Gastric Function
Functional Anatomy of the Stomach
The mucosa is composed of surface epithelial cells and glands
With increasing rates of secretion of gastric juice, the H+ concentration rises and the Na+ concentration falls
The proximal portion of the stomach secretes acid, pepsinogens, intrinsic factor, bicarbonate, and mucus, whereas the distal part releases gastrin and somatostatin
Corpus
Antrum
The stomach accommodates food, mixes it with gastric secretions, grinds it, and empties the chyme into the duodenum
Acid Secretion
The parietal cell has a specialized tubulovesicular structure that increases apical membrane area when the cell is stimulated to secrete acid
An H-K pump is responsible for gastric acid secretion by parietal cells
Three secretagogues (acetylcholine, gastrin, and histamine) directly and indirectly induce acid secretion by parietal cells
The three acid secretagogues act through either Ca2+/diacylglycerol or cAMP
Antral and duodenal G cells release gastrin, whereas ECL cells in the corpus release histamine
Gastric D cells release somatostatin, the central inhibitor of acid secretion
Several enteric hormones (“enterogastrone”) and prostaglandins inhibit gastric acid secretion
A meal triggers three phases of acid secretion
Basal State
Cephalic Phase
Gastric Phase
Intestinal Phase
Pepsinogen Secretion
Chief cells, triggered by both cAMP and Ca2+ pathways, secrete multiple pepsinogens that initiate protein digestion
Agonists Acting via cAMP
Agonists Acting via Ca2+
Low pH is required for both pepsinogen activation and pepsin activity
Protection of the Gastric Surface Epithelium and Neutralization of Acid in the Duodenum
Vagal stimulation and irritation stimulate gastric mucous cells to secrete mucins
Gastric surface cells secrete , stimulated by acetylcholine, acids, and prostaglandins
Mucus protects the gastric surface epithelium by trapping an -rich fluid near the apical border of these cells
Acid entry into the duodenum induces S cells to release secretin, triggering the pancreas and duodenum to secrete
Filling and Emptying of the Stomach
Gastric motor activity plays a role in filling, churning, and emptying
Filling of the stomach is facilitated by both receptive relaxation and gastric accommodation
The stomach churns its contents until the particles are small enough to be gradually emptied into the duodenum
References
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Chapter 43: Pancreatic and Salivary Glands
Overview of Exocrine Gland Physiology
The pancreas and major salivary glands are compound exocrine glands
Acinar cells are specialized protein-synthesizing cells
Duct cells are epithelial cells specialized for fluid and electrolyte transport
Goblet cells contribute to mucin production in exocrine glands
Pancreatic Acinar Cell
The acinar cell secretes digestive proteins in response to stimulation
Acetylcholine and cholecystokinin mediate the regulated secretion of proteins by pancreatic acinar cells
Ca2+ is the major second messenger for the secretion of proteins by pancreatic acinar cells
Ca2+
cAMP
Effectors
In addition to proteins, the pancreatic acinar cell secretes a plasma-like fluid
Pancreatic Duct Cell
The pancreatic duct cell secretes isotonic NaHCO3
Secretin (via cAMP) and ACh (via Ca2+) stimulate secretion by pancreatic ducts
Apical membrane chloride channels are important sites of neurohumoral regulation
Pancreatic duct cells may also secrete glycoproteins
Composition, Function, and Control of Pancreatic Secretion
Pancreatic juice is a protein-rich, alkaline secretion
In the fasting state, levels of secreted pancreatic enzymes oscillate at low levels
CCK from duodenal I cells stimulates acinar enzyme secretion, and secretin from S cells stimulates and fluid secretion by ducts
A meal triggers cephalic, gastric, and intestinal phases of pancreatic secretion
Cephalic Phase
Gastric Phase
Intestinal Phase
The pancreas has large reserves of digestive enzymes for carbohydrates and proteins, but not for lipids
Fat in the distal part of the small intestine inhibits pancreatic secretion
Several mechanisms protect the pancreas from autodigestion
Salivary Acinar Cell
Different salivary acinar cells secrete different proteins
Cholinergic and adrenergic neural pathways are the most important physiological activators of regulated secretion by salivary acinar cells
Both cAMP and Ca2+ mediate salivary acinar secretion
Salivary Duct Cell
Salivary duct cells produce a hypotonic fluid that is poor in NaCl and rich in KHCO3
Parasympathetic stimulation decreases Na+ absorption, whereas aldosterone increases Na+ absorption by duct cells
Salivary duct cells also secrete and take up proteins
Composition, Function, and Control of Salivary Secretion
Depending on protein composition, salivary secretions can be serous, seromucous, or mucous
At low flow rates, the saliva is hypotonic and rich in K+, whereas at higher flow rates, its composition approaches that of plasma
Parasympathetic stimulation increases salivary secretion
Parasympathetic Control
Sympathetic Control
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Chapter 44: Intestinal Fluid and Electrolyte Movement
Functional Anatomy
Both the small and large intestine absorb and secrete fluid and electrolytes, whereas only the small intestine absorbs nutrients
The small intestine has a villus-crypt organization, whereas the colon has surface epithelial cells with interspersed crypts
The surface area of the small intestine is amplified by folds, villi, and microvilli; amplification is less marked in the colon
Overview of Fluid and Electrolyte Movement in the Intestines
The small intestine absorbs ~6.5 L/day of an ~8.5-L fluid load that is presented to it, and the colon absorbs ~1.9 L/day
The small intestine absorbs net amounts of water, Na+, Cl−, and K+ and secretes , whereas the colon absorbs net amounts of water, Na+, and Cl− and secretes both K+ and
The intestines absorb and secrete solutes by both active and passive mechanisms
Intestinal fluid movement is always coupled to solute movement, and sometimes solute movement is coupled to fluid movement by solvent drag
The resistance of the tight junctions primarily determines the transepithelial resistance of intestinal epithelia
Cellular Mechanisms of Na+ Absorption
Na/glucose and Na/amino-acid cotransport in the small intestine is a major mechanism for postprandial Na+ absorption
Electroneutral Na-H exchange in the duodenum and jejunum is responsible for Na+ absorption that is stimulated by luminal alkalinity
Parallel Na-H and Cl-HCO3 exchange in the ileum and proximal part of the colon is the primary mechanism of Na+ absorption during the interdigestive period
Epithelial Na+ channels are the primary mechanism of “electrogenic” Na+ absorption in the distal part of the colon
Cellular Mechanisms of Cl− Absorption and Secretion
Voltage-dependent Cl− absorption represents coupling of Cl− absorption to electrogenic Na+ absorption in both the small intestine and the large intestine
Electroneutral Cl-HCO3 exchange results in Cl− absorption and secretion in the ileum and colon
Parallel Na-H and Cl-HCO3 exchange in the ileum and the proximal part of the colon mediates Cl− absorption during the interdigestive period
Electrogenic Cl− secretion occurs in crypts of both the small and the large intestine
Cellular Mechanisms of K+ Absorption and Secretion
Overall net transepithelial K+ movement is absorptive in the small intestine and secretory in the colon
K+ absorption in the small intestine probably occurs via solvent drag
Passive K+ secretion is the primary mechanism for net colonic secretion
Active K+ secretion is also present throughout the large intestine and is induced both by aldosterone and by cAMP
Aldosterone
cAMP and Ca2+
Active K+ absorption takes place only in the distal portion of the colon and is energized by an apical H-K pump
Regulation of Intestinal Ion Transport
Chemical mediators from the enteric nervous system, endocrine cells, and immune cells in the lamina propria may be either secretagogues or absorptagogues
Secretagogues can be classified by their type and by the intracellular second-messenger system that they stimulate
Mineralocorticoids, glucocorticoids, and somatostatin are absorptagogues
References
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Chapter 45: Nutrient Digestion and Absorption
Carbohydrate Digestion
Carbohydrates, providing ~45% of total energy needs of Western diets, require hydrolysis to monosaccharides before absorption
Luminal digestion begins with the action of salivary amylase and finishes with pancreatic amylase
“Membrane digestion” involves hydrolysis of oligosaccharides to monosaccharides by brush-border disaccharidases
Carbohydrate Absorption
SGLT1 is responsible for the Na+-coupled uptake of glucose and galactose across the apical membrane
The GLUT transporters mediate the facilitated diffusion of fructose at the apical membrane and of all three monosaccharides at the basolateral membrane
Protein Digestion
Proteins require hydrolysis to oligopeptides or amino acids before absorption in the small intestine
Luminal digestion of protein involves both gastric and pancreatic proteases, and yields amino acids and oligopeptides
Brush-border peptidases fully digest some oligopeptides to amino acids, whereas cytosolic peptidases digest oligopeptides that directly enter the enterocyte
Protein, Peptide, and Amino-Acid Absorption
Absorption of whole protein by apical endocytosis occurs primarily during the neonatal period
The apical absorption of dipeptides, tripeptides, and tetrapeptides occurs via an H+-driven cotransporter
Amino acids enter enterocytes via one or more group-specific apical transporters
At the basolateral membrane, amino acids exit enterocytes via Na+-independent transporters and enter via Na+-dependent transporters
Lipid Digestion
Natural lipids of biological origin are sparingly soluble in water
Dietary lipids are predominantly TAGs
Endogenous lipids are phospholipids and cholesterol from bile and membrane lipids from desquamated intestinal epithelial cells
The mechanical disruption of dietary lipids in the mouth and stomach produces an emulsion of lipid particles
Lingual and gastric (acid) lipase initiate lipid digestion
Pancreatic (alkaline) lipase, colipase, milk lipase, and other esterases—aided by bile salts—complete lipid hydrolysis in the duodenum and jejunum
Lipid Absorption
Products of lipolysis enter the bulk water phase of the intestinal lumen as vesicles, mixed micelles, and monomers
Lipids diffuse as mixed micelles and monomers through unstirred layers before crossing the jejunal enterocyte brush border
The enterocyte re-esterifies lipid components and assembles them into chylomicrons
The enterocyte secretes chylomicrons into the lymphatics during feeding and secretes VLDLs during fasting
Digestion and Absorption of Vitamins and Minerals
Intestinal absorption of fat-soluble vitamins follows the pathways of lipid absorption and transport
Dietary folate (PteGlu7) must be deconjugated by a brush-border enzyme before absorption by an anion exchanger at the apical membrane
Vitamin B12 (cobalamin) binds to haptocorrin in the stomach and then to intrinsic factor in the small intestine before endocytosis by enterocytes in the ileum
Ca2+ absorption, regulated primarily by vitamin D, occurs by active transport in the duodenum and by diffusion throughout the small intestine
Mg2+ absorption occurs by an active process in the ileum
Heme and nonheme iron are absorbed in the duodenum by distinct cellular mechanisms
Nonheme Iron
Heme Iron
Nutritional Requirements
No absolute daily requirement for carbohydrate or fat intake exists
The daily protein requirement for adult humans is typically 0.8 g/kg body weight but is higher in pregnant women, postsurgical patients, and athletes
Minerals and vitamins are not energy sources but are necessary for certain enzymatic reactions, for protein complexes, or as precursors for biomolecules
Minerals
Vitamins
Excessive intake of vitamins and minerals has mixed effects on bodily function
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Chapter 46: Hepatobiliary Function
Overview of Liver Physiology
The liver biotransforms and degrades substances taken up from blood and either returns them to the circulation or excretes them into bile
The liver stores carbohydrates, lipids, vitamins, and minerals; it synthesizes carbohydrates, protein, and intermediary metabolites
Functional Anatomy of the Liver and Biliary Tree
Hepatocytes are secretory epithelial cells separating the lumen of bile canaliculi from the fenestrated endothelium of sinusoids
The liver contains endothelial cells, macrophages (Kupffer cells), and stellate cells (Ito cells) within the sinusoidal spaces
The liver has a dual blood supply, but a single venous drainage system
Hepatocytes can be thought of as being arranged as classic hepatic lobules, portal lobules, or acinar units
Periportal hepatocytes specialize in oxidative metabolism, whereas pericentral hepatocytes detoxify drugs
Bile drains from canaliculi into small terminal ductules, then into larger ducts, and eventually, via a single common duct, into the duodenum
Uptake, Processing, and Secretion of Compounds by Hepatocytes
An Na-K pump at the basolateral membranes of hepatocytes provides the energy for transporting a wide variety of solutes via channels and transporters
Hepatocytes take up bile acids, other organic anions, and organic cations across their basolateral (sinusoidal) membranes
Bile Acids and Salts
Organic Anions
Bilirubin
Organic Cations
Neutral Organic Compounds
Inside the hepatocyte, the basolateral-to-apical movement of many compounds occurs by protein-bound or vesicular routes
Bile Salts
Bilirubin
In phase I of the biotransformation of organic anions and other compounds, hepatocytes use mainly cytochrome P-450 enzymes
In phase II of biotransformation, conjugation of phase I products makes them more water soluble for secretion into blood or bile
In phase III of biotransformation, hepatocytes excrete products of phase I and II into bile or sinusoidal blood
The interactions of xenobiotics with nuclear receptors control phase I, II, and III
Hepatocytes secrete bile acids, organic anions, organic cations, and lipids across their apical (canalicular) membranes
Bile Salts
Organic Anions
Organic Cations
Biliary Lipids
Hepatocytes take up proteins across their basolateral membranes by receptor-mediated endocytosis and fluid-phase endocytosis
Bile Formation
The secretion of canalicular bile is active and isotonic
Major organic molecules in bile include bile acids, cholesterol, and phospholipids
Canalicular bile flow has a constant component driven by the secretion of small organic molecules and a variable component driven by the secretion of bile acids
Bile Acid–Independent Flow in the Canaliculi
Bile Acid–Dependent Flow in the Canaliculi
Secretin stimulates the cholangiocytes of ductules and ducts to secrete a watery, -rich fluid
The gallbladder stores bile and delivers it to the duodenum during a meal
The relative tones of the gallbladder and sphincter of Oddi determine whether bile flows from the common hepatic duct into the gallbladder or into the duodenum
Enterohepatic Circulation of Bile Acids
The enterohepatic circulation of bile acids is a loop consisting of secretion by the liver, reabsorption by the intestine, and return to the liver in portal blood for repeat secretion into bile
Efficient intestinal conservation of bile acids depends on active apical absorption in the terminal ileum and passive absorption throughout the intestinal tract
The Liver as a Metabolic Organ
The liver can serve as either a source or a sink for glucose
The liver synthesizes a variety of important plasma proteins (e.g., albumin, coagulation factors, and carriage proteins) and metabolizes dietary amino acids
Protein Synthesis
Amino-Acid Uptake
Amino-Acid Metabolism
The liver obtains dietary triacylglycerols and cholesterol by taking up remnant chylomicrons via receptor-mediated endocytosis
Cholesterol, synthesized primarily in the liver, is an important component of cell membranes and serves as a precursor for bile acids and steroid hormones
Synthesis of Cholesterol
The liver is the central organ for cholesterol homeostasis and for the synthesis and degradation of LDL
The liver is the prime site for metabolism and storage of the fat-soluble vitamins A, D, E, and K
Vitamin A
Vitamin D
Vitamin E
Vitamin K
The liver stores copper and iron
Copper
Iron
References
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Section VIII: The Endocrine System
Chapter 47: Organization of Endocrine Control
Principles of Endocrine Function
Chemical signaling can occur through endocrine, paracrine, or autocrine pathways
Endocrine Glands
Paracrine Factors
Hormones may be peptides, metabolites of single amino acids, or metabolites of cholesterol
Hormones can circulate either free or bound to carrier proteins
Immunoassays allow measurement of circulating hormones
Hormones can have complementary and antagonistic actions
Endocrine regulation occurs through feedback control
Endocrine regulation can involve hierarchic levels of control
The anterior pituitary regulates reproduction, growth, energy metabolism, and stress responses
The posterior pituitary regulates water balance and uterine contraction
Peptide Hormones
Specialized endocrine cells synthesize, store, and secrete peptide hormones
Peptide hormones bind to cell-surface receptors and activate a variety of signal-transduction systems
G Proteins Coupled to Adenylyl Cyclase
G Proteins Coupled to Phospholipase C
G Proteins Coupled to Phospholipase A2
Guanylyl Cyclase
Receptor Tyrosine Kinases
Tyrosine Kinase–Associated Receptors
Amine Hormones
Amine hormones are made from tyrosine and tryptophan
Amine hormones act via surface receptors
Steroid and Thyroid Hormones
Cholesterol is the precursor for the steroid hormones: cortisol, aldosterone, estradiol, progesterone, and testosterone
Steroid hormones bind to intracellular receptors that regulate gene transcription
Thyroid hormones bind to intracellular receptors that regulate metabolic rate
Steroid and thyroid hormones can also have nongenomic actions
References
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Chapter 48: Endocrine Regulation of Growth and Body Mass
Growth Hormone
GH, secreted by somatotrophs in the anterior pituitary, is the principal endocrine regulator of growth
GH is in a family of hormones with overlapping activity
Somatotrophs secrete GH in pulses
GH secretion is under hierarchical control by GH–releasing hormone and somatostatin
GH-Releasing Hormone
GHRH Receptor
Ghrelin
Ghrelin Receptor
Somatostatin
SS Receptor
Both GH and IGF-1 negatively feed back on GH secretion by somatotrophs
GH has short-term anti-insulin metabolic effects as well as long-term growth-promoting effects mediated by IGF-1
GH Receptor
Short-Term Effects of GH
Long-Term Effects of GH via IGF-1
Growth-Promoting Hormones
IGF-1 is the principal mediator of the growth-promoting action of GH
IGF-2 acts similarly to IGF-1 but is less dependent on GH
Growth rate parallels plasma levels of IGF-1 except early and late in life
Thyroid hormones, steroids, and insulin also promote growth
Thyroid Hormones
Sex Steroids
Glucocorticoids
Insulin
The musculoskeletal system responds to growth stimuli of the GHRH–GH–IGF-1 axis
Regulation of Body Mass
The balance between energy intake and expenditure determines body mass
Energy expenditure comprises resting metabolic rate, activity-related energy expenditure, and diet-induced thermogenesis
Hypothalamic centers control the sensations of satiety and hunger
Leptin tells the brain how much fat is stored
Leptin and insulin are anorexigenic (i.e., satiety) signals for the hypothalamus
POMC Neurons
NPY/AgRP Neurons
Secondary Neurons
Ghrelin is an orexigenic signal for the hypothalamus
Plasma nutrient levels and enteric hormones are short-term factors that regulate feeding
References
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Chapter 49: The Thyroid Gland
Synthesis of Thyroid Hormones
T4 and T3, made by iodination of tyrosine residues on thyroglobulin, are stored as part of thyroglobulin molecules in thyroid follicles
Follicular cells take up iodinated thyroglobulin, hydrolyze it, and release T4 and T3 into the blood for binding to plasma proteins
Peripheral tissues deiodinate T4 to produce T3
Action of Thyroid Hormones
Thyroid hormones act through nuclear receptors in target tissues
Thyroid hormones can also act by nongenomic pathways
Thyroid hormones increase basal metabolic rate by stimulating futile cycles of catabolism and anabolism
Carbohydrate Metabolism
Protein Metabolism
Lipid Metabolism
Na-K Pump Activity
Thermogenesis
Thyroid hormones are essential for normal growth and development
Hypothalamic-Pituitary-Thyroid Axis
TRH from the hypothalamus stimulates thyrotrophs of the anterior pituitary to secrete TSH, which stimulates T4/T3 synthesis
Thyrotropin-Releasing Hormone
TRH Receptor
Thyrotropin
TSH Receptor
T3 exerts negative feedback on TSH secretion
References
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Chapter 50: The Adrenal Gland
The Adrenal Cortex: Cortisol
Cortisol is the primary glucocorticoid hormone in humans
Target Tissues
Actions
The adrenal zona fasciculata converts cholesterol to cortisol
Cortisol binds to a cytoplasmic receptor that translocates to the nucleus and modulates transcription in multiple tissues
Corticotropin-releasing hormone from the hypothalamus stimulates anterior pituitary corticotrophs to secrete ACTH, which stimulates the adrenal cortex to synthesize and secrete cortisol
Corticotropin-Releasing Hormone
CRH Receptor
Arginine Vasopressin
Adrenocorticotropic Hormone
ACTH Receptor
Cortisol exerts negative feedback on CRH and ACTH secretion, whereas stress acts through higher CNS centers to stimulate the axis
Feedback to the Anterior Pituitary
Feedback to the Hypothalamus
Control by a Higher CNS Center
The Adrenal Cortex: Aldosterone
The mineralocorticoid aldosterone is the primary regulator of salt balance and extracellular volume
The glomerulosa cells of the adrenal cortex synthesize aldosterone from cholesterol via progesterone
Aldosterone stimulates Na+ reabsorption and K+ excretion by the renal tubule
Angiotensin II, K+, and ACTH all stimulate aldosterone secretion
Angiotensin II
Potassium
Adrenocorticotropic Hormone
Aldosterone exerts indirect negative feedback on the renin-angiotensin axis by increasing effective circulating volume and by lowering plasma [K+]
Renin-Angiotensin Axis
Potassium
Role of Aldosterone in Normal Physiology
Role of Aldosterone in Disease
The Adrenal Medulla
The adrenal medulla bridges the endocrine and sympathetic nervous systems
Only chromaffin cells of the adrenal medulla have the enzyme for epinephrine synthesis
Catecholamines bind to α and β adrenoceptors on the cell surface and act through heterotrimeric G proteins
The CNS-epinephrine axis provides integrated control of multiple functions
References
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Chapter 51: The Endocrine Pancreas
The islets of Langerhans are endocrine and paracrine tissue
Insulin
Insulin replenishes fuel reserves in muscle, liver, and adipose tissue
β cells synthesize and secrete insulin
The Insulin Gene
Insulin Synthesis
Secretion of Insulin, Proinsulin, and C Peptide
Glucose is the major regulator of insulin secretion
Metabolism of glucose by the β cell triggers insulin secretion
Neural and humoral factors modulate insulin secretion
Exercise
Feeding
The insulin receptor is a receptor tyrosine kinase
High levels of insulin lead to downregulation of insulin receptors
In liver, insulin promotes conversion of glucose to glycogen stores or to triacylglycerols
Glycogen Synthesis and Glycogenolysis
Glycolysis and Gluconeogenesis
Lipogenesis
Protein Metabolism
In muscle, insulin promotes the uptake of glucose and its storage as glycogen
In adipocytes, insulin promotes glucose uptake and conversion to TAGs for storage
Glucagon
Pancreatic α cells secrete glucagon in response to ingested protein
Pancreatic α Cells
Intestinal L Cells
Glucagon, acting through cAMP, promotes the synthesis of glucose by the liver
Glucagon promotes oxidation of fat in the liver, which can lead to ketogenesis
Somatostatin
Somatostatin inhibits the secretion of growth hormone, insulin, and other hormones
References
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Chapter 52: The Parathyroid Glands and Vitamin D
Calcium and Phosphate Balance
The gut, kidneys, and bone regulate calcium balance
The gut, kidneys, and bone also regulate phosphate balance
Physiology of Bone
Dense cortical bone and the more reticulated trabecular bone are the two major bone types
The extracellular matrix forms the nidus for the nucleation of hydroxyapatite crystals
Bone remodeling depends on the closely coupled activities of osteoblasts and osteoclasts
Parathyroid Hormone
Plasma Ca2+ regulates the synthesis and secretion of PTH
PTH Synthesis and Vitamin D
Processing of PTH
Metabolism of PTH
High plasma [Ca2+] inhibits the synthesis and release of PTH
The PTH receptor couples via G proteins to either adenylyl cyclase or phospholipase C
In the kidney, PTH promotes Ca2+ reabsorption, phosphate loss, and 1-hydroxylation of 25-hydroxyvitamin D
Stimulation of Ca2+ Reabsorption
Inhibition of Phosphate Reabsorption
Stimulation of the Last Step of Synthesis of 1,25- Dihydroxyvitamin D
In bone, PTH can promote net resorption or net deposition
Bone Resorption by Indirect Stimulation of Osteoclasts
Bone Resorption by Reduction in Bone Matrix
Bone Deposition
Vitamin D
The active form of vitamin D is its 1,25-dihydroxy metabolite
Vitamin D, by acting on the small intestine and kidney, raises plasma [Ca2+] and thus promotes bone mineralization
Small Intestine
Kidney
Bone
Calcium ingestion lowers—whereas phosphate ingestion raises—levels of both PTH and 1,25-dihydroxyvitamin D
Calcium Ingestion
Phosphate Ingestion
Calcitonin and Other Hormones
Calcitonin inhibits osteoclasts, but its effects are transitory
Sex steroid hormones promote bone deposition, whereas glucocorticoids promote resorption
PTHrP, encoded by a gene that is entirely distinct from that for PTH, can cause hypercalcemia in certain malignancies
References
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Section IX: The Reproductive System
Chapter 53: Sexual Differentiation
Genetic Aspects of Sexual Differentiation
Meiosis occurs only in germ cells and gives rise to male and female gametes
Fertilization of an oocyte by an X- or Y-bearing sperm establishes the zygote’s genotypic sex
Genotypic sex determines differentiation of the indifferent gonad into either an ovary or a testis
The testis-determining gene is located on the Y chromosome
Endocrine and paracrine messengers modulate phenotypic differentiation
Differentiation of the Gonads
Primordial germ cells migrate from the yolk sac to the primordial gonad
The primitive testis develops from the medulla of the primordial gonad
The primitive ovary develops from the cortex of the primordial gonad
Development of the Accessory Sex Organs
The embryonic gonad determines the development of the internal genitalia and the external sexual phenotype
Embryos of both sexes have a double set of embryonic genital ducts
In males, the wolffian ducts become the epididymis, vas deferens, seminal vesicles, and ejaculatory duct
In females, the müllerian ducts become the fallopian tubes, the uterus, and the upper third of the vagina
In males, development of the wolffian ducts requires testosterone
In males, antimüllerian hormone causes regression of the müllerian ducts
Differentiation of the External Genitalia
The urogenital sinus develops into the urinary bladder, the urethra, and, in females, the vestibule of the vagina
The external genitalia of both sexes develop from common anlagen
Endocrine and Paracrine Control of Sexual Differentiation
The SRY gene triggers development of the testis, which makes the androgens and AMH necessary for male sexual differentiation
Testosterone Production
Androgen Receptor
DHT Formation
Antimüllerian Hormone
Androgens direct the male pattern of sexual differentiation of the internal ducts, the urogenital sinus, and the external genitalia
Differentiation of the Duct System
Differentiation of the Urogenital Sinus and External Genitalia
Androgens and estrogens influence sexual differentiation of the brain
Puberty
Puberty involves steroid hormones produced by the gonads and the adrenals
Hypothalamic gonadotropin-releasing hormone secretion controls puberty
Multiple factors control the timing of puberty
Androgens and estrogens influence secondary sex characteristics at puberty
Males
Females
The appearance of secondary sex characteristics at puberty completes sexual differentiation and development
References
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Chapter 54: The Male Reproductive System
Hypothalamic-Pituitary-Gonadal Axis
The hypothalamus secretes GnRH, which acts on gonadotrophs in the anterior pituitary
Under the control of GnRH, gonadotrophs in the anterior pituitary secrete LH and FSH
LH stimulates the Leydig cells of the testis to produce testosterone
FSH stimulates Sertoli cells to synthesize hormones that influence Leydig cells and spermatogenesis
The hypothalamic-pituitary-testicular axis is under feedback inhibition by testicular steroids and inhibins
Testosterone
Leydig cells convert cholesterol to testosterone
Adipose tissue, skin, and the adrenal cortex also produce testosterone and other androgens
Testosterone acts on target organs by binding to a nuclear receptor
Metabolism of testosterone occurs primarily in the liver and prostate
Biology of Spermatogenesis and Semen
Spermatogenesis includes mitotic divisions of spermatogonia, meiotic divisions of spermatocytes to spermatids, and maturation to spermatozoa N54-7
The Sertoli cells support spermatogenesis
Sperm maturation occurs in the epididymis
Spermatozoa are the only independently motile cells in the human body
The accessory male sex glands—the seminal vesicles, prostate, and bulbourethral glands—produce the seminal plasma
Male Sex Act
The sympathetic and parasympathetic divisions of the autonomic nervous system control the male genital system
Sympathetic Division of the ANS
Parasympathetic Division of the ANS
Visceral Afferents
Erection is primarily under parasympathetic control
Parasympathetic Innervation
Sympathetic Innervation
Somatic Innervation
Afferent Innervation
Emission is primarily under sympathetic control
Motor Activity of the Duct System
Secretory Activity of the Accessory Glands
Ejaculation is under the control of a spinal reflex
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Chapter 55: The Female Reproductive System
Female reproductive organs include the ovaries and accessory sex organs
Reproductive function in the human female is cyclic
Hypothalamic-Pituitary-Gonadal Axis and Control of the Menstrual Cycle
The human menstrual cycle coordinates changes in both the ovary and endometrium
Follicular/Proliferative Phase
Ovulation
Luteal/Secretory Phase
Menses
The hypothalamic-pituitary-ovarian axis drives the menstrual cycle
Neurons in the hypothalamus release GnRH in a pulsatile fashion
GnRH stimulates gonadotrophs in the anterior pituitary to secrete FSH and LH
The ovarian steroids (estrogens and progestins) feed back on the hypothalamic-pituitary axis
Negative Feedback by Ovarian Steroids
Positive Feedback by Ovarian Steroids
Ovaries produce peptide hormones—inhibins, activins, and follistatins—that modulate FSH secretion
Negative Feedback by the Inhibins
Positive Feedback by the Activins
Modulation of gonadotropin secretion by positive and negative ovarian feedback produces the normal menstrual rhythm
Ovarian Steroids
Starting from cholesterol, the ovary synthesizes estradiol, the major estrogen, and progesterone, the major progestin
Estrogen biosynthesis requires two ovarian cells and two gonadotropins, whereas progestin synthesis requires only a single cell
Estrogens stimulate cellular proliferation and growth of sex organs and other tissues related to reproduction
The Ovarian Cycle: Folliculogenesis, Ovulation, and Formation of the Corpus Luteum
Female reproductive life span is determined by the number of primordial follicles established during fetal life
Primary Oocytes
Primordial Follicles
Primary Follicles
Secondary Follicles
Tertiary Follicles
Graafian Follicles
The oocyte grows and matures during folliculogenesis
FSH and LH stimulate the growth of a cohort of follicles
Each month, one follicle achieves dominance
Estradiol secretion by the dominant follicle triggers the LH surge and thus ovulation
After ovulation, theca and granulosa cells of the follicle differentiate into theca-lutein and granulosa-lutein cells of the corpus luteum
Growth and involution of the corpus luteum produce the rise and fall in estradiol and progesterone during the luteal phase
The Endometrial Cycle
The ovarian hormones drive the morphological and functional changes of the endometrium during the monthly cycle
The Menstrual Phase
The Proliferative Phase
The Secretory Phase
The effective implantation window is 3 to 4 days
Female Sex Act
The female sex response occurs in four distinct phases
Excitement
Plateau
Orgasm
Resolution
Both the sympathetic and the parasympathetic divisions control the female sex response
The female sex response facilitates sperm transport through the female reproductive tract
Menopause
Only a few functioning follicles remain in the ovaries of a menopausal woman
During menopause, levels of the ovarian steroids fall, whereas gonadotropin levels rise
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Chapter 56: Fertilization, Pregnancy, and Lactation
Transport of Gametes and Fertilization
Cilia and smooth muscle transport the egg and sperm within the female genital tract
The “capacitation” of the spermatozoa that occurs in the female genital tract enhances the ability of the sperm cell to fertilize the ovum
Fertilization begins as the sperm cell attaches to the zona pellucida and undergoes the acrosomal reaction, and it ends with the fusion of the male and female pronuclei
Implantation of the Developing Embryo
The presence of an embryo leads to decidualization of the endometrium
Uterine secretions nourish the preimplantation embryo, promote growth, and prepare it for implantation
The blastocyst secretes substances that facilitate implantation
During implantation, the blastocyst apposes itself to the endometrium, adheres to epithelial cells, and finally invades the stroma
Apposition
Adhesion
Invasion
Physiology of the Placenta
At the placenta, the space between the fetus’s chorionic villi and the mother’s endometrial wall contains a continuously renewed pool of extravasated maternal blood
Maternal Blood Flow
Fetal Blood Flow
Gases and other solutes move across the placenta
O2 and CO2 Transport
Other Solutes
The placenta makes a variety of peptide hormones, including hCG and human chorionic somatomammotropin
The Maternal-Placental-Fetal Unit
During pregnancy, progesterone and estrogens rise to levels that are substantially higher than their peaks in a normal cycle
After 8 weeks of gestation, the maternal-placental-fetal unit maintains high levels of progesterone and estrogens
Response of the Mother to Pregnancy
Both maternal cardiac output and blood volume increase during pregnancy
Increased levels of progesterone during pregnancy increase alveolar ventilation
Pregnancy increases the demand for dietary protein, iron, and folic acid
Less than one third of the total maternal weight gain during pregnancy represents the fetus
Parturition
Human birth usually occurs at around the 40th week of gestation
Parturition occurs in distinct stages, numbered 0 to 3
Stage 0—Quiescence
Stage 1—Transformation/Activation
Stage 2—Active Labor
Stage 3—Involution
Reciprocal decreases in progesterone receptors and increases in estrogen receptors are critical for the onset of labor
Signals from the fetus may initiate labor
PGs initiate uterine contractions, and both PGs and OT sustain labor
Prostaglandins
Oxytocin
Relaxin
Mechanical Factors
Positive Feedback
Lactation
The epithelial alveolar cells of the mammary gland secrete the complex mixture of sugars, proteins, lipids, and other substances that constitute milk
PRL is essential for milk production, and suckling is a powerful stimulus for PRL secretion
OT and psychic stimuli initiate milk ejection (“let-down”)
Suckling inhibits the ovarian cycle
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Chapter 57: Fetal and Neonatal Physiology
Biology of Fetal Growth
Two distinct circulations—fetoplacental and uteroplacental—underlie the transfer of gases and nutrients
Growth occurs by hyperplasia and hypertrophy
Growth depends primarily on genetic factors during the first half of gestation and on epigenetic factors thereafter
Increases in placental mass parallel periods of rapid fetal growth
Insulin, the insulin-like growth factors, and thyroxine stimulate fetal growth
Glucocorticoids and Insulin
Insulin-Like Growth Factors
Epidermal Growth Factor
Thyroid Hormones
Peptide Hormones
Many fetal tissues produce red blood cells early in gestation
The fetal gastrointestinal and urinary systems excrete products into the amniotic fluid by midpregnancy
A surge in protein synthesis, with an increase in muscle mass, is a major factor in the rapid fetal weight gain during the third trimester
Fetal lipid stores increase rapidly during the third trimester
Development and Maturation of the Cardiopulmonary System
Fetal lungs develop by repetitive branching of both bronchial and pulmonary arterial trees
An increase in cortisol, with other hormones, triggers surfactant production in the third trimester
Fetal respiratory movements begin near the end of the first trimester but wane just before birth
The fetal circulation has four unique pathways—placenta, ductus venosus, foramen ovale, and ductus arteriosus—to facilitate gas and nutrient exchange
Placenta
Ductus Venosus
Foramen Ovale
Ductus Arteriosus
Cardiopulmonary Adjustments at Birth
Loss of the placental circulation requires the newborn to breathe on its own
Mild hypoxia and hypercapnia, as well as tactile stimuli and cold skin, trigger the first breath
At birth, removal of the placenta increases systemic vascular resistance, whereas lung expansion decreases pulmonary vascular resistance
Removal of the Placental Circulation
Increase in Pulmonary Blood Flow
Closure of the ductus venosus within the first days of life forces portal blood to perfuse the liver
Closure of the foramen ovale occurs as left atrial pressure begins to exceed right atrial pressure
Closure of the ductus arteriosus completes the separation between the pulmonary and systemic circulations
Neonatal Physiology
Although the newborn is prone to hypothermia, nonshivering thermogenesis in brown fat helps to keep the neonate warm
The neonate mobilizes glucose and FAs soon after delivery
Carbohydrate Metabolism
Fat Metabolism
Metabolic Rate
Breast milk from a mother with a balanced diet satisfies all of the infant’s nutritional requirements during the first several months of life
The neonate is at special risk of developing fluid and acid-base imbalances
Humoral and cellular immune responses begin at early stages of development in the fetus
Fetus
Neonate
In premature newborns, immaturity of organ systems and fragility of homeostatic mechanisms exacerbate postnatal risks
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Section X: Physiology of Everyday Life
Chapter 58: Metabolism
Forms of Energy
Energy Balance
Energy input to the body is the sum of energy output and storage
The inefficiency of chemical reactions leads to loss of the energy available for metabolic processes
Free energy, conserved as high-energy bonds in ATP, provides the energy for cellular functions
Energy Interconversion From Cycling between 6-Carbon and 3-Carbon Molecules
Glycolysis converts the 6-carbon glucose molecule to two 3-carbon pyruvate molecules
Gluconeogenesis converts nonhexose precursors to the 6-carbon glucose molecule
Reciprocal regulation of glycolysis and gluconeogenesis minimizes futile cycling
Allosteric Regulation
Transcriptional Regulation
Cells can convert glucose or amino acids into FAs
The body permits only certain energy interconversions
Energy Capture (Anabolism)
After a carbohydrate meal, the body burns some ingested glucose and incorporates the rest into glycogen or TAGs
Liver
Muscle
Adipose Tissue
After a protein meal, the body burns some ingested amino acids and incorporates the rest into proteins
After a fatty meal, the body burns some ingested FAs and incorporates the rest into TAGs
Energy Liberation (Catabolism)
The first step in energy catabolism is to break down glycogen or TAGs to simpler compounds
Skeletal Muscle
Liver
Adipocytes
The second step in TAG catabolism is β-oxidation of FAs
The final common steps in oxidizing carbohydrates, TAGs, and proteins to CO2 are the citric acid cycle and oxidative phosphorylation
Citric Acid Cycle
Oxidative Phosphorylation
Ketogenesis
Oxidizing different fuels yields similar amounts of energy per unit O2 consumed
Integrative Metabolism During Fasting
During an overnight fast, glycogenolysis and gluconeogenesis maintain plasma glucose levels
Requirement for Glucose
Gluconeogenesis versus Glycogenolysis
Gluconeogenesis: The Cori Cycle
Gluconeogenesis: The Glucose-Alanine Cycle
Lipolysis
Starvation beyond an overnight fast enhances gluconeogenesis and lipolysis
Enhanced Gluconeogenesis
Enhanced Lipolysis
Prolonged starvation moderates proteolysis but accelerates lipolysis, thereby releasing ketone bodies
Decreased Proteolysis
Decreased Hepatic Gluconeogenesis
Increased Renal Gluconeogenesis
Increased Lipolysis and Ketogenesis
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Chapter 59: Regulation of Body Temperature
Heat and Temperature: Advantages of Homeothermy
Homeotherms maintain their activities over a wide range of environmental temperatures
Body core temperature depends on time of day, physical activity, time in the menstrual cycle, and age
The body’s rate of heat production can vary from ~70 kcal/hr at rest to 600 kcal/hr during exercise
Modes of Heat Transfer
Maintaining a relatively constant body temperature requires a fine balance between heat production and heat losses
Heat moves from the body core to the skin, primarily by convection
Heat moves from the skin to the environment by radiation, conduction, convection, and evaporation
Radiation
Conduction
Convection
Evaporation
When heat gain exceeds heat loss, body core temperature rises
Clothing insulates the body from the environment and limits heat transfer from the body to the environment
Active Regulation of Body Temperature by the Central Nervous System
Thermoreceptors in the skin and temperature-sensitive neurons in the hypothalamus respond to changes in their local temperature
Skin Thermoreceptors
Hypothalamic Temperature-Sensitive Neurons
The CNS thermoregulatory network integrates thermal information and directs changes in efferent activity to modify rates of heat transfer and production
Thermal effectors include behavior, cutaneous circulation, sweat glands, and skeletal muscles responsible for shivering
Hypothermia, Hyperthermia, and Fever
Hypothermia or hyperthermia occurs when heat transfer to or from the environment overwhelms the body’s thermoregulatory capacity
Exercise raises heat production, which is followed by a matching rise in heat loss, but at the cost of a steady-state hyperthermia of exercise
Fever is a regulated hyperthermia
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Chapter 60: Exercise Physiology and Sports Science
Motor Units and Muscle Function
The motor unit is the functional element of muscle contraction
Muscle force rises with the recruitment of motor units and an increase in their firing frequency
Compared with type I motor units, type II units are faster and stronger but more fatigable
As external forces stretch muscle, series elastic elements contribute a larger fraction of total tension
The action of a muscle depends on the axis of its fibers and its origin and insertion on the skeleton
Fluid and energetically efficient movements require learning
Strength versus endurance training differentially alters the properties of motor units N60-3
Conversion of Chemical Energy to Mechanical Work
ATP and PCr provide immediate but limited energy
Anaerobic glycolysis provides a rapid but self-limited source of ATP
Oxidation of glucose, lactate, and fatty acids provides a slower but long-term source of ATP
Oxidation of Nonmuscle Glucose
Oxidation of Lactate
Gluconeogenesis
Oxidation of Nonmuscle Lipid
Choice of Fuel Sources
Muscle Fatigue
Fatigued muscle produces less force and has a reduced velocity of shortening
Changes in the CNS produce central fatigue
Impaired excitability and impaired Ca2+ release can produce peripheral fatigue
High-Frequency Fatigue
Low-Frequency Fatigue
Fatigue can result from ATP depletion, lactic acid accumulation, and glycogen depletion
ATP Depletion
Lactic Acid Accumulation
Glycogen Depletion
Determinants of Maximal O2 Uptake and Consumption
Maximal O2 uptake by the lungs can exceed resting O2 uptake by more than 20-fold
O2 uptake by muscle is the product of muscle blood flow and O2 extraction
O2 delivery by the cardiovascular system is the limiting step for maximal O2 utilization
Limited O2 Uptake by the Lungs
Limited O2 Delivery by the Cardiovascular System
Limited O2 Extraction by Muscle
Effective circulating volume takes priority over cutaneous blood flow for thermoregulation
Sweating
Eccrine, but not apocrine, sweat glands contribute to temperature regulation
Eccrine sweat glands are tubules comprising a secretory coiled gland and a reabsorptive duct
Secretion by Coil Cells
Reabsorption by Duct Cells
The NaCl content of sweat increases with the rate of secretion but decreases with acclimatization to heat
Flow Dependence
Cystic Fibrosis
Replenishment
Acclimatization
The hyperthermia of exercise stimulates eccrine sweat glands
Endurance (Aerobic) Training
Aerobic training requires regular periods of stress and recovery
Aerobic training increases maximal O2 delivery by increasing plasma volume and maximal cardiac output
Maximizing Arterial O2 Content
Maximizing Cardiac Output
Aerobic training enhances O2 diffusion into muscle
Aerobic training increases mitochondrial content
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Chapter 61: Environmental Physiology
The Environment
Voluntary feedback control mechanisms can modulate the many layers of our external environment
Environmental temperature provides conscious clues for triggering voluntary feedback mechanisms
Room ventilation should maintain , , and levels of toxic substances within acceptable limits
Acceptable Limits for and
Measuring Room Ventilation
Carbon Monoxide
Threshold Limit Values and Biological Exposure Indices
Tissues must resist the G force produced by gravity and other mechanisms of acceleration
The partial pressures of gases—other than water—inside the body depend on Pb
Diving Physiology
Immersion raises Pb, thereby compressing gases in the lungs
SCUBA divers breathe compressed air to maintain normal lung expansion
Increased alveolar can cause narcosis
Increased alveolar can lead to O2 toxicity
Using helium to replace inspired N2 and O2 avoids nitrogen narcosis and O2 toxicity
After an extended dive, one must decompress slowly to avoid decompression illness
High-Altitude Physiology
Pb and ambient on top of Mount Everest are approximately one third of their values at sea level
Everest Base Camp
Peak of Mount Everest
Air Travel
Up to modest altitudes, arterial O2 content falls relatively less than Pb due to the shape of the Hb-O2 dissociation curve
During the first few days at altitude, compensatory adjustments to hypoxemia include tachycardia and hyperventilation
Long-term adaptations to altitude include increases in hematocrit, pulmonary diffusing capacity, capillarity, and oxidative enzymes
Hematocrit
Pulmonary Diffusing Capacity
Capillary Density
Oxidative Enzymes
High altitude causes mild symptoms in most people and acute or chronic mountain sickness in susceptible individuals
Symptoms of Hypoxia
Acute Mountain Sickness
Chronic Mountain Sickness
Flight and Space Physiology
Acceleration in one direction shifts the blood volume in the opposite direction
“Weightlessness” causes a cephalad shift of the blood volume and an increase in urine output
Space flight leads to motion sickness and to decreases in muscle and bone mass
Exercise partially overcomes the deconditioning of muscles during space flight
Return to earth requires special measures to maintain arterial blood pressure
References
Books and Reviews
Journal Articles
Chapter 62: The Physiology of Aging
Concepts in Aging
During the 20th century, the age structure of populations in developed nations shifted toward older individuals
The definition, occurrence, and measurement of aging are fundamental but controversial issues
Aging is an evolved trait
Human aging studies can be cross-sectional or longitudinal
Cross-Sectional Design
Longitudinal Design
Whether age-associated diseases are an integral part of aging remains controversial
Cellular and Molecular Mechanisms of Aging
Oxidative stress and related processes that damage macromolecules may have a causal role in aging
Reactive Oxygen Species
Glycation and Glycoxidation
Mitochondrial Damage
Somatic Mutations
Inadequacy of repair processes may contribute to the aging phenotype
DNA Repair
Protein Homeostasis
Autophagy
Dysfunction of the homeostasis of cell number may be a major factor in aging
Limitations in Cell Division
Cell Removal
Aging of the Human Physiological Systems
Aging people lose height and lean body mass but gain and redistribute fat
Aging thins the skin and causes the musculoskeletal system to become weak, brittle, and stiff
Skin
Skeletal Muscle
Bone
Synovial Joints
The healthy elderly experience deficits in sensory transduction and speed of central processing
Sensory Functions
Motor Functions
Cognitive Functions
Aging causes decreased arterial compliance and increased ventilation-perfusion mismatching
Cardiovascular Function
Pulmonary Function
Exercise
Glomerular filtration rate falls with age in many but not all people
Aging has only minor effects on gastrointestinal function
Aging causes modest declines in most endocrine functions
Insulin
Growth Hormone and IGF-1
Adrenal Steroids
Thyroid Hormones
Parathyroid Hormone
Gonadal Hormones
Aging Slowly
Caloric restriction slows aging and extends life in several species, including some mammals
Genetic alterations can extend life in several species
Proposed interventions to slow aging and extend human life are controversial
References
Books and Reviews
Journal Articles

Citation preview

Medical Physiology

Medical Physiology THIRD

3

EDITION

WALTER F. BORON, MD, PhD

EMILE L. BOULPAEP, MD

Professor David N. and Inez Myers/Antonio Scarpa Chairman Department of Physiology and Biophysics Case Western Reserve University Cleveland, Ohio

Professor Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899

MEDICAL PHYSIOLOGY, THIRD EDITION INTERNATIONAL EDITION

ISBN: 978-1-4557-4377-3 ISBN: 978-0-323-42796-8

Copyright © 2017 by Elsevier, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Previous editions copyrighted 2012, 2009, 2005, and 2003. Library of Congress Cataloging-in-Publication Data Names: Boron, Walter F., editor. | Boulpaep, Emile L., editor. Title: Medical physiology / [edited by] Walter F. Boron, Emile L. Boulpaep. Other titles: Medical physiology (Boron) Description: Edition 3. | Philadelphia, PA : Elsevier, [2017] | Includes bibliographical references and index. Identifiers: LCCN 2016005260| ISBN 9781455743773 (hardcover : alk. paper) | ISBN 9780323427968 (International ed.) Subjects: | MESH: Physiological Phenomena | Cell Physiological Phenomena Classification: LCC QP34.5 | NLM QT 104 | DDC 612—dc23 LC record available at http://lccn.loc.gov/2016005260

Executive Content Strategist: Elyse O’Grady Senior Content Development Specialist: Marybeth Thiel Publishing Services Manager: Julie Eddy Senior Project Manager: David Stein Design Direction: Julia Dummitt

Printed in China Last digit is the print number:  9  8  7  6  5  4  3  2  1

CONTRIBUTORS Peter S. Aronson, MD

C.N.H. Long Professor of Internal Medicine Professor of Cellular and Molecular Physiology Section of Nephrology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Eugene J. Barrett, MD, PhD

Professor Departments of Medicine and Pharmacology University of Virginia School of Medicine Charlottesville, Virginia

Paula Q. Barrett, PhD

Professor Department of Pharmacology University of Virginia School of Medicine Charlottesville, Virginia

Henry J. Binder, MD

Professor Emeritus of Medicine Department of Internal Medicine—Digestive Diseases Yale University School of Medicine New Haven, Connecticut

Walter F. Boron, MD, PhD

Professor David N. and Inez Myers/Antonio Scarpa Chairman Department of Physiology and Biophysics Case Western Reserve University Cleveland, Ohio

Emile L. Boulpaep, MD

Professor Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Lloyd Cantley, MD, FASN

Professor Department of Internal Medicine Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Michael J. Caplan, MD, PhD

C.N.H. Long Professor and Chair Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Barry W. Connors, PhD

Professor and Chair Department of Neuroscience Alpert Medical School Brown University Providence, Rhode Island

Arthur DuBois, MD

Professor Emeritus of Epidemiology and Public Health and Cellular and Molecular Physiology John B. Pierce Laboratory New Haven, Connecticut

Gerhard Giebisch, MD

Professor Emeritus of Cellular and Molecular Physiology Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut

Fred S. Gorelick, MD

Professor Departments of Internal Medicine and Cell Biology Yale University School of Medicine New Haven, Connecticut

Peter Igarashi, MD

Nesbitt Chair and Head Department of Medicine University of Minnesota Minneapolis, Minnesota

Ervin E. Jones, MD, PhD

Retired Department of Obstetrics and Gynecology Yale University School of Medicine New Haven, Connecticut

W. Jonathan Lederer, MD, PhD

Director and Professor, Center for Biomedical Engineering and Technology and Department of Physiology University of Maryland School of Medicine Baltimore, Maryland

George Lister, MD

Jean McLean Wallace Professor of Pediatrics Professor of Cellular and Molecular Physiology Yale School of Medicine New Haven, Connecticut

v

vi

Contributors

Charles M. Mansbach II, MD†

Professor of Medicine and Physiology University of Tennessee Health Science Center Memphis, Tennessee

Christopher R. Marino, MD

Professor of Medicine University of Tennessee Health Science Center Chief of Staff VA Medical Center Memphis, Tennessee

Edward J. Masoro, PhD

Professor Emeritus of Physiology University of Texas Health Science Center at San Antonio San Antonio, Texas

Steven S. Segal, PhD

Professor Department of Medical Pharmacology and Physiology University of Missouri School of Medicine Columbia, Missouri

Gerald I. Shulman, MD, PhD, FACP, MACE

Investigator, Howard Hughes Medical Institute George R. Cowgill Professor of Physiological Chemistry Professor of Medicine (Endocrinology/Metabolism) and Cellular & Molecular Physiology Co-Director, Yale Diabetes Research Center Yale University School of Medicine New Haven, Connecticut

Frederick J. Suchy, MD

Professor Department of Reproductive Biology Case Western Reserve University Cleveland, Ohio

Chief Research Officer Director, Children’s Hospital Colorado Research Institute Professor of Pediatrics Associate Dean for Child Health Research University of Colorado School of Medicine Aurora, Colorado

Edward G. Moczydlowski, PhD

Erich E. Windhager, MD

Sam Mesiano, PhD

Senior Associate Dean of Academic Affairs & Professor of Physiology College of Health Sciences California Northstate University Elk Grove, California

Shaun F. Morrison, PhD

Professor Department of Neurological Surgery Oregon Health & Science University Portland, Oregon

Kitt Falk Petersen, MD

Professor Section of Endocrinology Department of Internal Medicine Yale University School of Medicine New Haven, Connecticut

Bruce R. Ransom, MD, PhD

Magnuson Professor and Chair Department of Neurology Department of Physiology and Biophysics University of Washington Health Sciences Center Seattle, Washington

George B. Richerson, MD, PhD

Professor & Chairman Department of Neurology University of Iowa Carver College of Medicine Iowa City, Iowa

†

Deceased.

Professor Department of Physiology and Biophysics Weill Medical College Cornell University New York, New York

VIDEO TABLE OF CONTENTS 7-1 8-1 9-1 10-1 13-1 22-1 27-1 38-1 41-1 55-1

Action Potential Chemical Synaptic Transmission The Cross Bridge Cycle Chemotaxis Chemical Synaptic Transmission The Cardiac Cycle Pressures during Respiration The Countercurrent Multiplier Peristalsis The Menstrual Cycle

PREFACE TO THE THIRD EDITION We are delighted that the physiological community so eagerly welcomed the Second Edition of our book. The 3-fold philosophy that has guided us in the previous editions has endured as we prepared the Third Edition. First, we combine the expertise of several authors with the consistency of a single pen. In the First Edition, we achieved this singleness of pen by sitting—shoulder to shoulder—at a computer as we rewrote the primary copy of our authors, line by line. By the time we began editing the Third Edition, one of us had moved from New Haven to Cleveland. Even so, we continued to edit jointly and in real time—monitor to monitor—using desktop-sharing software. After more than two decades, we have become so accustomed to each other’s writing styles that we can literally finish each other’s sentences. Second, we still integrate physiological concepts from the level of DNA and epigenetics to the human body, and everything in between. Third, we complete the presentation of important physiological principles by pairing them with illustrations from pathophysiology, thereby putting physiology in a clinical context. In this Third Edition, we have updated the entire book to reflect new molecular insights. In the process, we have shortened the printed version of the book by 40 pages. The Third Edition contains 20 new or redrawn figures as well as enhancements to 125 others. Similarly, we included over 190 tables. In the First Edition, we launched the concept of online-only Notes—electronic footnotes that were available on the Student Consult website. These Notes (indicated by icons in the print version of the book) amplify concepts in the text, provide details and derivations of equations, add clinical illustrations, and include interesting facts (e.g., biographies of famous physiologists). With the increased use of online materials and eBooks, our readers may welcome our updating of the previous Notes as well as a 13% increase in the total number of Notes for the Third Edition, for a total of about 750. In the Second Edition, we provided the reader with numerous crosslinks to explanatory materials within the book by providing chapter numbers. In the Third Edition, we greatly expand the number of such crosslinks—but now refer the reader to specific pages in the print, and link the reader to specific paragraphs in the eBook. The eBook provides references to scientific literature. In Section II (Physiology of Cells and Molecules), fresh insights led to substantial revisions in Chapter 4 (Regulation of Gene Expression), including the subchapter on epigenetics, and another on posttranslational modifications. Moreover, advances in physiological genomics and the understanding of genetic diseases led to major expansions of two tables— one on the SLC family of transporters (Table 5-4 in the chapter on Transport of Solutes and Water) and the other on ion channels (Table 6-2 in the chapter on Electrophysiology

of the Cell Membrane). In both tables, our updates help the reader navigate through what sometimes are multiple systems of terminology. In Section III (The Nervous System), new molecular developments led to major changes in Chapter 15 (Sensory Transduction), including the transduction of taste. In Section IV (The Cardiovascular System), we have improved the molecular underpinning of the ionic currents in Chapter 21 (Cardiac Electrophysiology and the Electrocardiogram). In Section VI (The Urinary System), we welcome Peter Aronson as a new co-author. Improved molecular insights led to major improvements in Chapter 36, including the subchapters on urea, urate, phosphate, and calcium. In Section VII (The Gastrointestinal System), Chapter 43 (Pancreatic and Salivary Glands) underwent significant modernization, including an expansion of the treatment of salivary glands. In Chapter 45 (Nutrient Digestion and Absorption), we welcome Charles Mansbach as a new co-author. Section VIII (The Endocrine System) underwent significant updating, including the treatment of phosphate handling in Chapter 52 (The Parathyroid Glands and Vitamin D). In Section IX (The Reproductive System), we welcome two new authors. Sam Mesiano extensively reworked Chapters 53 (Sexual Differentiation) through Chapter 56 (Fertilization, Pregnancy, and Lactation), and George Lister has similarly rewritten Chapter 57 (Fetal and Neonatal Physiology). Finally, in Section X (Physiology of Everyday Life), we welcome Shaun Morrison, who extensively rewrote Chapter 59 (Regulation of Body Temperature). Chapter 62 (The Physiology of Aging) underwent extensive changes, including new treatments of necroptosis and frailty.

THE eBOOK Although you can still enjoy our book while reading the print version, you can also access the extended content at your computer via the website www.StudentConsult.com. The eBook is also available through the Inkling app on tablets and smart phones. Regardless of the platform for accessing the eBook, the student may access Notes, crosslinks, and references as noted above, and also can “follow” professors and see their highlights and annotations within the text.

ACKNOWLEDGMENTS A textbook is the culmination of successful collaborations among many individuals. First, we thank our chapter authors, who are listed under Contributors on pages v and vi. We also thank other colleagues who wrote WebNotes, or provided other valuable materials or input. Roberto vii

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Preface to the Third Edition

Dominguez provided Figure 9-5A, and Slavek Filipek and Kris Palczewski provided Figure 15-12. Philine Wangemann made invaluable suggestions for the Vestibular and Auditory Transduction subchapter in Chapter 15. George Dubyak responded to numerous queries. We thank all our readers who sent us their suggestions or corrections; we list them in the accompanying  NP-1. At the art studio DNA Illustrations, Inc, we thank David and Alex Baker for developing new figures and updating others, while maintaining the textbook’s aesthetic appeal, originally established by JB Woolsey and Associates. At Elsevier, we are most grateful to Elyse O’Grady— Executive Content Strategist—for her trust and endurance. Marybeth Thiel—Senior Content Development Specialist— was the project’s communications hub, responsible for coordinating all parties working on the textbook, and for assembling the many elements that comprised the final product. Her meticulous care was indispensible. We thank David Stein—Senior Project Manager—for overseeing

production of the textbook. Striving for consistency, Elsevier did us the favor of assigning a single copyeditor—Janet E. Lincoln—to the entire project. We were especially impressed with her meticulous copyediting. Moreover, because she read the manuscript as a dedicated student, she identified several logical or scientific errors, including inconsistencies between chapters. Finally, we thank four editorial assistants. Charleen Bertolini used every ounce of her friendly, good-humored, and tenacious personality to keep our authors—and us—on track during the first few years as we prepared the Third Edition. Later, three students in the MS in Medical Physiology Program at Case Western Reserve University took the reins from Charleen—Evan Rotar, Alisha Bouzaher, and Anne Jessica Roe. As we did for the first two editions, we again invite the reader to enjoy learning physiology. If you are pleased with our effort, tell others. If not, tell us.

Preface to the Third Edition

viii.e1

NP-1  List of Readers Who Made Suggestions Faculty Raif Musa Aziz, PhD, Assistant Professor, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil Mark Borden, Colorado

PhD,

Associate

Professor,

University

of

Gerald DiBona, MD, Professor Emeritus of Medicine and Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa Roberto Dominguez, PhD, Professor of Physiology, Perelman School of Medicine, University of Pennsylvania George Dubyak, PhD, Professor, Department of Physiology and Biophysics, Case Western Reserve University

Andrea Romani, MD, PhD, Associate Professor, Department of Physiology and Biophysics, Case Western Reserve University Corey Smith, PhD, Professor, Department of Physiology and Biophysics, Case Western Reserve University Julian Stelzer, PhD, Assistant Professor, Department of Physiology and Biophysics, Case Western Reserve University Funabashi Toshiya, MD, PhD, Professor, Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Japan Philine Wangemann, PhD, University Distinguished Professor, Department of Anatomy & Physiology, Kansas State University Ernest Wright, PhD, Professor, David Geffen School of Medicine, University of California—Los Angeles

Mikael Esmann, PhD, Professor of Physiology and Biophysics, Aarhus University

Students

Slavek Filipek, PhD, Department of Pharmacology, School of Medicine, Case Western Reserve University

Taylor Burch

Gabriel Haddad, MD, Chairman of Pediatrics, University of California—San Diego Ulrich Hopfer, MD, PhD, Professor Emeritus, Department of Physiology and Biophysics, Case Western Reserve University Norman Javitt, MD, PhD, Professor of Medicine and Pediatrics, New York University Medical Center Bhanu Jena, PhD, DSc, Professor of Physiology, School of Medicine, Wayne State University Stephen Jones, PhD, Professor, Department of Physiology and Biophysics, Case Western Reserve University Alan Kay, PhD, Professor of Biology, University of Iowa Rossana Occhipinti, PhD, Department of Physiology and Biophysics, Case Western Reserve University

Natthew Arunthamakun

Tung Chu Xiaoke Feng Clare Fewtrell Trevor Hall Jeffery Jeong Hani Khadra Bob Lee Shannon Li Sarabjot Makkar Claire Miller

Krzysztof Palczewski, PhD, Professor and Chair, Department of Pharmacology, School of Medicine, Case Western Reserve University

Pamela Moorehead

Mark Parker, PhD, Assistant Professor, Department of Physiology and Biophysics, SUNY at Buffalo

Sarah Sheldon

D. Narayan Rao, PhD, Department of Physiology, Faculty of Medicine, Benghazi University

Amalia Namath

Sadia Tahir Eunji Yim

PREFACE TO THE FIRST EDITION

We were intrigued by an idea suggested to us by W.B. Saunders: write a modern textbook of physiology that combines the expertise of a multi-author book with the consistency of a single pen. Our approach has been, first, to recruit as writers mainly professors who teach medical physiology at the Yale University School of Medicine, and then to recast the professors’ manuscripts in a uniform style. After much effort, we now present our book, which we hope will bring physiology to life and at the same time be a reliable resource for students.

transmission in the nervous system, sensory transduction, and neural circuits. In addition, Part III also treats two subjects—the autonomic nervous system and the neuronal microenvironment—that are important for understanding other physiological systems. Finally, Part X (The Physiology of Everyday Life) is an integrated, multisystem approach to metabolism, temperature regulation, exercise, and adaptations to special environments.

TARGET AUDIENCE

Some important aspects of physiology remain as fundamentally important today as when the pioneers of physiology discovered them a century or more ago. These early observations were generally phenomenological descriptions that physiologists have since been trying to understand at a mechanistic level. Where possible, a goal of this textbook is to extend this understanding all the way to the cell and molecule. Moreover, although some areas are evolving rapidly, we have tried to be as up to date as practical. To make room for the cellular and molecular bricks, we have omitted some classic experimental observations, especially when they were of a “black-box” nature. Just as each major Part of the textbook begins with an introductory chapter, each chapter generally first describes— at the level of the whole body or organ system (e.g., the kidney)—how the body performs a certain task and/or controls a certain parameter (e.g., plasma K+ concentration). As appropriate, our discussion then progresses in a reductionistic fashion from organ to tissue to cell and organelles, and ultimately to the molecules that underlie the physiology. Finally, most chapters include a discussion of how the body regulates the parameter of interest at all levels of integration, from molecules to the whole body.

We wrote Medical Physiology primarily as an introductory text for medical students, although it should also be valuable for students in the allied health professions and for graduate students in the physiological sciences. The book should continue to be useful for the advanced medical student who is learning pathophysiology and clinical medicine. Finally, we hope that physicians in training, clinical fellows, and clinical faculty will find the book worthwhile for reviewing principles and becoming updated on new information pertinent for understanding the physiological basis of human disease.

CONTENT OF THE TEXTBOOK Aside from Part I, which is a brief introduction to the discipline of physiology, the book consists of nine major Parts. Part II (Physiology of Cells and Molecules) reflects that, increasingly, the underpinnings of modern physiology have become cellular and molecular. Chapters 2, 4, and 5 would not be present in a traditional physiology text. Chapter 2 (Functional Organization of the Cell), Chapter 4 (Signal Transduction), and Chapter 5 (Regulation of Gene Expression) provide the essentials of cell biology and molecular biology necessary for understanding cell and organ function. The other chapters in Part II cover the cellular physiology of transport, excitability, and muscle—all of which are classic topics for traditional physiology texts. In this book we have extended each of these subjects to the molecular level. The remainder of the book will frequently send the reader back to the principles introduced in Part II. Parts III to IX address individual organ systems. In each case, the first chapter provides a general introduction to the system. Part III (Cellular Physiology of the Nervous System) is untraditional in that it deliberately omits those aspects of the physiology of the central nervous system that neuroscience courses generally treat and that require extensive knowledge of neuroanatomical pathways. Rather, Part III focuses on cellular neurophysiology, including synaptic

EMPHASIS OF THE TEXTBOOK

CREATING THE TEXTBOOK The first draft of each chapter was written by authors with extensive research and/or teaching experience in that field. The editors, sitting shoulder to shoulder at a computer, then largely rewrote all chapters line by line. The goal of this exercise was for the reader to recognize, throughout the entire book, a single voice—a unity provided by consistency in style, in organization, in the sequence for presenting concepts, and in terminology and notation, as well as in consistency in the expression of standard values (e.g., a cardiac output of 5 liters/min). The editors also attempted to minimize overlap among chapters by making extensive use of cross references (by page, figure, or table number) to principles introduced elsewhere in the book. ix

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Preface to the First Edition

After the first round of editing, Dr. Malcolm Thaler—a practicing physician and accomplished author in his own right—improved the readability of the text and sometimes added clinical examples. Afterwards, the editors again went through the entire text line by line to decide on the material to be included in specific illustrations, and to match the main text of the book with the content of each figure. The editors then traveled to Philadelphia to visit the art studio of JB Woolsey and Associates. Over many visits, John Woolsey and the editors together developed the content and format for each of the approximately 760 full-color illustrations used in the textbook. These meetings were unique intellectual and pedagogical dialogues concerning the design of the figures. To a large extent, the figures owe their pedagogical style to the creativity of John Woolsey. The illustrations evolved through several iterations of figure editing, based on suggestions from both the editors and authors. This evolution, as well as text changes requested by authors, led to yet a third round of editing of the entire book, often line by line. Throughout this seemingly endless process, our goal has been to achieve the proper balance among reader friendliness, depth, and accuracy.

SPECIAL FEATURES Compared with other major textbooks of physiology, a much larger fraction of the space in this book is devoted to illustrations. Thus, although our textbook may appear thick, it actually has fewer text words than most other leading medical physiology books. Virtually all illustrations in our book are in full color, conceived de novo, with consistent style and pedagogy. Many of the figures feature “dialogue balloons” that tell a story. The illustrations are also available in digital format on the Evolve Web site (http:// evolve.elsevier.com/productPages/s_417.html) for use in the classroom. The textbook makes considerable use of clinical boxes— highlighted on a color background—that present examples of diseases illustrating important physiological principles. The text includes over 2000 cross references that send the reader from the current page to specific pages, figures, or tables elsewhere in the book for relevant concepts or data. The text also includes hundreds of web icons, which direct the reader to our website at http://www.wbsaunders.com/ MERLIN/BandB/. These web links provide derivations of mathematical equations, amplification of concepts, material that was deleted for the sake of brevity from earlier drafts of the textbook, and clinical illustrations not included in the clinical boxes.

The website will also contain several other features, including summaries for each subchapter, an expanded list of references (sometimes with direct links to the primary literature), other links that may be of interest to the physiology student (e.g., biographies of famous physiologists), latebreaking scientific developments that occur after publication of the book, and—alas—the correction of errors. Finally, we invite the reader to visit our website to comment on our book, to point out errors, and to make other helpful suggestions.

ACKNOWLEDGMENTS A textbook is the culmination of successful collaborations among many individuals. First, we would like to thank our authors. Second, we acknowledge the expert input of Dr. Malcolm Thaler, both in terms of style and clinical insight. We also thank Dr. Thaler for emphasizing the importance of telling a “good story.” The textbook’s aesthetic appeal is largely attributable to JB Woolsey and Associates, particularly John Woolsey and Joel Dubin. At W.B. Saunders, we are especially thankful to William R. Schmitt—Acquisitions Editor—for his trust and patience over the years that this book has been in gestation. At the times when the seas were rough, he steered a safe course. Melissa Dudlick—Developmental Editor at W.B. Saunders— was the project’s nerve center, responsible for day-to-day communication among all parties working on the textbook, and for assembling all of the many components that went into making the final product. Her good humor and careful attention to detail greatly facilitated the creation of the textbook. We thank Frank Polizzano—Publishing Services Manager at W.B. Saunders—for overseeing production of the textbook. Before this textbook was completed, the author of Part X (The Physiology of Everyday Life), Ethan Nadel, passed away. We are indebted to those who generously stepped up to carefully check the nearly finished manuscripts for the final four chapters: Dr. Gerald Shulman for Chapter 57, Dr. John Stitt for Chapter 58, the late Dr. Carl Gisolfi for Chapter 59, and Dr. Arthur DuBois for Chapter 60. In addition, Dr. George Lister provided expert advice for Chapter 56. We are also grateful to Dr. Bruce Davis for researching the sequences of the polypeptide hormones, to Mr. Duncan Wong for expert information-technology services, and to Mrs. Leisa Strohmaier for administrative assistance. We now invite the reader to enjoy the experience of learning physiology. If you are pleased with our effort, tell others. If not, tell us.

CHAPTER

1 

FOUNDATIONS OF PHYSIOLOGY Emile L. Boulpaep and Walter F. Boron

What is physiology? Physiology is the dynamic study of life. Physiology describes the “vital” functions of living organisms and their organs, cells, and molecules. For centuries, the discipline of physiology has been closely intertwined with medicine. Although physiology is not primarily concerned with structure—as is the case for anatomy, histology, and structural biology— structure and function are inextricably linked because the living structures perform the functions. For some, physiology is the function of the whole person (e.g., exercise physiology). For many practicing clinicians, physiology may be the function of an individual organ system, such as the cardiovascular, respiratory, or gastrointestinal system. For still others, physiology may focus on the cellular principles that are common to the function of all organs and tissues. This last field has traditionally been called general physiology, a term that is now supplanted by cellular and molecular physiology. Although one can divide physiology according to varying degrees of reductionism, it is also possible to define a branch of physiology—for example, comparative physiology—that focuses on differences and similarities among different species. Indeed, comparative physiology may deal with all degrees of reductionism, from molecule to whole organism. In a similar way, medical physiology deals with how the human body functions, which depends on how the individual organ systems function, which depends on how the component cells function, which in turn depends on the interactions among subcellular organelles and countless molecules. Thus, medical physiology takes a global view of the human body; but in doing so, it requires an integrated understanding of events at the level of molecules, cells, and organs. Physiology is the mother of several biological sciences, having given birth to the disciplines of biochemistry, biophysics, and neuroscience, as well as their corresponding scientific societies and journals. Thus, it should come as no surprise that the boundaries of physiology are not sharply delineated. Conversely, physiology has its unique attributes. For example, physiology has evolved over the centuries from a more qualitative to a more quantitative science. Indeed, many of the leading physiologists were— and still are—trained as chemists, physicists, mathematicians, or engineers. 2

Physiological genomics is the link between the organ and the gene The life of the human body requires not only that individual organ systems do their jobs but also that these organ systems work “hand in hand” with each other. They must share information. Their actions must be interdependent. The cells within an organ or a tissue often share information, and certainly the individual cells must act in concert to perform the proper function of the organ or tissue. In fact, cells in one organ must often share information with cells in another organ and make decisions that are appropriate for the health of the individual cell as well as for the health of the whole person. In most cases, the sharing of information between organs and between cells takes place at the level of atoms or molecules. Cell-to-cell messengers or intracellular messengers may be as simple as H+ or K+ or Ca2+. The messengers may also be more complex chemicals. A cell may release a molecule that acts on a neighboring cell or that enters the bloodstream and acts on other cells a great distance away. In other cases, a neuron may send an axon a centimeter or even a meter away and rapidly modulate, through a neurotransmitter molecule, the activity of another cell or another organ. Cells and organs must interact with one another, and the method of communication is almost always molecular. The grand organizer—the master that controls the molecules, the cells, and the organs and the way they interact—is the genome with its epigenetic modifications. Traditionally, the discipline of physiology has, in its reductionistic journey, always stopped at about the level of cells and certain subcellular organelles as well as their component and controlling molecules. The discipline of physiology left to molecular biology and molecular genetics the business of how the cell controls itself through its DNA. The modern discipline of physiology has become closely intertwined with molecular biology, however, because DNA encodes the proteins in which physiologists are most interested. Very often, physiologists painstakingly develop elegant strategies for cloning the genes relevant to physiology. Sometimes brute-force approaches, such as the Human Genome Project in the United States, hand the physiologist a candidate gene, homologous to one of known function, on a silver platter. In still other cases, molecular biologists may clone a gene with

CHAPTER 1  •  Foundations of Physiology

no known function. In this case, it may be up to the physiologist to determine the function of the gene product; that is, to determine its physiology. Physiological genomics (or functional genomics) is a new branch of physiology devoted to the understanding of the roles that genes play in physiology. Traditionally, physiologists have moved in a reductionistic direction from organ to cell to molecule to gene. One of the most fascinating aspects of physiological genomics is that it has closed the circle and linked organ physiology directly with molecular biology. Perhaps one of the most striking examples is the knockout mouse. Knocking out the gene encoding a protein that, according to conventional wisdom, is very important will sometimes have no obvious effect or sometimes unexpected effects. It is up to the physiologist, at least in part, to figure out why. It is perhaps rather sobering to consider that to truly understand the impact of a transgene or a knockout on the physiology of a mouse, one would have to carefully re-evaluate the totality of mouse physiology. To grasp the function of a gene product, the physiologist must retrace the steps up the reductionistic road and achieve an integrated understanding of that gene’s function at the level of the cells, organs, and whole body. Physiology is unique among the basic medical sciences in that it is both broad in its scope (i.e., it deals with multiple systems) and integrative in its outlook. In some cases, important physiological parameters, such as blood pressure, may be under the control of many genes. Certain polymorphisms in several of these many genes could have a cumulative effect that produces high blood pressure. How would one identify which polymorphisms of which genes may underlie high blood pressure? This sort of complex problem does not easily lend itself to a physiologist’s controlled studies. One approach would be to study a population of people, or strains of experimental animals, and use statistical tools to determine which polymorphisms correlate with high blood pressure in a population. Indeed, epidemiologists use statistical tools to study group effects in populations. However, even after the identification of variants in various genes, each of which may make a small contribution to high blood pressure, the physiologist has an important role. First, the physiologist, performing controlled experiments, must determine whether a particular genetic variant does indeed have at least the potential to modulate blood pressure. Second, the physiologist must determine the mechanism of the effect.

Cells live in a highly protected milieu intérieur In his lectures on the phenomena of life, Claude Bernard noted in 1878 on the conditions of the constancy of life, which he considered a property of higher forms of life. According to Bernard, animals have two environments: the “milieu extérieur” that physically surrounds the whole organism; and the “milieu intérieur,” in which the tissues and cells of the organism live. This internal environment is neither the air nor the water in which an organism lives but rather—in the case of the human body—the well-controlled liquid environment that Bernard called “the organic liquid that circulates and bathes all the anatomic elements of the tissues, the lymph or the plasma.” In short, this internal environment is what we today call the extracellular fluid. He

3

argued that physiological functions continue in a manner indifferent to the changing environment because the milieu intérieur isolates the organs and tissues of the body from the vagaries of the physical conditions of the environment. Indeed, Bernard described the milieu intérieur as if an organism had placed itself in a greenhouse. According to Bernard’s concept of milieu intérieur, some fluids contained within the body are not really inside the body at all. For example, the contents of the gastrointestinal tract, sweat ducts, and renal tubules are all outside the body. They are all continuous with the milieu extérieur. Bernard compares a complex organism to an ensemble of anatomical elements that live together inside the milieu intérieur. Therefore, in Section II of this textbook, we examine the physiology of these cells and molecules. In Chapter 2 (“Functional Organization of the Cell”), we begin our journey through physiology with a discussion of the biology of the cells that are the individual elements of the body. Chapter 3 (“Signal Transduction”) discusses how cells communicate directly through gap junctions or indirectly by molecules released into the extracellular fluid. These released molecules can bind to receptors on the cell membrane and initiate signal-transduction cascades that can modify gene transcription (a genomic response) and a wide range of other cell functions (nongenomic responses). Alternatively, these released molecules can bind to receptors in the cytoplasm or nucleus and alter the transcription of genes. In Chapter 4 (“Regulation of Gene Expression”), we examine the response of the nucleus. Chapter 5 (“Transport of Solutes and Water”) addresses how the plasma membrane separates the cell interior from Bernard’s milieu intérieur and establishes the composition of the cell interior. In the process of establishing the composition of the intracellular fluid, the plasma membrane also sets up ion and voltage gradients across itself. Excitable cells—mainly nerve and muscle cells—can exploit these gradients for the long-distance “electrical” transmission of information. The property of “excitability,” which requires both the perception of a change (a signal) and the reaction to it, is the topic of Chapters 6 to 9. In Section III, we examine how the nervous system exploits excitability to process information. Another theme developed by Bernard was that the “fixité du milieu intérieur” (the constancy of the extracellular fluid) is the condition of “free, independent life.” He explains that organ differentiation is the exclusive property of higher organisms and that each organ contributes to “compensate and equilibrate” against changes in the external environment. In that sense, each of the systems discussed in Sections IV to VIII permits the body to live within an adverse external environment because the cardiovascular system, the respiratory system, the urinary system, the gastrointestinal system, and the endocrine system create and maintain a constant internal environment. Individual cell types in various organ systems act in concert to support the constancy of the internal milieu, and the internal milieu in turn provides these cells with a culture medium in which they can thrive. The discipline of physiology also deals with those characteristics that are the property of a living organism as opposed to a nonliving organism. Four fundamental properties distinguish the living body. First, only living organisms exchange matter and energy with the environment to continue their

4

SECTION I  •  Introduction

existence. Several organ systems of the body participate in these exchanges. Second, only living organisms can receive signals from their environment and react accordingly. The principles of sensory perception, processing by the nervous system, and reaction are discussed in the chapters on excitability and the nervous system. Third, what distinguishes a living organism is the life cycle of growth and reproduction, as discussed in the chapters on reproduction (Section IX). Finally, the living organism is able to adapt to changing circumstances. This is a theme that is developed throughout this textbook but especially in the chapters on everyday life (Section X).

Homeostatic mechanisms—operating through sophisticated feedback control mechanisms— are responsible for maintaining the constancy of the milieu intérieur Homeostasis is the control of a vital parameter. The body carefully controls a seemingly endless list of vital parameters. Examples of tightly controlled parameters that affect nearly the whole body are arterial pressure and blood volume. At the level of the milieu intérieur, tightly regulated parameters include body core temperature and plasma levels of oxygen, glucose, potassium ions (K+), calcium ions (Ca2+), and hydrogen ions (H+). Homeostasis also occurs at the level of the single cell. Thus, cells regulate many of the same parameters that the body as a whole regulates: volume, the concentrations of many small inorganic ions (e.g., Na+, Ca2+, H+), and energy levels (e.g., ATP). One of the most common themes in physiology is the negative-feedback mechanism responsible for homeostasis. Negative feedback requires at least four elements. First, the system must be able to sense the vital parameter (e.g., glucose level) or something related to it. Second, the system must be able to compare the input signal with some internal reference value called a set-point, thereby forming a difference signal. Third, the system must multiply the error signal by some proportionality factor (i.e., the gain) to produce some sort of output signal (e.g., release of insulin). Fourth, the output signal must be able to activate an effector mechanism (e.g., glucose uptake and metabolism) that opposes the source of the input signal and thereby brings the vital parameter closer to the set-point (e.g., decrease of blood glucose levels back to normal).  N1-1  Sometimes the body controls a parameter, in part, by cleverly employing positive-feedback loops. A single feedback loop often does not operate in isolation but rather as part of a larger network of controls. Thus, a complex interplay may exist among feedback loops within single cells, within a tissue, within an organ or organ system, or at the level of the whole body. After studying these individual feedback loops in isolation, the physiologist may find that two feedback loops act either synergistically or antagonistically. For example, insulin lowers blood glucose levels, whereas epinephrine and cortisol have the opposite effect. Thus, the physiologist must determine the relative weights of feedback loops in competition with one another. Finally, the physiologist must also establish hierarchy among various feedback loops. For example, the hypothalamus controls the anterior pituitary, which controls the adrenal

cortex, which releases cortisol, which helps control blood glucose levels. Another theme of homeostasis is redundancy. The more vital a parameter is, the more systems the body mobilizes to regulate it. If one system should fail, others are there to help maintain homeostasis. It is probably for this reason that genetic knockouts sometimes fail to have their expected deleterious effects. The result of many homeostatic systems controlling many vital parameters is a milieu intérieur with a stable composition. Whether at the level of the milieu intérieur or the cytoplasm of a single cell, homeostasis occurs at a price: energy. When a vital parameter (e.g., the blood glucose level) is well regulated, that parameter is not in equilibrium. Equilibrium is a state that does not involve energy consumption. Instead, a well-regulated parameter is generally in a steady state. That is, its value is constant because the body or the cell carefully matches actions that lower the parameter value with other actions that raise it. The net effect is that the vital parameter is held at a constant value. An important principle in physiology, to which we have already alluded, is that each cell plays a specialized role in the overall function of the body. In return, the body—which is the sum of all these cells—provides the milieu intérieur appropriate for the life of each cell. As part of the bargain, each cell or organ must respect the needs of the body as a whole and not run amok for its own greedy interests. For example, during exercise, the system that controls body core temperature sheds heat by elaborating sweat for evaporation. However, the production of sweat ultimately reduces blood volume. Because the body as a whole places a higher priority on the control of blood volume than on the control of body core temperature, at some point the system that controls blood volume will instruct the system that controls body core temperature to reduce the production of sweat. Unfortunately, this juggling of priorities works only if the individual stops exercising; if not, the result may be heat stroke. The adaptability of an organism depends on its ability to alter its response. Indeed, flexible feedback loops are at the root of many forms of physiological adaptation. For instance, at sea level, experimentally lowering the level of oxygen (the sensory stimulus) in the inspired air causes an increase in breathing (the response). However, after acclimatization at high altitude to low oxygen levels, the same low level of oxygen (the same sensory stimulus) causes one to breathe much faster (a greater response). Thus, the response may depend on the previous history and therefore the “state” of the system. In addition to acclimatization, genetic factors can also contribute to the ability to respond to an environmental stress. For example, certain populations of humans who have lived for generations at high altitude withstand hypoxia better than lowlanders do, even after the lowlanders have fully acclimatized.

Medicine is the study of “physiology gone awry” Medicine borrows its physicochemical principles from physiology. Medicine also uses physiology as a reference state: it is essential to know how organs and systems function in the healthy person to grasp which components may be

CHAPTER 1  •  Foundations of Physiology

4.e1

N1-1  Feedback Control Contributed by Arthur DuBois In proportional control, the set-point is not reached because the difference signal would disappear, and control would come to an end. Engineers devised a way around this. They took the time integral of the difference signal and used that to activate the effector mechanism to achieve integral control that would allow return to the set-point. There was another problem. Since there is a time delay in processing the input signal, there is a delay in returning to the set-point. Engineers also had a way around that. They took the time-derivative of the difference signal and added that to the corrective signal, speeding up the return toward the set-point. Another problem turned up. If you have a heater and a cooler, each with its own thermostat, and you want the room to be 23°C to 25°C, you must set one thermostat to turn on the heater at

temperatures 25°C but shut it off at ≤25°C to avoid running the heater and cooler both at once. If the room is cold, the heater will warm it up to 23°C, then shut off. If the room is warm, the cooler will cool it down to 25°C, then shut off. By analogy, the body has separate systems for shivering and sweating, so both do not occur at once. One can picture that anabolic and catabolic pathways should cycle separately and not simultaneously. Many body systems such as respiratory and circulatory controls oscillate between slightly above and slightly below the desired average, hunting for it rather than sitting on a single ideal value. In a case in which the control system is less precise, the swings become wider, as they do when a drunk driver wanders back and forth across the road proceeding home.

CHAPTER 1  •  Foundations of Physiology

malfunctioning in a patient. A large part of clinical medicine is simply dealing with the abnormal physiology brought about by a disease process. One malfunction (e.g., heart failure) can lead to a primary pathological effect (e.g., a decrease in cardiac output) that—in chain-reaction style— leads to a series of secondary effects (e.g., fluid overload) that are the appropriate responses of physiological feedback loops. Indeed, as clinician-physiologists have explored the basis of disease, they have discovered a great deal about physiology. For this reason, we have tried to illustrate physiological principles with clinical examples, some of which are displayed in clinical boxes in this text. Physiologists have developed many tools and tests to examine normal function. A large number of functional tests—used in diagnosis of a disease, monitoring of the evolution of an illness, and evaluation of the progress of therapy—are direct transfers of technology developed in

5

the physiology laboratory. Typical examples are cardiac monitoring, pulmonary function tests, and renal clearance tests as well as the assays used to measure plasma levels of various ions, gases, and hormones. Refinements of such technology in the hospital environment, in turn, benefit the study of physiology. Thus, the exchange of information between medicine and physiology is a two-way street. The understanding of physiology summarized in this book comes from some experiments on humans but mostly from research on other mammals and even on squids and slime molds. However, our ultimate focus is on the human body.

REFERENCES The reference list is available at www.StudentConsult.com.

CHAPTER 1  •  Foundations of Physiology

REFERENCES Bernard C: Leçons sur les phénomènes de la vie communs aux animaux et aux végétaux. Cours de physiologie générale du Museum d’Histoire Naturelle. Paris, Baillière et Fils, 1878. Cannon WB: The Wisdom of the Body. New York, WW Norton, 1932. Smith HW: From Fish to Philosopher. New York, Doubleday, 1961.

5.e1

CHAPTER

2 

FUNCTIONAL ORGANIZATION OF THE CELL Michael J. Caplan

In the minds of many students, the discipline of physiology is linked inextricably to images from its past. This prejudice is not surprising because many experiments from physiology’s proud history, such as those of Pavlov on his dogs, have transcended mere scientific renown and entered the realm of popular culture. Some might believe that the science of physiology devotes itself exclusively to the study of whole animals and is therefore an antique relic in this era of molecular reductionism. Nothing could be further from the truth. Physiology is and always has been the study of the homeostatic mechanisms that allow an organism to persist despite the ever-changing pressures imposed by a hostile environment. These mechanisms can be appreciated at many different levels of resolution. Certainly it would be difficult to understand how the body operates unless one appreciates the functions of its organs and the communication between these organs that allows them to influence one another’s behaviors. It would also be difficult to understand how an organ performs its particular tasks unless one is familiar with the properties of its constituent cells and molecules. The modern treatment of physiology that is presented in this textbook is as much about the interactions of molecules in cells as it is about the interactions of organs in organisms. It is necessary, therefore, at the outset to discuss the structure and characteristics of the cell. Our discussion focuses first on the architectural and dynamic features of a generic cell. We then examine how this generic cell can be adapted to serve in diverse physiological capacities. Through adaptations at the cellular level, organs acquire the machinery necessary to perform their individual metabolic tasks.

STRUCTURE OF BIOLOGICAL MEMBRANES The surface of the cell is defined by a membrane The chemical composition of the cell interior is very different from that of its surroundings. This observation applies equally to unicellular paramecia that swim freely in a freshwater pond and to neurons that are densely packed in the cerebral cortex of the human brain. The biochemical processes involved in cell function require the maintenance of a precisely regulated intracellular environment. The cytoplasm is an extraordinarily complex solution, the constituents of which include myriad proteins, nucleic acids, nucleotides, 8

and sugars that the cell synthesizes or accumulates at great metabolic cost. The cell also expends tremendous energy to regulate the intracellular concentrations of numerous ions. If there were no barrier surrounding the cell to prevent exchange between the intracellular and extracellular spaces, all of the cytoplasm’s hard-won compositional uniqueness would be lost by diffusion in a few seconds. The requisite barrier is provided by the plasma membrane, which forms the cell’s outer skin. The plasma membrane is impermeable to large molecules such as proteins and nucleic acids, thus ensuring their retention within the cytosol. It is selectively permeable to small molecules such as ions and metabolites. However, the metabolic requirements of the cell demand a plasma membrane that is much more sophisticated than a simple passive barrier that allows various substances to leak through at different rates. Frequently, the concentration of a nutrient in the extracellular fluid (ECF) is several orders of magnitude lower than that required inside the cell. If the cell wishes to use such a substance, therefore, it must be able to accumulate it against a concentration gradient. A simple pore in the membrane cannot concentrate anything; it can only modulate the rate at which a gradient dissipates. To accomplish the more sophisticated feat of creating a concentration gradient, the membrane must be endowed with special machinery that uses metabolic energy to drive the uphill movements of substances— active transport—into or out of the cell. In addition, it would be useful to rapidly modulate the permeability properties of the plasma membrane in response to various metabolic stimuli. Active transport and the ability to control passive permeabilities underlie a wide range of physiological processes, from the electrical excitability of neurons to the resorptive and secretory functions of the kidney. In Chapter 5, we will explore how cells actively transport solutes across the plasma membrane. The mechanisms through which the plasma membrane’s dynamic selectivity is achieved, modified, and regulated are discussed briefly below in this chapter and in greater detail in Chapter 7.

The cell membrane is composed primarily of phospholipids Our understanding of biological membrane structure is based on studies of red blood cells, or erythrocytes, that were conducted in the early part of the 20th century. The

CHAPTER 2  •  Functional Organization of the Cell

A—PHOSPHATIDYLETHANOLAMINE

B—PHOSPHOLIPID ICON This icon is used in this text to represent this and other phospholipid molecules.

+

NH3

Ethanolamine

CH2 CH2

C—MONOLAYER

O

Phosphate O

P

Hydrophobic lipid tails Hydrophilic head groups

–

O

O

Glycerol

CH2

CH

O

O

C CH2

9

O

C

CH2

Water

O

CH2

D—PHOSPHOLIPID BILAYER

Fatty acid

In an aqueous environment, polar hydrophilic head groups orient toward the polar water… …and nonpolar (hydrophobic) tails orient away from the water. Thus, a phospholipid bilayer is formed.

R1

R2

Figure 2-1  Phospholipids.

erythrocyte lacks the nucleus and other complicated intracellular structures that are characteristic of most animal cells. It consists of a plasma membrane surrounding a cytoplasm that is rich in hemoglobin. It is possible to break open erythrocytes and release their cytoplasmic contents. The plasma membranes can then be recovered by centrifugation to provide a remarkably pure preparation of cell surface membrane. Biochemical analysis reveals that this membrane is composed of two principal constituents: lipid and protein. Most of the lipid associated with erythrocyte plasma membranes belongs to the molecular family of phospholipids. In general, phospholipids share a glycerol backbone, two hydroxyl groups of which are esterified to various fatty-acid or acyl groups (Fig. 2-1A). These acyl groups may have different numbers of carbon atoms and also may have double bonds between carbons. For glycerol-based phospholipids, the third glycerolic hydroxyl group is esterified to a phosphate group, which is in turn esterified to a small molecule referred to as a head group. The identity of the head group determines the name as well as many of the properties of the individual phospholipids. For instance, glycerol-based phospholipids that bear an ethanolamine molecule in the head group position are categorized as phosphatidylethanolamines (see Fig. 2-1A).

Phospholipids form complex structures in aqueous solution The unique structure and physical chemistry of each phospholipid (see Fig. 2-1B) underlie the formation of biological membranes and explain many of their most important properties. Fatty acids are nonpolar molecules. Their long carbon

chains lack the charged groups that would facilitate interactions with water, which is polar. Consequently, fatty acids dissolve poorly in water but readily in organic solvents; thus, fatty acids are hydrophobic. On the other hand, the head groups of most phospholipids are charged or polar. These head groups interact well with water and consequently are very water soluble. Thus, the head groups are hydrophilic. Because phospholipids combine hydrophilic heads with hydrophobic tails, their interaction with water is referred to as amphipathic. When mixed with water, phospholipids organize themselves into structures that prevent their hydrophobic tails from making contact with water while simultaneously permitting their hydrophilic head groups to be fully dissolved. When added to water at fairly low concentrations, phospholipids form a monolayer (see Fig. 2-1C) on the water’s surface at the air-water interface. It is energetically less costly to the system for the hydrophobic tails to stick up in the air than to interact with the solvent. At higher concentrations, phospholipids assemble into micelles. The hydrophilic head groups form the surfaces of these small spheres, whereas the hydrophobic tails point toward their centers. In this geometry, the tails are protected from any contact with water and instead are able to participate in energetically favorable interactions among themselves. At still higher concentrations, phospholipids spontaneously form bilayers (see Fig. 2-1D). In these structures, the phospholipid molecules arrange themselves into two parallel sheets or leaflets that face each other tail to tail. The hydrophilic head groups form the surfaces of the bilayer; the hydrophobic tails form the center of the sandwich. The hydrophilic surfaces insulate the hydrophobic tails from

10

SECTION II  •  Physiology of Cells and Molecules

contact with the solvent, leaving the tails free to associate exclusively with one another. The physical characteristics of a lipid bilayer largely depend on the chemical composition of its constituent phospholipid molecules. For example, the width of the bilayer is determined by the length of the fatty-acid side chains. Dihexadecanoic phospholipids (whose two fatty-acid chains are each 16 carbons long) produce bilayers that are 2.47 nm wide; ditetradecanoic phospholipids (bearing 14-carbon fatty acids) generate 2.3-nm bilayers. Similarly, the nature of the head groups determines how densely packed adjacent phospholipid molecules are in each leaflet of the membrane. Detergents can dissolve phospholipid membranes because, like the phospholipids themselves, they are amphipathic. They possess very hydrophilic head groups and hydrophobic tails and are water soluble at much higher concentrations than are the phospholipids. When mixed together in aqueous solutions, detergent and phospholipid molecules interact through their hydrophobic tails, and the resulting complexes are water soluble, either as individual dimers or in mixed micelles. Therefore, adding sufficient concentrations of detergent to phospholipid bilayer membranes disrupts the membranes and dissolves the lipids. Detergents are extremely useful tools in research into the structure and composition of lipid membranes.

The diffusion of individual lipids within a leaflet of a bilayer is determined by the chemical makeup of its constituents Despite its highly organized appearance, a phospholipid bilayer is a fluid structure. An individual phospholipid molecule is free to diffuse within the entire leaflet in which it resides. The rate at which this two-dimensional diffusion occurs is extremely temperature dependent. At high temperatures, the thermal energy of any given lipid molecule is greater than the interaction energy that would tend to hold adjacent lipid molecules together. Under these con­ ditions, lateral diffusion can proceed rapidly, and the lipid is said to be in the sol state. At lower temperatures, inter­ action energies exceed the thermal energies of most individual molecules. Thus, phospholipids diffuse slowly because they lack the energy to free themselves from the embraces of their neighbors. This behavior is characteristic of the gel state. The temperature at which the bilayer membrane converts from the gel to the sol phase (and vice versa) is referred to as the transition temperature. The transition temperature is another characteristic that depends on the chemical makeup of the phospholipids in the bilayer. Phospholipids with long, saturated fatty-acid chains can extensively interact with one another. Consequently, a fair amount of thermal energy is required to overcome these interactions and permit diffusion. Not surprisingly, such bilayers have relatively high transition temperatures. For example, the transition temperature for dioctadecanoic phosphatidylcholine (which has two 18-carbon fatty-acid chains, fully saturated) is 55.5°C. In contrast, phospholipids that have shorter fatty-acid chains or double bonds (which introduce kinks) cannot line up next to each other as well and hence do not interact as well.

Considerably less energy is required to induce them to participate in diffusion. For example, if we reduce the length of the carbon chain from 18 to 14, the transition temperature falls to 23°C. If we retain 18 carbons but introduce one double bond (making the fatty-acid chains monounsaturated), the transition temperature also falls dramatically. By mixing other types of lipid molecules into phos­ pholipid bilayers, we can markedly alter the membrane’s fluidity properties. The glycerol-based phospholipids, the most common membrane lipids, include the phosphatidylethanolamines described above (see Fig. 2-1A), as well as the phosphatidylinositols (Fig. 2-2A), phosphatidylserines (see Fig. 2-2B), and phosphatidylcholines (see Fig. 2-2C). The second major class of membrane lipids, the sphingo­ lipids (derivatives of sphingosine), is made up of three subgroups: sphingomyelins (see Fig. 2-2D),  N2-1  gly­ cosphingolipids such as the galactocerebrosides (see Fig. 2-2E), and gangliosides (not shown in figure). Cholesterol (see Fig. 2-2F) is another important membrane lipid. Because these other molecules are not shaped exactly like the glycerolbased phospholipids, they participate to different degrees in intermolecular interactions with phospholipid side chains.  N2-2  The presence of these alternative lipids changes the strength of the interactions that prevents lipid molecules from diffusing. Consequently, the membrane has a different fluidity and a different transition temperature. This behavior is especially characteristic of the cholesterol molecule, whose rigid steroid ring binds to and partially immobilizes fattyacid side chains. Therefore, at modest concentrations, cholesterol decreases fluidity. However, when it is present in high concentrations, cholesterol can substantially disrupt the ability of the phospholipids to interact among themselves, which increases fluidity and lowers the gel-sol transition temperature. This issue is significant because animal cell plasma membranes can contain substantial quantities of cholesterol. Bilayers composed of several different lipids do not undergo the transition from gel to sol at a single, well-defined temperature. Instead, they interconvert more gradually over a temperature range that is defined by the composition of the mixture. Within this transition range in such multicomponent bilayers, the membrane can become divided into compositionally distinct zones. The phospholipids with long-chain, saturated fatty acids will adhere to one another relatively tightly, which results in the formation of regions with gel-like properties. Phospholipids bearing short-chain, unsaturated fatty acids will be excluded from these regions and migrate to sol-like regions. Hence, “lakes” of lipids with markedly different physical properties can exist side by side in the plane of a phospholipid membrane. Thus, the same thermodynamic forces that form the elegant bilayer structure can partition distinct lipid domains within the bilayer. As discussed below, the segregation of lipid lakes in the plane of the membrane may be important for sorting membrane proteins to different parts of the cell. Although phospholipids can diffuse in the plane of a lipid bilayer membrane, they do not diffuse between adjacent leaflets (Fig. 2-3). The rate at which phospholipids spontaneously “flip-flop” from one leaflet of a bilayer to the other is extremely low. As mentioned above, the center of a bilayer membrane consists of the fatty-acid tails of the phospholipid

CHAPTER 2  •  Functional Organization of the Cell

N2-1  Sphingomyelins Contributed by Emile Boulpaep and Walter Boron The polar head group of sphingomyelins can be either phosphocholine, as shown in Figure 2-2D, or phosphoethanolamine (analogous to the phosphoethanolamine moiety in Fig. 2-1A). Note that sphingomyelins are both (1) sphingolipids because they contain sphingosine, and (2) phospholipids because they contain a phosphate group as do the glycerol-based phospholipids shown in Figures 2-1A and 2-2A–C.

N2-2  Diversity of Lipids in a Bilayer Contributed by Michael Caplan

Hydrophilic heads

Hydrophobic tails

Cholesterol aids in stiffening the membrane.

eFigure 2-1  The upper leaflet of this lipid bilayer contains, from left to right, phosphatidylinositol, phosphatidylserine, cholesterol, phosphatidylinositol, phosphatidylcholine, and cholesterol.

10.e1

11

CHAPTER 2  •  Functional Organization of the Cell

A

B

PHOSPHATIDYLINOSITOL

PHOSPHATIDYLSERINE

C

PHOSPHATIDYLCHOLINE CH3

+

NH3 OH

Inositol

OH

H

Serine

N +

O

OH O

Choline

Phosphate

–

O

O

CH

O

O O

C

O O

P

O

CH2

CH2

O

P

–

O

O

CH2

CH

O

O O

C

C

–

O

P

O

CH2

CH3

CH2

CH2

HO

C

–

COO

C

OH

Glycerol

H3C

O

CH2

O

CH2

CH

O

O

C

O

C

CH2

CH2

CH2

CH2

CH2

CH2

R1

R2

R1

R2

R1

R2

CH2

O

Fatty acid

D

E

SPHINGOMYELIN CH3

Choline

+

N

CH2OH

Galactose

OH

Sphingosine

Sphingosine

OH

H

H

CH

CH

N

CH

–

O CH2

OH

O CH2

O

P

OH

OH

CH2 O

CH

CH

N

CH

C

O

CHOLESTEROL

O

HO

CH3

CH2

O

F

GALACTOCEREBROSIDE

CH3

C

O

CH3

CH3 CH3

CH

CH

CH2

CH2 CH2 CH2

CH H3C

CH2

CH CH3

Figure 2-2  Structures of some common membrane lipids.

molecules and is an extremely hydrophobic environment. For a phospholipid molecule to jump from one leaflet to the other, its highly hydrophilic head group would have to transit this central hydrophobic core, which would have an extremely high energy cost. This caveat does not apply to cholesterol (see Fig. 2-3), whose polar head is a single hydroxyl group. The energy cost of dragging this small polar hydroxyl group through the bilayer is relatively low, which permits relatively rapid cholesterol flip-flop.

Phospholipid bilayer membranes are impermeable to charged molecules The lipid bilayer is ideally suited to separate two aqueous compartments. Its hydrophilic head groups interact well with water at both membrane surfaces, whereas the hydrophobic center ensures that the energetic cost of crossing the membrane is prohibitive for charged atoms or molecules. Pure phospholipid bilayer membranes are extremely

12

SECTION II  •  Physiology of Cells and Molecules

PM

Phospholipids can move laterally, rotate, or flex. Rarely do they flip to the other leaflet.

ER M

PM

Cholesterol aids in stiffening the membrane and can flip easily. Figure 2-3  Mobility of lipids within a bilayer.

impermeable to almost any charged water-soluble substance. Ions such as Na+, K+, Cl–, and Ca2+ are insoluble in the hydrophobic membrane core and consequently cannot travel from the aqueous environment on one side of the membrane to the aqueous environment on the opposite side. The same is true of large water-soluble molecules, such as proteins, nucleic acids, sugars, and nucleotides. Whereas phospholipid membranes are impermeable to water-soluble molecules, small uncharged polar molecules can cross fairly freely. This is often true for O2, CO2, NH3, and, remarkably, water itself. Water molecules may, at least in part, traverse the membrane through transient cracks between the hydrophobic tails of the phospholipids without having to surmount an enormous energetic barrier. The degree of permeability of water (and perhaps that of CO2 and NH3 as well) varies extensively with lipid composition; some phospholipids (especially those with short or kinked fattyacid chains) permit a much greater rate of transbilayer water diffusion than others do.

E

Figure 2-4  Transmission electron micrograph of a cell membrane. The

The plasma membrane is a bilayer

photograph shows two adjacent cells of the pancreas of a frog (original magnification ×43,000). The inset is a high-magnification view (original magnification ×216,000) of the plasma membranes (PM) of the cells. Note that each membrane includes two dense layers with an intermediate layer of lower density. The dense layers represent the interaction of the polar head groups of the phospholipids with the OsO4 used to stain the preparation. E, nuclear envelope; M, mitochondrion. (From Porter KR, Bonneville MR: Fine Structure of Cells and Tissues, 4th ed. Philadelphia, Lea & Febiger, 1973.)

As may be inferred from the preceding discussion, the membrane at the cell surface is, in fact, a phospholipid bilayer. The truth of this statement was established by a remarkably straightforward experiment. In 1925, Gorter and Grendel measured the surface area of the lipids they extracted from erythrocyte plasma membranes. They used a device called a Langmuir trough in which the lipids are allowed to line up at an air-water interface (see Fig. 2-1C) and are then packed together into a continuous monolayer by a sliding bar that decreases the surface available to them. The area of the monolayer that was created by the erythrocyte lipids was exactly twice the surface area of the erythrocytes from which they were derived. Therefore, the plasma membrane must be a bilayer. Confirmation of the bilayer structure of biological membranes has come from x-ray diffraction studies performed on the repetitive whorls of membrane that form the myelin sheaths surrounding neuronal axons (see pp. 292–293). The membrane’s bilayer structure can be visualized directly in the high-magnification electron micrograph depicted in Figure 2-4. The osmium tetraoxide molecule (OsO4) with which the membrane is stained binds to the head groups of phospholipids. Thus, both surfaces of a phospholipid bilayer appear black in electron micrographs,

whereas the membrane’s unstained central core appears white. The phospholipid compositions of the two leaflets of the plasma membrane are not identical. Labeling studies performed on erythrocyte plasma membranes reveal that the surface that faces the cytoplasm contains phospha­ tidylethanolamine and phosphatidylserine, whereas the outward-facing leaflet is composed almost exclusively of phosphatidylcholine. As is discussed below in this chapter, this asymmetry is created during the biosynthesis of the phospholipid molecules. It is not entirely clear what advantage this distribution provides to the cell. The interactions between certain proteins and the plasma membrane may require this segregation. The lipid asymmetry may be especially important for those phospholipids that are involved in second-messenger cascades. Phosphatidylinositols, for example, give rise to phosphoinositides, which play critical roles in signaling pathways (see pp. 58–61). In addition, the phosphatidylinositol composition of the cytoplasmic face of an organelle helps to define the identity of the organelle and to govern its trafficking and targeting properties. Finally, the phospholipids that are characteristic of animal cell plasma membranes generally have one saturated and one

CHAPTER 2  •  Functional Organization of the Cell

Peripheral protein

13

Integral proteins

Extracellular space

Some proteins are linked to membrane phospholipids via an oligosaccharide...

Most integral membrane proteins have membrane-spanning α-helical domains of about 20 amino acids.

Peripheral proteins are noncovalently bonded with integral proteins.

Some have multiple membranespanning domains.

A

B

C

E

D H N

Integral protein

P

C O

P

F

…or are linked directly to fatty acids or prenyl groups.

Cytosol Figure 2-5  Classes of membrane proteins. In E, protein is coupled via a GPI linkage.

unsaturated fatty-acid residue. Consequently, they are less likely to partition into sol-like or gel-like lipid domains than are phospholipids that bear identical fatty-acid chains.  N2-3

Membrane proteins can be integrally or peripherally associated with the plasma membrane The demonstration that the plasma membrane’s lipid components form a bilayer leaves open the question of how the membrane’s protein constituents are organized. Membrane proteins can belong to either of two broad classes, peripheral or integral. Peripherally associated membrane proteins are neither embedded within the membrane nor attached to it by covalent bonds; instead, they adhere tightly to the cytoplasmic or extracellular surfaces of the plasma membrane (Fig. 2-5A). They can be removed from the membrane, however, by mild treatments that disrupt ionic bonds (very high salt concentrations) or hydrogen bonds (very low salt concentrations). In contrast, integral membrane proteins are intimately associated with the lipid bilayer. They cannot be eluted from the membrane by these high- or low-salt washes. For integral membrane proteins to be dislodged, the membrane itself must be dissolved by adding detergents. Integral membrane proteins can be associated with the lipid bilayer in any of three ways. First, some proteins actually span the lipid bilayer once or several times (see Fig. 2-5B, C) and hence are referred to as transmembrane proteins. Experiments performed on erythrocyte membranes reveal that these proteins can be labeled with protein-tagging reagents applied to either side of the bilayer.

The second group of integral membrane proteins is embedded in the bilayer without actually crossing it (see Fig. 2-5D). A third group of membrane-associated proteins is not actually embedded in the bilayer at all. Instead, these lipid-anchored proteins are attached to the membrane by a covalent bond that links them either to a lipid component of the membrane or to a fatty-acid derivative that intercalates into the membrane. For example, proteins can be linked to a special type of glycosylated phospholipid molecule (see Fig. 2-5E), which is most often glycosylphosphatidylinositol (GPI), on the outer leaflet of the membrane. This family is referred to collectively as the glycophospholipidlinked proteins. Another example is a direct linkage to a fatty acid (e.g., a myristyl group) or a prenyl (e.g., farnesyl) group that intercalates into the inner leaflet of the membrane (see Fig. 2-5F).

The membrane-spanning portions of transmembrane proteins are usually hydrophobic α helices How can membrane-spanning proteins remain stably associated with the bilayer in a conformation that requires at least some portion of their amino-acid sequence to be in continuous contact with the membrane’s hydrophobic central core? The answer to this question can be found in the special structures of those protein domains that actually span the membrane. The side chains of the eight amino acids listed in the upper portion of Table 2-1 are hydrophobic. These aromatic or uncharged aliphatic groups are almost as difficult to solvate in water as are the fatty-acid side chains of the membrane phospholipids themselves. Not surprisingly, therefore,

CHAPTER 2  •  Functional Organization of the Cell

13.e1

N2-3  Membrane Microdomains Contributed by Michael Caplan According to current models (see Anderson and Jacobson, 2002; Edidin, 2003), lipids and proteins are not uniformly distributed in the plane of the membranes that surround cells and organelles. Instead, certain lipids and associated proteins cluster to form microdomains that differ in composition, structure, and function from the rest of the membrane that surrounds them. These microdomains can be thought of as small islands bordered by the “lake” of lipids and proteins that constitute the bulk of the membrane. These two-dimensional structures are composed of lipids that tend to form close interactions with one another, resulting in the self-assembly of organized domains that include specific types of lipids and exclude others. The lipids that tend to be found in microdomains include sphingomyelin, cholesterol, and glycolipids. Proteins that are able to interact closely with microdomainforming lipids can also become selectively incorporated into these microdomains. A number of different names are used to refer to these microdomains, the most common of which are caveolae and rafts. Caveolae (see pp. 42–43) were originally identified in the electron microscope as flask-shaped invaginations of the plasma membrane. They carry a coat composed of proteins called caveolins, and they tend to be at least 50 to 80 nm in diameter. Caveolae have been shown to participate in endocytosis of specific subsets of proteins and are also richly endowed with signaling molecules, such as receptor tyrosine kinases. Rafts are less well understood structures, which are defined by the biochemical behaviors of their constituents when the surrounding membrane is dissolved in nonionic detergents. Lipid microdomains rich in sphingomyelin, cholesterol, and glycolipids tend to resist solubilization in these detergents under certain

conditions and can be recovered intact by density centrifugation. Once again, a number of interesting proteins involved in cell signaling and communication, including kinases, ion channels, and G proteins, tend to be concentrated in rafts, or to become associated with rafts upon the activation of specific signaltransduction pathways. Rafts are thought to collect signaling proteins into small, highly concentrated zones, thereby facilitating their interactions and hence their ability to activate particular pathways. Rafts are also involved in membrane trafficking processes. In polarized epithelial cells, the sorting of a number of proteins to the apical plasma membrane is dependent upon their ability to partition into lipid rafts that form in the plane of the membrane of the trans-Golgi network. Little is known about what lipid rafts actually look like in cell membranes in situ. It is currently thought that they are fairly small (1000 members either known or predicted from genome sequences. GPCRs mediate cellular responses to a diverse array of signaling molecules, such as hormones, neurotransmitters, vasoactive peptides, odorants, tastants, and other local mediators. Despite the chemical diversity of their ligands, most receptors of this class have a similar structure (Fig. 3-3). They consist of a single polypeptide chain with seven membranespanning α-helical segments, an extracellular N terminus that is glycosylated, a large cytoplasmic loop that is composed mainly of hydrophilic amino acids between helices 5 and 6, and a hydrophilic domain at the cytoplasmic C terminus. Most small ligands (e.g., epinephrine) bind in the plane of the membrane at a site that involves several membrane-spanning segments. In the case of larger protein ligands, a portion of the extracellular N terminus also participates in ligand binding. The 5,6-cytoplasmic loop appears to be the major site of interaction with the intracellular G protein, although the 3,4-cytoplasmic loop and the cytoplasmic C terminus also contribute to binding in some cases. Binding of the GPCR to its extracellular ligand regulates this interaction between the receptor and the G proteins, thus transmitting a signal to downstream effectors. In the next four sections of this subchapter, we discuss the general

CHAPTER 3  •  Signal Transduction

N3-4  Compartmentalization of Second-Messenger Effects Contributed by Laurie Roman In the textbook, we referred only to whole-cell levels of intracellular second messengers (e.g., cAMP), as if these messengers were uniformly distributed throughout the cell. However, some cell physiologists and cell biologists believe that local effects of intracellular second messengers may be extremely important in governing how signal-transduction processes work. One piece of evidence for such local effects is that the receptors for hormones and other extracellular agonists often are a part of macromolecular clusters of proteins that share a common physiological role. For example, a hormone receptor, its downstream heterotrimeric G protein, an amplifying enzyme (e.g., adenylyl cyclase) that generates the intracellular second messenger (e.g. cAMP), other proteins (e.g., the A kinase anchoring protein [or AKAP]), and the effector molecule (e.g., protein kinase A) may all reside in a microdomain at the cell membrane. Thus, it is possible that a particular hormone could act by locally raising [cAMP]i to levels much higher than in neighboring areas, so that—of all the cellular proteins potentially sensitive to cAMP—only a local subset of these targets may be activated by the newly formed cAMP. A second piece of evidence for the local effects of cAMP is the wide distribution of phosphodiesterases, which would be expected to break down cAMP and limit its ability to spread throughout the cell.

51.e1

52

SECTION II  •  Physiology of Cells and Molecules

Extracellular space

BOX 3-1  Action of Toxins on Heterotrimeric G Proteins

I

N

C G protein binding Cytosol Figure 3-3  G protein–coupled receptor.

principles of how G proteins function and then consider three major second-messenger systems that G proteins trigger.

GENERAL PROPERTIES OF G PROTEINS

nfectious diarrheal disease has a multitude of causes. Cholera toxin, a secretory product of the bacterium Vibrio cholerae, is responsible in part for the devastating characteristics of cholera. The toxin is an oligomeric protein composed of one A subunit and five B subunits (AB5). After cholera toxin enters intestinal epithelial cells, the A subunit separates from the B subunits and becomes activated by proteolytic cleavage. The resulting active A1 fragment catalyzes the ADP ribosylation of Gαs. This ribosylation, which involves transfer of the ADP-ribose moiety from the oxidized form of nicotinamide adenine dinucleotide (NAD+) to the α subunit, inhibits the GTPase activity of Gαs. As a result of this modification, Gαs remains in its activated, GTP-bound form and can activate adenylyl cyclase. In intestinal epithelial cells, the constitutively activated Gαs elevates levels of cAMP, which causes an increase in Cl− conductance and water flow and thereby contributes to the large fluid loss characteristic of this disease. A related bacterial product is pertussis toxin, which is also an AB5 protein. It is produced by Bordetella pertussis, the causative agent of whooping cough. Pertussis toxin ADPribosylates Gαi. This ADP-ribosylated Gαi cannot exchange its GDP (inactive state) for GTP. Thus, αi remains in its GDPbound inactive state. As a result, receptor occupancy can no longer release the active αi-GTP, so adenylyl cyclase cannot be inhibited. Thus, both cholera toxin and pertussis toxin increase the generation of cAMP.

G proteins are heterotrimers that exist in many combinations of different α, β, and γ subunits G proteins are members of a superfamily of GTP-binding proteins. This superfamily includes the classic heterotrimeric G proteins that bind to GPCRs as well as the so-called small GTP-binding proteins, such as Ras. Both the heterotrimeric and small G proteins can hydrolyze GTP and switch between an active GTP-bound state and an inactive GDPbound state. Heterotrimeric G proteins are composed of three subunits, α, β, and γ. At least 16 different α subunits (~42 to 50 kDa), 5 β subunits (~33 to 35 kDa), and 11 γ subunits (~8 to 10 kDa) are present in mammalian tissue. The α subunit binds and hydrolyzes GTP and also interacts with “downstream” effector proteins such as adenylyl cyclase. Historically, the α subunits were thought to provide the principal specificity to each type of G protein, with the βγ complex functioning to anchor the trimeric complex to the membrane. However, it is now clear that the βγ complex also functions in signal transduction by interacting with effector molecules distinct from those regulated by the α subunits. Moreover, both the α and γ subunits are involved in anchoring the complex to the membrane. The α subunit is held to the membrane by either a myristyl or a palmitoyl group, whereas the γ subunit is held via a prenyl group. The multiple α, β, and γ subunits demonstrate distinct tissue distributions and interact with different receptors and effectors (Table 3-2). Because of the potential for several hundred combinations of the known α, β, and γ subunits, G proteins are ideally suited to link a diversity of receptors to

a diversity of effectors. The many classes of G proteins, in conjunction with the presence of several receptor types for a single ligand, provide a mechanism whereby a common signal can elicit the appropriate physiological response in different tissues. For example, when epinephrine binds β1 adrenergic receptors in the heart, it stimulates adenylyl cyclase, which increases heart rate and the force of contraction. However, in the periphery, epinephrine acts on α2 adrenergic receptors coupled to a G protein that inhibits adenylyl cyclase, thereby increasing peripheral vascular resistance and consequently increasing venous return and blood pressure. Among the first effectors found to be sensitive to G proteins was the enzyme adenylyl cyclase. The heterotrimeric G protein known as Gs was so named because it stimulates adenylyl cyclase. A separate class of G proteins was given the name Gi because it is responsible for the ligand-dependent inhibition of adenylyl cyclase. Identification of these classes of G proteins was greatly facilitated by the observation that the α subunits of individual G proteins are substrates for ADP ribosylation catalyzed by bacterial toxins. The toxin from Vibrio cholerae activates Gs, whereas the toxin from Bordetella pertussis inactivates the cyclase-inhibiting Gi (Box 3-1). For their work in identifying G proteins and elucidating the physiological role of these proteins, Alfred Gilman and Martin Rodbell received the 1994 Nobel Prize in Physiology or Medicine.  N3-5

CHAPTER 3  •  Signal Transduction

N3-5  Alfred Gilman and Martin Rodbell For more information about Alfred Gilman and Martin Rodbell and the work that led to their Nobel Prize, visit http:// www.nobel.se/medicine/laureates/1994/index.html (accessed October 2014).

52.e1

CHAPTER 3  •  Signal Transduction

53

TABLE 3-2  Families of G Proteins FAMILY/SUBUNIT

TOXIN

DISTRIBUTION

RECEPTOR

EFFECTOR/ROLE

100

CTX

Ubiquitous

β adrenergic, TSH, glucagon, others

↑ Adenylyl cyclase ↑ Ca2+ channel

88

CTX

Olfactory epithelium

Odorant

↑ Adenylyl cyclase Open K+ channel

100 88

PTX PTX PTX

~Ubiquitous Ubiquitous ~Ubiquitous

M2, α2 adrenergic, others

↑ IP3, DAG, Ca2+, and AA, ↓ adenylyl cyclase

αO1A αO1B

73 73

PTX PTX

Brain, others Brain, others

Met-enkephalin, α2 adrenergic, others

αt1 αt2

68 68

PTX, CTX PTX, CTX

Retinal rods Retinal cones

Rhodopsin Cone opsin

αg αz

67 60

PTX, CTX

Taste buds Brain, adrenal, platelet

Taste ?

Gq αq α11 α14 α15 α16

100 88 79 57 58

~Ubiquitous ~Ubiquitous Lung, kidney, liver B cell, myeloid T cell, myeloid

M1, α1 adrenergic, others

↑ PLCβ1, PLC β2, PLC β3

Several receptors

↑ PLCβ1, PLC β2, PLC β3

G12 α12 α13

100 67

Ubiquitous Ubiquitous

Gs (αs) αs(s) αs(l) αolf Gi (αi) αi1 αi2 αi3

% IDENTITY

↑ cGMP-phosphodiesterase

↓ Adenylyl cyclase

CTX, cholera toxin; PTX, pertussis toxin.

G-protein activation follows a cycle In their inactive state, heterotrimeric G proteins are a complex of α, β, and γ subunits in which GDP occupies the guanine nucleotide–binding site of the α subunit. After ligand binding to the GPCR (Fig. 3-4, step 1), a conformational change in the receptor–G protein complex facilitates the release of bound GDP and simultaneous binding of GTP to the α subunit (see Fig. 3-4, step 2). This GDP-GTP exchange stimulates dissociation of the complex from the receptor (see Fig. 3-4, step 3) and causes disassembly of the trimer into a free GTP-bound α subunit and separate βγ complex (see Fig. 3-4, step 4). The GTP-bound α subunit interacts in the plane of the membrane with downstream effectors such as adenylyl cyclase and phospholipases (see Fig. 3-4, step 5), or cleavage of its myristoyl or palmitoyl group can release the α subunit from the membrane. Similarly, the βγ subunit can activate ion channels or other effectors. The α subunit is itself an enzyme that catalyzes the hydrolysis of GTP to GDP and inorganic phosphate (Pi). The result is an inactive α-GDP complex that dissociates from its downstream effector and reassociates with a βγ subunit (see Fig. 3-4, step 6); this reassociation terminates signaling and brings the system back to resting state (see Fig. 3-4, step 1). The βγ subunit stabilizes α-GDP and thereby substantially slows the rate of GDP-GTP exchange (see Fig. 3-4, step 2) and dampens signal transmission in the resting state.

The RGS (for “regulation of G-protein signaling”) family of proteins appears to enhance the intrinsic GTPase activity of some but not all α subunits. Investigators have identified at least 19 mammalian RGS proteins and shown that they interact with specific α subunits. RGS proteins promote GTP hydrolysis and thus the termination of signaling.

Activated α subunits couple to a variety of downstream effectors, including enzymes and ion channels Activated α subunits can couple to a variety of enzymes. A major enzyme that acts as an effector downstream of activated α subunits is adenylyl cyclase (Fig. 3-5A), which catalyzes the conversion of ATP to cAMP. This enzyme can be either activated or inhibited by G-protein signaling, depending on whether it associates with the GTP-bound form of Gαs (stimulatory) or Gαi (inhibitory). Thus, different ligands—acting through different combinations of GPCRs and G proteins—can have opposing effects on the same intracellular signaling pathway.  N3-4 G proteins can also activate enzymes that break down cyclic nucleotides. For example, the G protein called transducin contains an αt subunit that activates the cGMP phosphodiesterase, which in turn catalyzes the breakdown of cGMP to GMP (see Fig. 3-5B). This pathway plays a key role in phototransduction in the retina (see p. 368). G proteins can also couple to phospholipases. These enzymes catabolize phospholipids, as discussed in detail below in the section on G-protein second messengers.

54

SECTION II  •  Physiology of Cells and Molecules

N

Receptor (R) consists of seven membranespanning segments.

C 1 In the resting state, the receptor associates with the inactive G protein heterotrimer.

2 Upon ligand binding, the receptor-G protein complex undergoes a conformational change that promotes the exchange of GDP for GTP.

Extracellular space E1

E1 E1

γ

β

R

α

E2

G protein

γ

β

R

α

E2

Cytosol

3 G protein dissociates from the receptor.

4

α-GTP and βγ

subunits dissociate.

E1

R R

γ

β

α

E2

γ

R R

E1

α

β

E2

6

5 Both α-GTP and βγ can now interact with their appropriate effectors (E1, E2).

α-catalyzed hydrolysis of GTP to GDP inactivates α and promotes reassembly of the trimer.

E1

E1

R R

α

γ

β

E2

R R

γ

α

β

Pi

Figure 3-4  Enzymatic cycle of heterotrimeric G proteins.

RGS

E2

Members of the RGS family of G-protein regulators stimulate GTP hydrolysis with some but not all α subunits.

CHAPTER 3  •  Signal Transduction

A G PROTEINS ACTING VIA ADENYLYL CYCLASE Extracellular space

Adenylyl cyclase

γ

αs

β

αs

αi

AC

G protein complex (stimulatory)

cAMP

β

γ

G protein complex (inhibitory) NH2

Cyclic AMP activates protein kinase A.

Cytosol B

PKA

Adenine N

N

N

N CH2 O

G PROTEIN ACTING VIA A PHOSPHODIESTERASE Light

H

O O

Extracellular space

P

H

H

H

OH

O –

Phosphodiesterase

O

Cyclic AMP γ

Cytosol

αt

αt

β

G protein complex (transducin)

PDE

cGMP

GMP O

The breakdown of cGMP leads to the closure of cGMP-dependent channels. O O

–

H2N

P O

N

CH2 O H

H

H

H

GMP

Phospholipase C

β

N

OH OH

G PROTEIN ACTING VIA A PHOSPHOLIPASE

γ

O

–

Extracellular space C

N

N

cGMP

Guanine

C

αq

αq

PIP2

PLC

DAG

PKC

DAG activates the enzyme protein kinase C.

PKC

Ca2+

G protein complex

IP3

IP3 signals the release of Ca2+ from the ER. ER Figure 3-5  Downstream effects of activated G-protein α subunits. A, When a ligand binds to a receptor

coupled to αs, adenylyl cyclase (AC) is activated, whereas when a ligand binds to a receptor coupled to αi, the enzyme is inhibited. The activated enzyme converts ATP to cAMP, which then can activate PKA. B, In phototransduction, a photon interacts with the receptor and activates the G protein transducin. The αt activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP; this lowers the intracellular concentrations of cGMP and therefore closes the cGMP-activated channels. C, In this example, the ligand binds to a receptor that is coupled to αq, which activates PLC. This enzyme converts PIP2 to IP3 and DAG. The IP3 leads to the release of Ca2+ from intracellular stores, whereas the DAG activates PKC.

55

56

SECTION II  •  Physiology of Cells and Molecules

This superfamily of phospholipases can be grouped into phospholipases A2, C, or D on the basis of the site at which the enzyme cleaves the phospholipid. G proteins that include the αq subunit activate phospholipase C, which breaks phosphatidylinositol 4,5-bisphosphate into two intracellular messengers, membrane-associated diacylglycerol and cytosolic IP3 (see Fig. 3-5C). Diacylglycerol stimulates protein kinase C, whereas IP3 binds to a receptor on the endoplasmic reticulum (ER) membrane and triggers the release of Ca2+ from intracellular stores. Some G proteins interact with ion channels. Agonists that bind to the β adrenergic receptor activate the L-type Ca2+ channel (see pp. 190–193) in the heart and skeletal muscle. The α subunit of the G protein Gs binds to and directly stimulates L-type Ca2+ channels and also indirectly stimulates this channel via a signal-transduction cascade that involves cAMP-dependent phosphorylation of the channel.

βγ subunits can activate downstream effectors Following activation and disassociation of the heterotrimeric G protein, βγ subunits can also interact with downstream effectors. The neurotransmitter ACh released from the vagus nerve reduces the rate and strength of heart contraction. This action in the atria of the heart is mediated by muscarinic M2 AChRs, members of the GPCR family (see p. 341). These receptors can be activated by muscarine, an alkaloid found in certain poisonous mushrooms. Muscarinic AChRs are very different from the nicotinic AChRs discussed above, which are ligand-gated ion channels. Binding of ACh to the muscarinic M2 receptor in the atria activates a heterotrimeric G protein, which results in the generation of both activated Gαi as well as a free βγ subunit complex. The βγ complex then interacts with a particular class of K+ channels, increasing their permeability. This increase in K+ permeability keeps the membrane potential relatively negative and thus renders the cell more resistant to excitation. The βγ subunit complex also modulates the activity of adenylyl cyclase and phospholipase C and stimulates phospholipase A2. Such effects of βγ can be independent of, synergize with, or antagonize the action of the α subunit. For example, studies using various isoforms of adenylyl cyclase have demonstrated that purified βγ stimulates some isoforms, inhibits others, and has no effect on still others. Different combinations of βγ isoforms may have different activities. For example, β1γ1 is one tenth as efficient at stimulating type II adenylyl cyclase as is β1γ2. Some βγ complexes can bind to a special protein kinase called the β adrenergic receptor kinase (βARK). As a result of this interaction, βARK translocates to the plasma membrane, where it phosphorylates the ligand-receptor complex (but not the unbound receptor). This phosphorylation results in the recruitment of β-arrestin to the GPCR, which in turn mediates disassociation of the receptor-ligand complex and thus attenuates the activity of the same β adrenergic receptors that gave rise to the βγ complex in the first place. This action is an example of receptor desensitization. These phosphorylated receptors eventually undergo endocytosis, which transiently reduces the number of receptors that are available on the cell surface. This endocytosis is an important step in resensitization of the receptor system.

Small GTP-binding proteins are involved in a vast number of cellular processes A distinct group of proteins that are structurally related to the α subunit of the heterotrimeric G proteins are the small GTP-binding proteins. More than 100 of these have been identified to date, and they have been divided into five groups: Ras, Rho, Rab, Arf, and Ran families. These 21-kDa proteins can be membrane associated (e.g., Ras) or may translocate between the membrane and the cytosol (e.g., Rho). The three isoforms of Ras (NRas, HRas, and KRas) relay signals from the plasma membrane to the nucleus via an elaborate kinase cascade (see pp. 89–90), thereby regulating gene transcription. In some tumors, mutation of the genes encoding Ras proteins results in constitutively active Ras. These mutated genes are called oncogenes because the altered Ras gene product promotes the malignant transformation of a cell and can contribute to the development of cancer (oncogenesis). In contrast, Rho family members are primarily involved in rearrangement of the actin cytoskeleton. Rab and Arf regulate vesicle trafficking, whereas Ran regulates nucleocytoplasmic transport. Similarly to the α subunit of heterotrimeric G proteins, the small GTP-binding proteins switch between an inactive GDP-bound form and an active GTP-bound form. Two classes of regulatory proteins modulate the activity of these small GTP-binding proteins. The first of these includes the GTPase-activating proteins (GAPs) and neurofibromin (a product of the neurofibromatosis type 1 gene). GAPs increase the rate at which small GTP-binding proteins hydrolyze bound GTP and thus result in more rapid inactivation. Counteracting the activity of GAPs are guanine nucleotide exchange factors (GEFs) such as “son of sevenless” or SOS (see p. 69), which promote the conversion of inactive Ras-GDP to active Ras-GTP. Interestingly, cAMP directly activates several GEFs, such as Epac (exchange protein activated by cAMP); this demonstrates crosstalk between a classical heterotrimeric G-protein signaling pathway and the small Ras-like G proteins.

G-PROTEIN SECOND MESSENGERS: CYCLIC NUCLEOTIDES cAMP usually exerts its effect by increasing the activity of protein kinase A Activation of Gs-coupled receptors results in the stimulation of adenylyl cyclase, which can cause [cAMP]i to rise 5-fold in ~5 seconds (see Fig. 3-5A). This sudden rise is counteracted by cAMP breakdown to AMP by cAMP phosphodiesterase. The downstream effects of this increase in [cAMP]i depend on the cellular microdomains in which [cAMP]i rises as well as the specialized functions that the responding cell carries out in the organism. For example, in the adrenal cortex, ACTH stimulation of cAMP production results in the secretion of aldosterone and cortisol (see p. 1023); in the kidney, a vasopressin-induced rise in cAMP levels facilitates water reabsorption (see p. 818). Excess cAMP is also responsible for certain pathological conditions, such as cholera (see Box 3-1). Another pathological process associated with

CHAPTER 3  •  Signal Transduction

excess cAMP is McCune-Albright syndrome, characterized by a triad of (1) variable hyperfunction of multiple endocrine glands, including precocious puberty in girls; (2) bone lesions; and (3) pigmented skin lesions (café au lait spots). This disorder is caused by a somatic mutation during development that constitutively activates the G-protein αs subunit in a mosaic pattern. cAMP exerts many of its effects through cAMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate of ATP to specific serine or threonine residues on substrate proteins. PKA phosphorylation sites are present in a multitude of intracellular proteins, including ion channels, receptors, metabolic enzymes, and signaling pathway proteins. Phosphorylation of these sites can influence either the localization or the activity of the substrate. For example, phosphorylation of the β2 adrenergic receptor by PKA causes receptor desensitization in neurons, whereas phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) increases its Cl− channel activity. To enhance regulation of phosphorylation events, the cell tightly controls the activity of PKA so that the enzyme can respond to subtle—and local—variations in cAMP levels. One important control mechanism is the use of regulatory subunits that constitutively inhibit PKA. In the absence of cAMP, two catalytic subunits of PKA associate with two of these regulatory subunits; the result is a heterotetrameric protein complex that has a low level of catalytic activity (Fig. 3-6). Binding of cAMP to the regulatory subunits induces a conformational change that diminishes their affinity for the catalytic subunits, and the subsequent dissociation of the complex results in activation of kinase activity. Not only can PKA activation have the short-term effects noted above, but the free catalytic subunit of PKA can also enter the nucleus, where substrate phosphorylation can activate the transcription of specific PKA-dependent genes (see p. 89). Although most cells use the same catalytic subunit, different regulatory subunits are found in different cell types.

cAMP-dependent kinase (PKA) is composed of two regulatory (R) and 2 catalytic (C) subunits. Binding of cAMP to the regulatory subunits induces a conformational change that reduces their affinity for the catalytic subunits. cAMP cAMP

R

C

C cAMP

cAMP

C

R

R

R cAMP

cAMP cAMP

PKA

C

cAMP

The complex dissociates and the catalytic subunits are free to catalyze the phosphorylation of protein substrates. Figure 3-6  Activation of PKA by cAMP.

57

Another mechanism that contributes to regulation of PKA is the targeting of the enzyme to specific subcellular locations. Such targeting promotes the preferential phosphorylation of substrates that are confined to precise locations within the cell. PKA targeting is achieved by the association of a PKA regulatory subunit with an A kinase anchoring protein (AKAP), which in turn binds to cytoskeletal elements or to components of cellular subcompartments. More than 35 AKAPs are known. The specificity of PKA targeting is highlighted by the observation that, in neurons, PKA is localized to postsynaptic densities through its association with AKAP79. This anchoring protein also targets calcineurin—a protein phosphatase—to the same site. This targeting of both PKA and calcineurin to the same postsynaptic site makes it possible for the cell to tightly regulate the phosphorylation state of important neuronal substrates. The cAMP generated by adenylyl cyclase can interact with effectors other than PKA. For example, olfactory receptors (see pp. 358–359) activate a member of the Gs family called Golf. The subsequent rise in [cAMP]i activates a cyclic nucleotide–gated (CNG) ion channel (see Table 6-2, family No. 4). Na+ influx through this channel leads to membrane depolarization and the initiation of a nerve impulse. For his work in elucidating the role played by cAMP as a second messenger in regulating glycogen metabolism (see Fig. 58-9), Earl Sutherland received the 1971 Nobel Prize in Physiology or Medicine.  N3-6  In 1992, Edmond Fischer and Edwin Krebs shared the prize for their part in demonstrating the role of protein phosphorylation in the signaltransduction process.  N3-7 This coordinated set of phosphorylation and dephosphorylation reactions has several physiological advantages. First, it allows a single molecule (e.g., cAMP) to regulate a range of enzymatic reactions. Second, it affords a large amplification to a small signal. The concentration of epinephrine needed to stimulate glycogenolysis in muscle is ~10−10 M. This subnanomolar level of hormone can raise [cAMP]i to ~10−6 M. Thus, the catalytic cascades amplify the hormone signal 10,000-fold, which results in the liberation of enough glucose to raise blood glucose levels from ~5 to ~8 mM. Although the effects of cAMP on the synthesis and degradation of glycogen are confined to muscle and liver, a wide variety of cells use cAMP-mediated acti­vation cascades in the response to a wide variety of hormones.

Protein phosphatases reverse the action of kinases As discussed above, one way that the cell can terminate a cAMP signal is to use a phosphodiesterase to degrade cAMP. In this way, the subsequent steps along the signaling pathway can also be terminated. However, because the downstream effects of cAMP often involve phosphorylation of effector proteins at serine and threonine residues by kinases such as PKA, another powerful way to terminate the action of cAMP is to dephosphorylate these effector proteins. Such dephosphorylation events are mediated by enzymes called serine/ threonine phosphoprotein phosphatases. Four groups of serine/threonine phosphoprotein phosphatases (PPs) are known: 1, 2a, 2b, and 2c. These enzymes themselves are regulated by phosphorylation at their serine, threonine, and tyrosine residues. The balance between kinase

CHAPTER 3  •  Signal Transduction

N3-6  Earl W. Sutherland, Jr. For more information about Earl W. Sutherland, Jr., and the work that led to his Nobel Prize, visit http://www.nobel.se/ medicine/laureates/1971/index.html (accessed October 2014).

57.e1

N3-7  Edmond H. Fischer and Edwin S. Krebs For more information about Edmond H. Fischer and Edwin S. Krebs and the work that led to their Nobel Prize, visit http:// www.nobel.se/medicine/laureates/1992/index.html (accessed October 2014).

58

SECTION II  •  Physiology of Cells and Molecules

phosphorylated tyrosine groups and thus acts to recruit the phosphatase to its target substrate. Many of the PTPs are themselves regulated by phosphorylation.

PKA (active) C cAMP

R

cAMP

cGMP exerts its effect by stimulating a nonselective cation channel in the retina

cAMP

cGMP is another cyclic nucleotide that is involved in G-protein signaling events. In the outer segments of rods and cones in the visual system, the G protein does not couple to an enzyme that generates cGMP but, as noted above, couples to an enzyme that breaks it down. As discussed beginning on page 367, light activates a GPCR called rhodopsin, which activates the G protein transducin (see p. 368), which in turn activates the cGMP phosphodiesterase (see p. 368) that lowers [cGMP]i. The fall in [cGMP]i closes cGMP-gated nonselective cation channels that are members of the same family of CNG ion channels that cAMP activates in olfactory signaling (see pp. 358–359).

R

cAMP

C

I-1

I-1

P

PP1

I-1

P

Inactive PP1

Phosphoprotein phosphatase (active)

Figure 3-7  Inactivation of PP1 by PKA.

and phosphatase activity plays a major role in the control of signaling events. PP1 dephosphorylates many proteins phosphorylated by PKA, including those phosphorylated in response to epinephrine (see Fig. 58-9). Another protein, phosphoprotein phosphatase inhibitor 1 (I-1), can bind to and inhibit PP1. Interestingly, PKA phosphorylates and induces I-1 binding to PP1 (Fig. 3-7), thereby inhibiting PP1 and preserving the phosphate groups added by PKA in the first place. PP2a, which is less specific than PP1, appears to be the main phosphatase responsible for reversing the action of other protein serine/threonine kinases. The Ca2+-dependent PP2b, also known as calcineurin, is prevalent in the brain, muscle, and immune cells and is also the pharmacological target of the immunosuppressive reagents FK-506 (tacrolimus) and cyclosporine. The substrates for PP2c include the DNA checkpoint regulators Chk1 and Chk2, which normally sense DNA damage in the setting of organ injury and temporarily stop cell proliferation. Dephosphorylation of these kinases by PP2c inactivates them and allows the cell to re-enter the cell cycle during the repair process. In addition to serine/threonine kinases such as PKA, a second group of kinases involved in regulating signaling pathways (discussed beginning on pp. 68–70) are tyrosine kinases that phosphorylate their substrate proteins on tyrosine residues. The enzymes that remove phosphates from these tyrosine residues—phosphotyrosine phosphatases (PTPs)—are much more variable than the serine and threonine phosphatases. The first PTP to be characterized was the cytosolic enzyme PTP1B from human placenta. PTP1B has a high degree of homology with CD45, a membrane protein that is both a receptor and a tyrosine phosphatase. The large family of PTPs can be divided into two classes: membrane-spanning receptor-like proteins such as CD45 and cytosolic tyrosine phosphatases such as PTP1B. A number of intracellular PTPs contain Src homology 2 (SH2) domains, a peptide sequence or motif that interacts with

G-PROTEIN SECOND MESSENGERS: PRODUCTS OF PHOSPHOINOSITIDE BREAKDOWN Many messengers bind to receptors that activate phosphoinositide breakdown Although the phosphatidylinositols (PIs) are minor constituents of cell membranes, they are largely distributed in the internal leaflet of the membrane and play an important role in signal transduction. The inositol sugar moiety of PI molecules (see Fig. 2-2A) can be phosphorylated to yield two major phosphoinositides involved in signal transduction: phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3 or PIP3; see p. 69).  N3-8 Certain membrane-associated receptors act through G proteins (e.g., Gq) that stimulate phospholipase C (PLC) to cleave PIP2 into inositol 1,4,5-trisphosphate (or P3) and diacylglycerol (DAG), as shown in Figure 3-8A. PLCs are classified into three families (β, γ, δ) that differ in their catalytic properties, cell-type–specific expression, and modes of activation. PLCβ is typically activated downstream of certain G proteins (e.g., Gq), whereas PLCγ contains an SH2 domain and is activated downstream of certain tyrosine kinases. Stimulation of PLCβ results in a rapid increase in cytosolic IP3 levels as well as an early peak in DAG levels (see Fig. 3-8B). Both products are second messengers. The watersoluble IP3 travels through the cytosol to stimulate Ca2+ release from intracellular stores (see next section). DAG remains in the plane of the membrane to activate protein kinase C, which migrates from the cytosol and binds to DAG in the membrane (see pp. 60–61). Phosphatidylcholines (PCs), which—unlike PI—are an abundant phospholipid in the cell membrane, are also a source of DAG. The cell can produce DAG from PC by either of two mechanisms (see Fig. 3-8C). First, PLC can directly convert PC to phosphocholine and DAG. Second, phospholipase D (PLD), by cleaving the phosphoester bond on the other side of the phosphate, can convert PC to choline and phosphatidic acid (PA; also phospho-DAG). This PA

CHAPTER 3  •  Signal Transduction

N3-8  Acyl Groups Contributed by Emile Boulpaep and Walter Boron As noted in the text, phosphatidylinositols (PIs) (see p. 10) and phosphatidylcholines (PCs) (see p. 10) can each contain a variety of acyl groups. Therefore, the phosphoinositides derived from them can also contain a variety of acyl groups. A phosphoinositide is a PI derivative containing one, two, or three additional phosphate groups. Because there are three possible attachment sites (at sites 3, 4, or 5), there are a total of seven combinations possible.

Seven Combinations • Three monophosphates: • PI3P • PI4P • PI5P • Three bisphosphates called PIP2 • PI(3,4)P2 • PI(4,5)P2 • PI(3,5)P2 • One trisphosphate called PIP3 • PI(3,4,5)P3

58.e1

CHAPTER 3  •  Signal Transduction

PIP2

DAG 13p6

A

PRODUCTION OF IP3 AND DAG

Binding of a hormone to a cell surface G protein–coupled receptor activates phospholipase Cβ.

O

PLC cleaves the polar head group here.

C

P

O

O

O

Cytosol

O CH2

O O

–

–

O

OH HO

3 O

O

P

5

O OH

1

4

2

–

5 4 O

3

IP3

O

–

OH HO

O P

O

P O

6

O

OH

2

Phospholipase Cβ hydrolyzes PIP2 into IP3 and DAG.

Plasma membrane

O

OH O

P O

6

O

O C

CH2 CH

O

1

P

O

O

–

O

O

DAG

Plasma membrane Cytosol

O

CH2

O –

C O

CH2 CH

O

Extracellular space

O

C

γ

α

β

α

PKC

PLCβ PKC

PIP2

Receptor–G protein complex

Active

IP3

C IP3 interacts with its receptor (ITPR) in the membrane of the ER, which allows the release + of Ca2 into the cytosol.

ER H+

Ca

BREAKDOWN OF PHOSPHATIDYLCHOLINE BY PLC AND PLD R1

R2

The SERCA Ca pump transports the + Ca2 back into the ER.

2+

CH2 C

B

TIME COURSE OF IP3 AND DAG LEVELS

Response

DAG

IP3 The early DAG peak is caused by DAG released from PIP2 by PLCβ.

H2C

PLC

C

O

O

CH

CH2

P

O

O

PLD

CH2

Choline

CH2 H3C

N

+

CH3

CH3

Seconds

Minutes

Hours

Figure 3-8  Second messengers in the DAG/IP3 pathway. ER, endoplasmic reticulum; SERCA, sarcoplasmic and endoplasmic reticulum Ca-ATPase.

O

O –

O

The slow DAG wave is caused by DAG released by PLCβ and PLD from phosphatidylcholine (PC).

CH2 O

59

60

SECTION II  •  Physiology of Cells and Molecules

can then be converted to DAG via PA-phosphohydrolase. Production of DAG from PC, either directly (via PLC) or indirectly (via PLD), produces the slow wave of increasing cytosolic DAG shown in Figure 3-8B. Thus, in some systems, the formation of DAG is biphasic and consists of an early peak that is transient and parallels the formation of IP3, followed by a late phase that is slow in onset but sustained for several minutes.

IP3 liberates Ca2+ from intracellular stores As discussed on page 126, three major transport mechanisms keep free intracellular Ca2+ ([Ca2+]i) below ~100 nM. Increases in [Ca2+]i from this extremely low baseline allow Ca2+ to function as an important second messenger. IP3 generated by the metabolism of membrane phospholipids travels through the cytosol and binds to the IP3 receptor, a ligandgated Ca2+ channel located in the membrane of the endoplasmic reticulum (see Fig. 3-8A). The result is a release of Ca2+ from intracellular stores and a rise in [Ca2+]i. Indeed, it was within this system that Ca2+ was first identified as a messenger mediating the stimulus-response coupling of endocrine cells. The IP3 receptor (ITPR) is a tetramer composed of subunits of ~260 kDa. At least three genes encode the subunits of the receptor. These genes are subject to alternative splicing, which further increases the potential for receptor diversity. The receptor is a substrate for phosphorylation by protein kinases A and C as well as calcium-calmodulin (Ca2+-CaM)–dependent protein kinases.  N3-9 Interaction of IP3 with its receptor results in passive efflux of Ca2+ from the ER and thus a rapid rise in the free cytosolic Ca2+ concentration. The IP3-induced changes in [Ca2+]i exhibit complex temporal and spatial patterns. The rise in [Ca2+]i, which can be brief or persistent, can oscillate repetitively or spread across groups of cells coupled by gap junctions. In at least some systems, the frequency of [Ca2+]i oscillations seems to be physiologically important. For example, in isolated pancreatic acinar cells, graded increases in the concentration of ACh produce graded increases in the frequency—but not the magnitude—of repetitive [Ca2+]i spikes. The mechanisms responsible for [Ca2+]i oscillations and waves are complex. It appears that both propagation and oscillation depend on positive-feedback mechanisms, in which high [Ca2+]i facilitates Ca2+ release, as well as on negative-feedback mechanisms, in which high [Ca2+]i inhibits further Ca2+ release. Structurally related to ITPRs are the Ca2+-release channels known as ryanodine receptors (RYRs; see p. 230). Because cytosolic Ca2+ activates RYRs, these channels play an important role in elevating [Ca2+]i in certain cells by a process known as calcium-induced Ca2+ release (CICR; see pp. 242–243)—an example of the positive feedback noted above. For example, RYRs are responsible for releasing Ca2+ from the sarcoplasmic reticulum of muscle and thereby switching on muscle contraction (see pp. 229–230). Moreover, cyclic ADP ribose (cADPR), the product of ADP-ribosylcyclases, increases the sensitivity of RYR to cytosolic Ca2+, thereby augmenting CICR. [Ca2+]i can increase as the result not only of Ca2+ release from intracellular stores, but also of enhanced influx through Ca2+ channels in the plasma membrane. By whatever mecha-

nism, increased [Ca2+]i exerts its effects by binding to cellular proteins and changing their activity, as discussed in the next two sections. Some Ca2+-dependent signaling events are so sensitive to Ca2+ that a [Ca2+]i increase of as little as 100 nM can trigger a vast array of cellular responses. These responses include secretion of digestive enzymes by pancreatic acinar cells, release of insulin by β cells, contraction of vascular smooth muscle, conversion of glycogen to glucose in the liver, release of histamine by mast cells, aggregation of platelets, and DNA synthesis and cell division in fibroblasts. The same mechanisms that normally keep [Ca2+]i at extremely low levels (see p. 126) are also responsible for reversing the increases in [Ca2+]i that occur during signaling events. Increases in [Ca2+]i activate an ATP-fueled Ca pump (SERCA; see p. 118) that begins pumping Ca2+ back into the ER. In addition, a Ca pump (see p. 118) and Na-Ca exchanger (see pp. 123–124) at the plasma membrane extrude excess Ca2+ from the cell. These processes are much slower than Ca2+ release, so [Ca2+]i remains high until IP3 is dephosphorylated, terminating Ca2+ release via ITPR and thereby allowing the transporters to restore [Ca2+]i to basal levels.

Calcium activates calmodulin-dependent protein kinases How does an increase in [Ca2+]i lead to downstream responses in the signal-transduction cascade? The effects of changes in [Ca2+]i are mediated by Ca2+-binding proteins, the most important of which is calmodulin (CaM). CaM is a highaffinity cytoplasmic Ca2+-binding protein of 148 amino acids. Each molecule of CaM cooperatively binds four calcium ions. Ca2+ binding induces a major conformational change in CaM that allows it to bind to other proteins (Fig. 3-9). Although CaM does not have intrinsic enzymatic activity, it forms a complex with a number of enzymes and thereby confers a Ca2+ dependence on their activity. For example, binding of the Ca2+-CaM complex activates the enzyme that degrades cAMP, cAMP phosphodiesterase. Many of the effects of CaM occur as the Ca2+-CaM complex binds to and activates a family of Ca2+-CaM–dependent kinases known as CaM kinases (CaMKs). These kinases phosphorylate specific serine and threonine residues of a variety of proteins. An important CaMK in smooth-muscle cells is myosin light-chain kinase (MLCK) (see p. 247). Another CaMK is glycogen phosphorylase kinase (PK), which plays a role in glycogen degradation (see p. 1182). MLCK, PK, and some other CaMKs have a rather narrow substrate specificity. The ubiquitous CaM kinase II (CaMKII), on the other hand, has a broad substrate specificity. Especially high levels of this multifunctional enzyme are present at the synaptic terminals of neurons. One of the actions of CaMKII is to phosphorylate and thereby activate the rate-limiting enzyme (tyrosine hydroxylase; see Fig. 13-8) in the synthesis of catecholamine neurotransmitters. CaMKII can also phosphorylate itself, which allows it to remain active in the absence of Ca2+.

DAGs and Ca2+ activate protein kinase C As noted above, hydrolysis of PIP2 by PLC yields not only the IP3 that leads to Ca2+ release from internal stores but also

CHAPTER 3  •  Signal Transduction

N3-9  IP3 Receptor Diversity Contributed by Laurie Roman As noted in the text, the IP3 receptor (IPTR) is a tetramer composed of subunits of ~260 kDa, and at least four different genes encode the receptor subunits. These genes are subject to alternative splicing, further increasing the potential for receptor diversity. IP3 receptors bind their ligand with high affinity (the dissociation constant KD = 2–10 nM) or low affinity (KD = 40 nM). However, the extent to which these different affinities correlate with particular forms of the receptor has not been established.

60.e1

CHAPTER 3  •  Signal Transduction

61

Inactive protein

2+

Ca

Active protein

Calmodulin

Ca2+-Calmodulin

Ca2+-Calmodulin−dependent protein kinase

Figure 3-9  CaM. After four intracellular Ca2+ ions bind to CaM, the Ca2+-CaM complex can bind to and activate another protein. In this example, the activated protein is a Ca2+-CaM–dependent kinase.

DAG (see Fig. 3-8A). The most important function of DAG is to activate protein kinase C (PKC), an intracellular serine/ threonine kinase. In mammals, the PKC family comprises at least 10 members that differ in their tissue and cellular localization. This family is further subdivided into three groups that all require membrane-associated phosphatidylserine but have different requirements for Ca2+ and DAG. The classical PKC family members PKCα, PKCβ, and PKCγ require both DAG and Ca2+ for activation, whereas the novel PKCs (such as PKCδ, PKCε, and PKCη) require DAG but are independent of Ca2+, and the atypical PKCs (PKCζ and PKCλ) appear to be independent of both DAG and Ca2+. As a consequence, the signals generated by the PKC pathway depend on the isoforms of the enzyme that a cell expresses as well as on the levels of Ca2+ and DAG at specific locations at the cell membrane. Moreover, proteins such as receptor for activated C-kinase (RACK) and receptor for inactivated C-kinase (RICK) can target specific PKC isoforms to specific cellular compartments. In its basal state, PKCα is an inactive, soluble cytosolic protein. When a GPCR activates PLC, both DAG (generated in the inner leaflet of the plasma membrane) and Ca2+ (released in response to IP3) bind to the PKC regulatory domain; this results in translocation of PKCα to the membrane and activation of the PKC kinase domain. Even though the initial Ca2+ signal is transient, PKCα activation can be sustained, resulting in activation of physiological responses, such as proliferation and differentiation. Elevated levels of active PKCα are maintained by a slow wave of elevated DAG (see Fig. 3-8B), which is due to the hydrolysis of PC by PLC and PLD. Physiological stimulation of the classical and novel PKCs by DAG can be mimicked by the exogenous application of a class of tumor promoters called phorbol esters. These plant products bind to the regulatory domain of PKCs and thus specifically activate them even in the absence of DAG. Among the major substrates of PKC are the myristoylated, alanine-rich C-kinase substrate proteins, known as MARCKS proteins. These acidic proteins contain consensus sites for PKC phosphorylation as well as CaM- and actin-binding

sites. MARCKS proteins cross-link actin filaments and thus appear to play a role in translating extracellular signals into actin plasticity and changes in cell shape. Unphosphorylated MARCKS proteins are associated with the plasma membrane, and they cross-link actin. Phosphorylation of the MARCKS proteins causes them to translocate into the cytosol, where they are no longer able to cross-link actin. Thus, mitogenic growth factors that activate PKC may produce morphological changes and anchorage-independent cell proliferation in part by modifying the activity of MARCKS proteins. PKC can also directly or indirectly modulate transcription factors and thereby enhance the transcription of specific genes (see p. 86). Such genomic actions of PKC explain why phorbol esters are tumor promoters.

G-PROTEIN SECOND MESSENGERS: ARACHIDONIC ACID METABOLITES In addition to DAG, other hydrolysis products of membrane phospholipids can act as signaling molecules.  N3-10  The best characterized of these hydrolysis products is arachidonic acid (AA), which is attached by an ester bond to the second carbon of the glycerol backbone of membrane phospholipids (Fig. 3-10). Phospholipase A2 initiates the cellular actions of AA by releasing this fatty acid from glycerol-based phospholipids.  N3-11  A series of enzymes subsequently convert AA into a family of biologically active metabolites that are collectively called eicosanoids (from the Greek eikosi [20]) because, like AA, they all have 20 carbon atoms. Three major pathways can convert AA into these eicosanoids (Fig. 3-11). In the first pathway, cyclooxygenase (COX) enzymes produce thromboxanes (TXs), prostaglandins (PGs), and prostacyclins. In the second pathway, 5-lipoxygenase enzymes produce leukotrienes (LTs) and some hydroxyeicosatetraenoic acid (HETE) compounds. In the third pathway, the epoxygenase enzymes, which are members of the cytochrome P-450 class, produce other HETE compounds as well as cis-epoxyeicosatrienoic acid (EET) compounds. These

CHAPTER 3  •  Signal Transduction

N3-10  Platelet-Activating Factor

61.e1

N3-11  Phospholipase A2

Contributed by Ed Moczydlowski

Contributed by Laurie Roman

Although it is not a member of the arachidonic acid (AA) family, platelet-activating factor (PAF) is an important lipid signaling molecule. PAF is an ether lipid that the cell synthesizes either de novo or by remodeling of a membrane-bound precursor. PAF occurs in a wide variety of organisms and mediates many biological activities. In mammals, PAF is a potent inducer of platelet aggregation and stimulates the chemotaxis and degranulation of neutrophils, thereby facilitating the release of LTB4 and 5-HETE. PAF is involved in several aspects of allergic reactions; for example, it stimulates histamine release and enhances the secretion of immunoglobulin E, immunoglobulin A, and tumor necrosis factor. Endothelial cells are also an important target of PAF; PAF causes a negative shift of Vm in these cells by activating Ca2+-dependent K+ channels. PAF also enhances vascular permeability and the adhesion of neutrophils and platelets to endothelial cells. PAF exerts its effects by binding to a specific receptor on the plasma membrane. A major consequence of PAF binding to its GPCR is formation of IP3 and stimulation of a group of MAPKs. PAF acetylhydrolase terminates the action of this signaling lipid.

Phospholipase A2 (PLA2) catalyzes the hydrolytic cleavage of glycerol-based phospholipids (see Fig. 2-2A–C) at the second carbon of the glycerol backbone, yielding AA and a lysophospholipid (see Fig. 3-10). Some of the cytosolic PLA2 enzymes require Ca2+ for activity. In addition, raising [Ca2+]i from the physiological level of ~100 nM to ~300 nM facilitates the association of cytoplasmic PLA2 with cell membranes, where the PLA2 can be activated by specific G proteins.

62

SECTION II  •  Physiology of Cells and Molecules

BOX 3-2  Eicosanoid Nomenclature

Polar head group

T

O O–

O

P O

CH2

H C

O

OH

C

CH2

O

Polar head group O O–

P

O

he nomenclature of the eicosanoids is not as arcane as it might first appear. The numerical subscript 2 (as in PGH2) or 4 (as in LTA4) refers to the number of double bonds in the eicosanoid backbone. For example, AA has four double bonds, as do the leukotrienes. For the cyclooxygenase metabolites, the letter (A to I) immediately preceding the 2 refers to the structure of the 5-carbon ring that is formed about halfway along the 20-carbon chain of the eicosanoid. For the leukotrienes, the letters A and B that immediately precede the 4 refer to differences in the eicosanoid backbone. For the cysteinyl leukotrienes, the letter C refers to the full glutathione conjugate (see Fig. 46-8). Removal of glutamate from LTC4 yields LTD4, and removal of glycine from LTD4 yields LTE4, leaving behind only cysteine. For 5-HPETE and 5-HETE, the fifth carbon atom (counting the carboxyl group as number 1) is derivatized with a hydroperoxy or hydroxy group, respectively.

O

Phospholipase A2 cleaves here.

O

CH2

CH2

O

O

C

C

Phospholipase A2 is the primary enzyme responsible for releasing AA

CH2

O

Lysophospholipid

COOH

The arachidonic acid is always found esterified to the second carbon atom of the glycerol backbone. Phospholipid

COOH

Arachidonic acid Figure 3-10  Release of AA from membrane phospholipids by PLA2. AA

is esterified to membrane phospholipids at the second carbon of the glycerol backbone. PLA2 cleaves the phospholipid at the indicated position and releases AA as well as a lysophospholipid.

three enzymes catalyze the stereospecific insertion of molecular O2 into various positions in AA. The cyclooxygenases, lipoxygenases, and epoxygenases are selectively distributed in different cell types, which further increases the complexity of eicosanoid biology. Eicosanoids have powerful biological activities, including effects on allergic and inflammatory processes, platelet aggregation, vascular smooth muscle, and gastric acid secretion.

The first step in the phospholipase A2 (PLA2) signaltransduction cascade is binding of an extracellular agonist to a membrane receptor (see Fig. 3-11). These receptors include those for serotonin (5-HT2 receptors), glutamate (mGLUR1 receptors), fibroblast growth factor-β, interferon-α (IFN-α), IFN-β, and IFN-γ. Once the receptor is occupied by its agonist, it can activate a G protein that belongs to the Gi/Go family. The mechanism by which this activated G protein stimulates PLA2 is not well understood. It does not appear that a G-protein α subunit is involved. The G-protein βγ dimer may stimulate PLA2 either directly or via mitogenactivated protein kinases (MAPKs) (see p. 69), which phosphorylates PLA2 at a serine residue. The result is rapid hydrolysis of phospholipids that contain AA. In contrast to the direct pathway just mentioned, agonists acting on other receptors may promote AA release indirectly. First, a ligand may bind to a receptor coupled to PLC, which would lead to the release of DAG (see Fig. 3-11). As noted above, DAG lipase can cleave DAG to yield AA and a monoacylglycerol (MAG). Agonists that act via this pathway include dopamine (D2 receptors), adenosine (A1 receptors), norepinephrine (α2 adrenergic receptors), and serotonin (5-HT1 receptors). Second, any agonist that raises [Ca2+]i can promote AA formation because Ca2+ can stimulate some cytosolic forms of PLA2. Third, any signal-transduction pathway that activates MAPK can also enhance AA release because MAPK phosphorylates PLA2.

Cyclooxygenases, lipoxygenases, and epoxygenases mediate the formation of biologically active eicosanoids Once it is released from the membrane, AA can diffuse out of the cell, be reincorporated into membrane phospholipids, or be metabolized (see Fig. 3-11). In the first pathway of AA metabolism (see Fig. 3-11), cyclooxygenases  N3-12  catalyze the stepwise conversion of AA into the intermediates prostaglandin G2 (PGG2) and

CHAPTER 3  •  Signal Transduction

N3-12  Cyclooxygenase Contributed by Laurie Roman Cyclooxygenase catalyzes the stepwise conversion of AA into the intermediates PGG2 and PGH2. Thus, this enzyme is also referred to as prostaglandin H synthetase (PGHS). As noted in Box 3-3, it is the same enzyme that catalyzes both reactions. Cyclooxygenase exists in three isoforms, COX-1 (a transcript of 2.8 kilobases [kb]), COX-2 (a 4.1-kb transcript), and COX-3 (a splice variant of COX-1 that is also known as COX-1b).

62.e1

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CHAPTER 3  •  Signal Transduction

INDIRECT PATHWAYS

DIRECT PATHWAY Extracellular space Phospholipase A2

γ

α

β

Phospholipid

Lysophospholipid

MAG

DAG

Phospholipase Cβ

DAG lipase

PLA2

PLCβ

α

α

Receptor-G protein complex 1

γ

Receptor-G protein complex 2

Reincorporation of AACoA

Cytosol

IP3

Ca2+

ARACHIDONIC ACID Cyclooxygenase (COX)

ASA

ER

Epoxygenase (Cytochrome P450)

COOH 5-Lipoxygenase

Other HETEs EETs

5-HPETE PGG2

COOH OOH

COX

Dehydrase

LTA4

PGH2

Peroxidase

5-HETE LTA4 Hydrolase

LTB4

OH

O

COOH

O Thromboxane synthase

Prostacyclin synthase

Glutathione-S-transferase

TXA2 (active, unstable)

PGI2 (active, unstable)

TXB2 (inactive)

6-keto-PGF1α (weak)

PGD2

PGE2

HO

LTD4

LTE4

LEUKOTRIENES

NH2

PGE2 HO

O

LTC4

LTE4

OH

OH

COOH

PGF2α

PROSTAGLANDINS

PGI2

TXA2 O O

β

COOH

OH

S COOH

COOH O

COOH OH

Figure 3-11  AA signaling pathways. In the direct pathway, an agonist binds to a receptor that activates PLA2, which releases AA from a membrane phospholipid (see Fig. 3-10). In one of three indirect pathways, an agonist binds to a different receptor that activates PLC and thereby leads to the formation of DAG and IP3, as in Figure 3-8; DAG lipase then releases the AA from DAG. In a second indirect pathway, the IP3 releases Ca2+ from internal stores, which leads to the activation of PLA2 (see the direct pathway). In a third indirect pathway (not shown), MAPK stimulates PLA2. Regardless of its source, the AA may follow any of three pathways to form a wide array of eicosanoids. The cyclooxygenase pathway produces thromboxanes (TXA2 and TXB2), prostacyclin (i.e., PGI2), and prostaglandins. The 5-lipoxygenase pathway produces 5-HETE and the leukotrienes. The epoxygenase pathway leads to the production of other HETEs and EETs. AACoA, arachidonic-Acid–coenzyme A; ASA, acetylsalicylic acid.

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SECTION II  •  Physiology of Cells and Molecules

BOX 3-3  Therapeutic Inhibition of Cyclooxygenase Isoforms

C

yclooxygenase is a bifunctional enzyme that first oxidizes AA to PGG2 through its cyclooxygenase activity and then peroxidizes this compound to PGH2. X-ray crystallographic studies of COX-1 reveal that the sites for the two enzymatic activities (i.e., cyclooxygenase and peroxidase) are adjacent but spatially distinct. The cyclooxygenase site is a long hydrophobic channel. Aspirin (acetylsalicylic acid) irreversibly inhibits COX-1 by acetylating a serine residue at the top of this channel. Several of the other NSAIDs interact, via their carboxyl groups, with other amino acids in the same region. COX-1 activation plays an important role in intravascular thrombosis because it leads to TXA2 synthesis by platelets. Inhibition of this process by low-dose aspirin is a mainstay for prevention of coronary thrombosis in patients with atherosclerotic coronary artery disease. However, COX-1 activation is also important for producing the cytoprotective prostanoids PGE2 (a prostaglandin) and PGI2 (a prostacyclin) in the gastric mucosa. It is the loss of these compounds that can lead to the unwanted side effect of gastrointestinal bleeding after long-term aspirin use.  N3-15 Inflammatory stimuli induce COX-2 in a number of cell types, and it is inhibition of COX-2 that provides the antiinflammatory actions of high-dose aspirin (a weak COX-2 inhibitor) and other nonselective cyclooxygenase inhibitors such as ibuprofen. Because the two enzymes are only 60% homologous, pharmaceutical companies have now generated compounds that specifically inhibit COX-2, such as celecoxib. COX-2 inhibitors work well as anti-inflammatory agents and have a reduced likelihood of causing gastrointestinal bleeding because they do not inhibit COX-1–dependent prostacyclin production. COX-2 inhibitors have been reported to increase the risk of thrombotic cardiovascular events when they are taken for long periods.

prostaglandin H2 (PGH2). PGH2 is the precursor of the other prostaglandins, the prostacyclins and the thromboxanes. As noted in Box 3-3, cyclooxygenase exists in two predominant isoforms, cyclooxygenase 1 (COX-1) and COX-2, as well as the COX-1b spice variant of COX-1. In many cells, COX-1 is expressed in a constitutive fashion, whereas COX-2 levels can be induced by specific stimuli. For example, in monocytes stimulated by inflammatory agents such as interleukin-1β (IL-1β), only levels of COX-2 increase. These observations have led to the concept that expression of COX-1 is important for homeostatic prostaglandin functions such as platelet aggregation and regulation of vascular tone, whereas upregulation of COX-2 is primarily important for mediating prostaglandin-dependent inflammatory responses. However, as selective inhibitors of COX-2 have become available, it has become clear that this is an oversimplification. In the second pathway of AA metabolism, 5-lipoxygenase initiates the conversion of AA into biologically active leu­ kotrienes. For example, in myeloid cells, 5-lipoxygenase converts AA to 5-hydroperoxyeicosatetraenoic acid (5-HPETE),  N3-13  which is short-lived and rapidly degraded by a peroxidase to the corresponding alcohol 5-HETE. Alter­natively, a dehydrase can convert 5-HPETE to

an unstable epoxide, leukotriene A4 (LTA4), which can be either further metabolized by LTA4 hydrolase to LTB4 or coupled (“conjugated”) by LTC4 synthase to the tripeptide glutathione (see p. 955). This conjugation—via the cysteine residue of glutathione—yields LTC4. Enzymes sequentially remove portions of the glutathione moiety to produce LTD4 and LTE4. LTC4, LTD4, and LTE4 are the “cysteinyl” leukotrienes; they participate in allergic and inflammatory responses and make up the mixture previously described as the slow-reacting substance of anaphylaxis. The third pathway of AA metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase).  N3-14  Molecular O2 is a substrate in this reaction. The epoxygenase pathway converts AA into two major products, HETEs and EETs. Members of both groups display a diverse array of biological activities. Moreover, the cells of different tissues (e.g., liver, kidney, eye, and pituitary) use different biosynthetic pathways to generate different epoxygenase products.

Prostaglandins, prostacyclins, and thromboxanes (cyclooxygenase products) are vasoactive, regulate platelet action, and modulate ion transport  N3-16 The metabolism of PGH2 to generate selected prostanoid derivatives is cell specific. For example, platelets convert PGH2 to thromboxane A2 (TXA2), a short-lived compound that can aggregate platelets, bring about the platelet release reaction, and constrict small blood vessels. In contrast, endothelial cells convert PGH2 to prostacyclin I2 (PGI2), which inhibits platelet aggregation and dilates blood vessels. Many cell types convert PGH2 to prostaglandins. Acting locally in a paracrine or autocrine fashion, prostaglandins are involved in such processes as platelet aggregation, airway constriction, renin release, and inflammation.  N3-16  Prostaglandin synthesis has also been implicated in the pathophysiological mechanism of cardiovascular disease, cancer, and inflammatory diseases. Nonsteroidal antiinflammatory drugs (NSAIDs) such as aspirin, acetaminophen, ibuprofen, indomethacin, and naproxen directly target cyclooxygenase. NSAID inhibition of cyclooxygenase is a useful tool in the treatment of inflammation and fever and, at least in the case of aspirin, in the prevention of heart disease. The diverse cellular responses to prostanoids are mediated by a family of G protein–coupled prostanoid receptors. This family currently has nine proposed members, including receptors for thromboxane/PGH2 (TP), PGI2 (IP), PGE2 (EP1 to EP4), PGD2 (DP and CRTH2), and PGF2α (FP). These prostanoid receptors signal via Gq, Gi, or Gs, depending on cell type. These in turn regulate intracellular adenylyl cyclase (see p. 53) and phospholipases (see p. 58).

The leukotrienes (5-lipoxygenase products) play a major role in inflammatory responses Many lipoxygenase metabolites of AA have a role in allergic and inflammatory diseases (Table 3-3).  N3-17  LTB4 is produced by inflammatory cells such as neutrophils and macrophages. The cysteinyl leukotrienes including LTC4 and LTE4 are synthesized by mast cells, basophils, and

CHAPTER 3  •  Signal Transduction

N3-13  Names of Arachidonic Acid Metabolites

64.e1

N3-14  Epoxygenase Contributed by Emile Boulpaep and Walter Boron

Contributed by Emile Boulpaep and Walter Boron 5-HPETE = 5-S-hydroperoxy-6-8-trans-11,14-ciseicosatetraenoic acid 5-HETE = 5-hydroxyeicosatetraenoic acid EET = cis-epoxyeicosatrienoic acid

As shown in Figure 3-11, one pathway of arachidonic-acid (AA) metabolism begins with the transformation of AA by epoxygenase (a cytochrome P-450 oxidase) to two major products: HETEs and EETs. Epoxygenase requires molecular oxygen (i.e., it is an oxidase) and has several required cofactors, including cytochrome P-450 reductase, NADPH/NADP+ (reduced/oxidized forms of nicotinamide adenine dinucleotide phosphate), or NADH/NAD+ (reduced/oxidized forms of nicotinamide adenine dinucleotide).

N3-15  Side Effects of Cyclooxygenase Inhibitors Contributed by Emile Boulpaep and Walter Boron Both COX-1 and COX-2 appear to be required for production of PGE2 in the renal glomerulus, a process that is important in maintaining normal glomerular perfusion in the event of decreased renal blood flow. Thus, another risk of cyclooxygenase inhibitors is diminished renal function in patients with heart failure or volume depletion. Similar to the nonselective cyclooxygenase inhibitors, COX-2 inhibitors have been shown to decrease renal perfusion and increase the risk of hemodynamic acute renal failure in susceptible individuals.

REFERENCE Schnermann J, Chou C-L, Ma T, et al: Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci U S A 95:9660–9664, 1998.

N3-16  Actions of Prostanoids Contributed by Laurie Roman The prostanoids may participate in regulation of the Na-K pump, which plays a central role in salt and water transport in the kidney and the maintenance of ion gradients in all cell types. For example, the inhibition of the Na-K pump produced by IL-1 appears to be mediated by the formation of PGE2. Indeed, IL-1 stimulates the formation of PGE2, and application of exogenous PGE2 inhibits Na-K pump activity directly. Moreover, cyclooxygenase blockers prevent the Na-K pump inhibition induced by IL-1. This action on the Na-K pump is not limited to the kidney; AA metabolites also inhibit the pump in the brain. Prostaglandins also are vasoactive and are important in the regulation of renal blood flow.

N3-17  Actions of Leukotrienes Contributed by Laurie Roman LTC4, LTD4, LTE4, and LTF4 are often referred to as the “cysteinyl leukotrienes” or sometimes as the “peptidyl leukotrienes.” As summarized in Figure 3-11, the enzyme glutathione-Stransferase (GST) conjugates LTA4, which is unstable, to the sulfhydryl group of the cysteine in glutathione (glutathione, also abbreviated GSH, is the branched tripeptide Glu-Cys-Gly) to produce LTC4. (See page 955 to learn how the liver uses GSH for conjugation reactions.) The enzyme γ-glutamyl transferase clips off the glutamate residue of LTC4 to produce LTD4 (which is conjugated to -Cys-Gly). A dipeptidase clips the dipeptide bond between Cys and Gly to release the terminal Gly as well as LTE4 (which is conjugated to only the -Cys). Leukotrienes have multiple effects on the vascular endothelium during inflammation. Various regulatory processes may interact at the level of the small blood vessels to increase the margination (i.e., the attachment to the vessel wall) of subgroups of leukocytes, increase the permeability at the postcapillary venule, and evoke diapedesis (i.e., the migration of the cell through the endothelium) of the adherent leukocytes to create a focus of interstitial inflammation. Each of these steps can be affected by leukotrienes as well as other agents. The infiltration of leukocytes begins when the cells adhere to the endothelium of the postcapillary venule. Mediators that can increase the adhesiveness of leukocytes include LTB4 and several of the cysteinyl leukotrienes. Increased vascular permeability,

influenced by the pulling apart of adjacent endothelial cells, can occur in response to LTC4, LTD4, and LTE4. After adherent leukocytes accumulate—and the size of the interendothelial cell pores increases—a stimulus for diapedesis produces an influx of leukocytes into the interstitial space. Once in the interstitial space, the leukocytes come under the influence of LTB4, a potent chemotactic factor (i.e., chemical attractant) for neutrophils (a type of white blood cell that phagocytoses invading organisms) and less so for eosinophils (another type of white blood cell). LTB4 is also chemokinetic (i.e., speeds up chemotaxis) for eosinophils. In the lungs, the cysteinyl leukotrienes appear to stimulate the secretion of mucus by the bronchial mucosa. Nanomolar concentrations of LTC4 and LTD4 stimulate the contraction of the smooth muscles of bronchi as well as smaller airways. Both LTB4 (generated by a hydrolase from the unstable LTA4) and the cysteinyl leukotrienes (i.e., LTC4, LTD4, and LTE4) act as growth or differentiation factors for a number of cell types in vitro. LTB4 stimulates myelopoiesis (formation of white blood cells) in human bone marrow, whereas LTC4 and LTD4 stimulate the proliferation of glomerular epithelial cells in the kidney. Picomolar concentrations of LTB4 stimulate the differentiation of a particular type of T lymphocytes referred to as competent suppressor or CD8+ lymphocytes. Additional immunological regulatory functions that may be subserved by LTB4 include the stimulation of IFN-γ and IL-2 production by T cells.

CHAPTER 3  •  Signal Transduction

65

TABLE 3-3  Involvement of Leukotrienes in Human Disease DISEASE

EVIDENCE

Asthma

Bronchoconstriction from inhaled LTE4; identification of LTC4, LTD4, and LTE4 in serum or urine or both

Psoriasis

Detection of LTB4 and LTE2 in fluids from psoriatic lesions

Adult respiratory distress syndrome (ARDS)

Elevated levels of LTB4 in plasma

Allergic rhinitis

Elevated levels of LTB4 in nasal fluids

Gout

Detection of LTB4 in joint fluid

Rheumatoid arthritis

Elevated levels of LTB4 in joint fluids and serum

Inflammatory bowel disease (ulcerative colitis and Crohn disease)

Identification of LTB4 in gastrointestinal fluids and LTE4 in urine

BOX 3-4  Role of Leukotrienes in Disease

S

ince the original description of the slow-reacting substance of anaphylaxis, which is generated during antigenic challenge of a sensitized lung, leukotrienes have been presumed to play a part in allergic disease of the airways (see Table 3-3). The involvement of cells (mast cells, basophils, and eosinophils) that produce cysteinyl leukotrienes (LTC4 through LTF4) in these pathobiological processes supports this concept. In addition, the levels of LTC4, LTD4, and LTE4 are increased in lavage fluid from the nares of patients with allergic rhinitis after the application of specific antigens to the nasal airways. Introducing LTC4 or LTD4 into the airways as an aerosol (nebulizer concentration of only 10 µM) causes maximal expiratory airflow (a rough measure of airway resistance; see p. 602) to decline by ~30%. This bronchoconstrictor effect is 1000-fold more potent than that of histamine, the “reference” agonist. Leukotrienes affect both large and small airways; histamine affects relatively smaller airways. Activation of the cysLT1 receptor in mast cells and eosinophils results in the chemotaxis of these cells to sites of inflammation. Because antagonists of the cysLT1 receptor (e.g., montelukast sodium) can partially block these bronchoconstrictive and proinflammatory effects, these agents are useful in the treatment of allergen-induced asthma and rhinitis. In addition to being involved in allergic disease, several of the leukotrienes are associated with other inflammatory disorders. Synovial fluid from patients with rheumatoid arthritis contains 5-lipoxygenase products. Another example is the skin disease psoriasis. In patients with active psoriasis, LTB4, LTC4, and LTD4 have been recovered from skin chambers overlying abraded lesions. Leukotrienes also appear to be involved in inflammatory bowel disease. LTB4 and other leukotrienes are generated and released in vitro from intestinal mucosa obtained from patients with ulcerative colitis or Crohn disease.

the receptor in mast cells and eosinophils causes release of the proinflammatory cytokines histamine and tumor necrosis factor-alpha (TNF-α). In addition to playing a role in the inflammatory response, the lipoxygenase metabolites can also influence the activity of many ion channels, either directly or by regulating protein kinases. For example, in synaptic nerve endings, lipoxygenase metabolites decrease the excitability of cells by activating K+ channels. Lipoxygenase products may also regulate secretion. In pancreatic islet cells, free AA generated in response to glucose appears to be part of a negative-feedback loop that prevents excess insulin secretion by inhibiting CaM kinase II.

The HETEs and EETs (epoxygenase products) tend to enhance Ca2+ release from intracellular stores and to enhance cell proliferation The epoxygenase pathway leads to the production of HETEs other than 5-HETE as well as EETs. HETEs and EETs have been implicated in a wide variety of processes, some of which are summarized in Table 3-4. For example, in stimulated mononuclear leukocytes, HETEs enhance Ca2+ release from intracellular stores and promote cell proliferation. In smoothmuscle cells, HETEs increase proliferation and migration; these AA metabolites may be one of the primary factors involved in the formation of atherosclerotic plaque. In blood vessels, HETEs can be potent vasoconstrictors. EETs enhance the release of Ca2+ from intracellular stores, increase Na-H exchange, and stimulate cell proliferation. In blood vessels, EETs primarily induce vasodilation and angiogenesis, although they have vasoconstrictive properties in the smaller pulmonary blood vessels.

Degradation of the eicosanoids terminates their activity eosinophils, cells that are commonly associated with allergic inflammatory responses such as asthma and urticaria. The cysteinyl leukotriene receptors cysLT1 and cysLT2 are GPCRs found on airway smooth-muscle cells as well as on eosinophils, mast cells, and lymphocytes. CysLT1, which couples to both pertussis toxin–sensitive and pertussis toxin–insensitive G proteins, mediates phospholipasedependent increases in [Ca2+]i. In the airways, these events produce a potent bronchoconstriction, whereas activation of

Inactivation of the products of eicosanoids is an important mechanism for terminating their biological action. In the case of COX products, the enzyme 15-hydroxyprostaglandin dehydrogenase catalyzes the initial reactions that convert biologically active prostaglandins into their inactive 15-keto metabolites. This enzyme also appears to be active in the catabolism of thromboxanes. As far as the 5-lipoxygenase products are concerned, the specificity and cellular distribution of the enzymes that metabolize leukotrienes parallel the diversity of the enzymes

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SECTION II  •  Physiology of Cells and Molecules

TABLE 3-4  Actions of Epoxygenase Products HETEs

CELL/TISSUE

ACTION

Stimulated mononuclear leukocytes

↑ Cell proliferation ↑ Ca2+ release from intracellular stores ↓TNFα production Implicated in the destruction of these cells in type 1 (juvenileonset) diabetes mellitus ↓ Release of fibrinolytic factors ↓ Binding of antithrombin ↑ Cell proliferation ↑ Migration Formation of atherosclerotic plaque? Potent vasoconstrictors “Myogenic” vasoconstrictive response of renal and cerebral arteries

β cells of pancreatic islets Endothelial cells Vascular smooth-muscle cells Blood vessels

EETs

Cells, general

Endocrine cells Toad bladder

Blood vessels Endothelium Platelets

↑ Ca release from intracellular stores ↑ Na-H exchange ↑ Cell proliferation ↓ Cyclooxygenase activity ↓ Release of somatostatin, insulin, glucagon ↓ Vasopressin-stimulated H2O permeability ↓ Renin release Vasodilation Angiogenesis ↑ Tumor cell adhesion ↓ Aggregation 2+

involved in their synthesis. For example, 20-hydrolase-LTB4, a member of the P-450 family, catalyzes the ω-oxidation of LTB4, thereby terminating its biological activity. LTC4 is metabolized through two pathways. One oxidizes the LTC4. The other pathway first removes the glutamic acid residue of the conjugated glutathione, which yields LTD4, and then removes the glycine residue, which yields LTE4; the latter is readily excreted into the urine. The metabolic breakdown of the HETE and EET products of epoxygenase (cytochrome P-450) is rapid and complex. The predominant pathway of inactivation appears to be hydrolysis via soluble epoxide hydrolase to form dihydroxyeicosatrienoic acids (DHETs), which themselves can induce biological responses, such as vasodilation. Once formed, DHETs can be excreted intact in the urine or can form conjugates with reduced glutathione (GSH). In addition, both EETs and DHETs can undergo β-oxidation to form epoxy fatty acids or can be metabolized by cyclooxygenase to generate various prostaglandin analogs.

RECEPTORS THAT ARE CATALYTIC A number of hormones and growth factors bind to cellsurface proteins that have—or are associated with— enzymatic activity on the cytoplasmic side of the membrane.

Here we discuss five classes of such catalytic receptors (Fig. 3-12): 1. Receptor guanylyl cyclases catalyze the generation of cGMP from GTP. 2. Receptor serine/threonine kinases phosphorylate serine or threonine residues on cellular proteins 3. Receptor tyrosine kinases (RTKs) phosphorylate tyrosine residues on themselves and other proteins. 4. Tyrosine kinase–associated receptors interact with cytosolic (i.e., not membrane-bound) tyrosine kinases. 5. Receptor tyrosine phosphatases cleave phosphate groups from tyrosine groups of cellular proteins.

The receptor guanylyl cyclase transduces the activity of atrial natriuretic peptide, whereas a soluble guanylyl cyclase transduces the activity of nitric oxide Receptor (Membrane-Bound) Guanylyl Cyclase  Some of the best-characterized examples of a transmembrane protein with guanylyl cyclase activity (see Fig. 3-12A) are the receptors for the natriuretic peptides.  N3-18  These ligands are a family of related small proteins (~28 amino acids) including atrial natriuretic peptide (ANP), B-type or brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). For example, in response to atrial and ventricular stretch that occur with intravascular volume expansion, cardiac myocytes release ANP and BNP, which act through receptor guanylyl cyclases. Their action is to relax vascular smooth muscle and dilate blood vessels (see ANP in Table 20-7, and p. 553) as well as to enhance Na+ excretion into urine (natriuresis; see p. 843). Both activities contribute to lowering of effective circulating blood volume and thus blood pressure (see pp. 554–555). Natriuretic peptide receptors NPRA and NPRB are membrane proteins with an extracellular ligand-binding domain and a single membrane-spanning segment (see Fig. 3-12A). The intracellular domain has two consensus catalytic domains for guanylyl cyclase activity. Binding of a natriuretic peptide induces a conformational change in the receptor that causes receptor dimerization and activation. Thus, binding of ANP to its receptor causes the conversion of GTP to cGMP and raises intracellular levels of cGMP. In turn, cGMP activates a cGMP-dependent kinase (PKG or cGK) that phosphorylates proteins at certain serine and threonine residues. In the renal medullary collecting duct, the cGMP generated in response to ANP may act not only via PKG but also by directly modulating ion channels (see p. 768). Soluble Guanylyl Cyclase  In contrast to the receptor guanylyl cyclase, which is activated by ANP, the cytosolic soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO). This sGC is unrelated to the receptor guanylyl cyclase and contains a heme moiety that binds NO. NO is a highly reactive, short-lived free radical. This dissolved gas arises from a family of NO synthase (NOS) enzymes that catalyze the reaction L-arginine + 1.5 NADPH + H + + 2O2 (3-1) → NO + citrulline + 1.5 NADP +

Here, NADPH and NADP+ are the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate,

CHAPTER 3  •  Signal Transduction

66.e1

N3-18  Atrial Natriuretic Peptide Contributed by Emile Boulpaep Granular inclusions in atrial myocytes, called Palade bodies, contain pro-ANP, the precursor of atrial natriuretic peptide (ANP; also called atrial natriuretic factor, ANF). Pro-ANP, comprising 126 amino acids, is derived from the precursor known as prepro-ANP (151 residues in the human). The converting enzyme corin—a cardiac transmembrane serine protease—cleaves the pro-ANP during or after release from the atria, which yields the inactive N-terminal fragment of 98 residues and the active C-terminal 28–amino-acid peptide called ANP. Release is primarily caused by stretch of the atrial myocytes. Hormones such as angiotensin, endothelins, arginine vasopressin, and glucocorticoid modulate ANP expression and release. It is noteworthy that expression of corin is reduced in heart failure, which blunts the release of ANP in the failing heart. This blunting might contribute to the inappropriate increase of extracellular fluid volume in heart failure. ANP is a member of the NP (natriuretic peptide) family of peptides. The biological effects of ANP are potent vasodilation, diuresis, natriuresis, and kaliuresis, as well as inhibition of the renin-angiotensin-aldosterone system. At least three types of natriuretic peptide receptors (NPRs) exist: NPRA (also called GC-A—GC for guanylyl cyclase), NPRB (also called GC-B), and NPR-C. NPRA and NPRB are receptors

with a single transmembrane domain coupled to a cytosolic guanylyl cyclase (see p. 66). Activation of NPRA or NPRB leads to the intracellular generation of cGMP. In smooth muscle, intracellular cGMP activates the cGMP-dependent protein kinase that phosphorylates MLCK. Phosphorylation of MLCK inactivates MLCK; this leads to the dephosphorylation of myosin light chains, which allows muscle relaxation. The ANP C-type receptor NPRC is not coupled to a messenger system but serves mainly to clear the natriuretic peptides from the circulation. The heart, brain, pituitary, and lung synthesize an ANP-like compound termed BNP, originally known as brain natriuretic peptide (32 residues in the human). The biological actions of BNP are similar to those of ANP. The hypothalamus, pituitary, and kidney synthesize C-type natriuretic peptide or CNP, which is highly homologous to ANP and BNP. CNP binds only to NPRB and is only a weak natriuretic but a strong vasodilator. The kidney also synthesizes an ANP-like natriuretic compound known as urodilatin or URO. URO has four additional amino acids compared to ANP and also binds to the ANP A-type receptor. Its biological effect in the target tissue is also transduced by cGMP.

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CHAPTER 3  •  Signal Transduction

A

C

RECEPTOR GUANYLYL CYCLASES

B

Extracellular space N

RECEPTOR SERINE/ THREONINE KINASES

RECEPTOR TYROSINE KINASES (RTKs)

D

TYROSINE-KINASE– ASSOCIATED RECEPTORS N

Ligand

N

RECEPTOR TYROSINE PHOSPHATASES

Carbohydrate groups

N

Ligand N

E

N N

N

Ligand

Serinethreonine kinase domain C C

C

Type I

Guanylyl cyclase domains Cytosol ANP RECEPTOR

JAK2

JAK2

C

Type II TGF- RECEPTOR

C

C

Tyrosine kinase domains

This is the kinase that phosphorylates NGF downstream RECEPTOR effectors.

Tyrosine kinases

C

C

GROWTH HORMONE RECEPTOR

Tyrosine phosphatase domain

C

CD45

Figure 3-12  Catalytic receptors. A, Receptor guanylyl cyclases have an extracellular ligand-binding domain. B, Receptor serine/threonine kinases have two subunits. The ligand binds only to the type II subunit. C, RTKs similar to the NGF receptor dimerize on binding a ligand. D, Tyrosine kinase–associated receptors have no intrinsic enzyme activity but associate noncovalently with soluble nonreceptor tyrosine kinases. E, Receptor tyrosine phosphatases have intrinsic tyrosine phosphatase activity.

respectively. Tetrahydrobiopterin is a cofactor. The NOS family includes neuronal or nNOS (NOS1), inducible or iNOS (NOS2), and endothelial or eNOS (NOS3). nNOS and iNOS are soluble enzymes, whereas eNOS is linked to the plasma membrane. The activation of NOS begins as an extracellular agonist (e.g., ACh) binds to a plasmamembrane receptor, triggering the entry of Ca2+, which binds to cytosolic CaM and then stimulates NOS. In smooth muscle, NO stimulates the sGC, which then converts GTP to cGMP, activating PKG, which leads to smooth-muscle relaxation. Why NO is so ubiquitous and when its release is important are not known. However, abnormalities of the NO system are involved in the pathophysiological processes of adult respiratory distress syndrome, high-altitude pulmonary edema, stroke, and other diseases. For example, the importance of NO in the control of blood flow had long been exploited unwittingly to treat angina pectoris. Angina is the classic chest pain that accompanies inadequate blood flow to the heart muscle, usually as a result of coronary artery atherosclerosis. Nitroglycerin relieves this pain by spontaneously breaking down and releasing NO, which relaxes the smooth muscles of peripheral arterioles, thereby reducing the work of the heart and relieving the associated pain. Understanding the physiological and pathophysiological

roles of NO has led to the introduction of clinical treatments that modulate the NO system. In addition to the use of NO generators for treatment of angina, examples include the use of gaseous NO for treatment of pulmonary edema and inhibitors of cGMP phosphodiesterase (see p. 53) such as sildenafil (Viagra) for treatment of erectile dysfunction. In addition to acting as a chemical signal in blood vessels, NO generated by iNOS appears to play an important role in the destruction of invading organisms by macrophages and neutrophils. NO generated by nNOS also serves as a neurotransmitter (see pp. 315–317) and may play a role in learning and memory. The importance of the NO signaling pathway was recognized by the awarding of the 1998 Nobel Prize for Physiology or Medicine to R.F. Furchgott, L.J. Ignarro, and F. Murad for their discoveries concerning NO as a signaling molecule in the cardiovascular system.  N3-18A

Some catalytic receptors are serine/threonine kinases We have already discussed how activation of various G protein–linked receptors can initiate a cascade that eventually activates kinases (e.g., PKA, PKC) that phosphorylate proteins at serine and threonine residues. In addition, some receptors are themselves serine/threonine kinases—such as

CHAPTER 3  •  Signal Transduction

N3-18A  Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad For more information about Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad and the work that led to their Nobel Prize, visit http://www.nobelprize.org/nobel_prizes/medicine/ laureates/1998/ (Accessed March 2015).

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SECTION II  •  Physiology of Cells and Molecules

the one for transforming growth factor-β (TGF-β)—and are thus catalytic receptors. The TGF-β superfamily includes a large group of cytokines, including five TGF-βs, antimüllerian hormone (see p. 1080), as well as the inhibins and activins (see p. 1113), bone morphogenic proteins, and other glycoproteins, all of which control cell growth and differentiation. Members of this family participate in embryogenesis, suppress epithelialcell growth, promote wound repair, and influence immune and endocrine functions. Unchecked TGF-β signaling is important in progressive fibrotic disorders (e.g., liver cirrhosis, idiopathic pulmonary fibrosis) that result in replacement of normal organ tissue by deposits of collagen and other matrix components. The receptors for TGF-β and related factors are glycoproteins with a single membrane-spanning segment and intrinsic serine/threonine-kinase activity. Receptor types I and II (see Fig. 3-12B) are required for ligand binding and catalytic activity. The type II receptor first binds the ligand, and this binding is followed by the formation of a stable ternary complex of ligand, type II receptor, and type I receptor. After recruitment of the type I receptor into the complex, the type II receptor phosphorylates the type I receptor, thereby activating the serine/threonine kinase activity of the type I receptor. The principal targets of this kinase activity are SMAD proteins, which fall into three groups.  N3-19  The largest group is the receptor-activated SMADs (SMADs 1, 2, 3, 5, and 8), which—after phosphorylation by activated type I receptors—association with SMAD4, the only member of the second group. This heterodimeric complex translocates to the nucleus, where it regulates transcription of target genes. The third group (SMAD6, SMAD7) is the inhibitory SMADs, which can bind to type I receptors and prevent the phosphorylation of the receptor-activated SMADs.

RTKs produce phosphotyrosine motifs recognized by SH2 and phosphotyrosine-binding domains of downstream effectors In addition to the class of receptors with intrinsic serine/ threonine kinase activity, other plasma-membrane receptors have intrinsic tyrosine kinase activity. All RTKs discovered to date phosphorylate themselves in addition to other cellular proteins. Epidermal growth factor (EGF), plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin and insulin-like growth factor type 1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF) can all bind to receptors that possess intrinsic tyrosine kinase activity. Creation of Phosphotyrosine Motifs  Most RTKs are singlepass transmembrane proteins that have an extracellular ligand-binding domain and a single intracellular kinase domain (see Fig. 3-12C). Binding of a ligand, such as NGF, facilitates the formation of receptor dimers that in turn promote the direct association and trans-phosphorylation of the adjacent kinase domains; the result is activation of the receptor complex. The activated receptors then catalyze the addition of phosphate to tyrosine (Y) residues on the receptor itself as well as specific membrane-associated and cytoplasmic proteins. The resulting phosphotyrosine (pY)

TABLE 3-5  Tyrosine Phosphopeptides of the PDGF Receptor That Are Recognized by SH2 Domains on Various Proteins TYROSINE (Y) THAT IS PHOSPHORYLATED IN PDGF RECEPTOR

pY MOTIF RECOGNIZED BY SH2-CONTAINING PROTEIN

SH2CONTAINING PROTEIN

Y579

pYIYVD

Src family kinases

Y708

pYMDMS

p85

Y719

pYVPML

p85

Y739

pYNAPY

GTPase-activating protein

Y1021

pYIIPY

PLCγ

motifs serve as high-affinity binding sites for the recruitment of a number of intracellular signaling molecules, discussed in the next paragraph. These interactions lead to the for­ mation of a signaling complex and the activation of downstream effectors. Some RTKs, such as the insulin and IGF-1 receptors,  N3-20  exist as dimers even before ligand binding but undergo a conformational change that promotes autophosphorylation and activation of the kinase domains (see pp. 1041–1042). Recognition of pY Motifs by SH2 and PhosphotyrosineBinding Domains  The pY motifs created by tyrosine kinases

serve as high-affinity binding sites for the recruitment of cytoplasmic or membrane-associated proteins that contain either an SH2 domain or PTB (phosphotyrosine-binding) domain. SH2 domains are ~100 amino acids in length. They are composed of relatively well conserved residues that form the binding pocket for pY motifs as well as more variable residues that are implicated in binding specificity. These residues that confer binding specificity primarily recognize the three amino acids located on the C-terminal side of the phosphotyrosine. For example, the activated PDGF receptor has five such pY motifs (Table 3-5), each of which interacts with a specific SH2-containing protein. In contrast to SH2 and PTB domains, which interact with highly regulated pY motifs, Src homology 3 (SH3) domains interact constitutively with proline-rich regions in other proteins in a manner that does not require phosphorylation of the motif. However, phosphorylation at distant sites can change the conformation near the proline-rich region and thereby regulate the interaction. Like SH2 interactions, SH3 interactions appear to be responsible for targeting of signaling molecules to specific subcellular locations. SH2- or SH3-containing proteins include growth factor receptor– bound protein 2 (GRB2), PLCγ, and the p85 subunit of the phosphatidylinositol-3-kinase. The MAPK Pathway  A common pathway by which activated RTKs transduce their signal to cytosol and even to the nucleus is a cascade of events that increase the activity of the small GTP-binding protein Ras. This Ras-dependent signaling pathway involves the following steps (Fig. 3-13):

Step 1: A ligand binds to the extracellular domain of a specific RTK, thus causing receptor dimerization.

CHAPTER 3  •  Signal Transduction

N3-19  SMADs

68.e1

N3-20  Insulin and IGF-1 Receptors

Contributed by Ed Moczydlowski

Contributed by Emile Boulpaep and Walter Boron

The largest group of SMAD proteins is the receptor-activated SMADs (SMADs 1, 2, 3, 5, and 8), which have a type I receptor–interacting domain that is phosphorylated by the activated type I receptor; this phosphorylation results in their disassociation from the receptor and subsequent association with the regulatory SMAD, SMAD4. This heterodimeric complex translocates to the nucleus where it can regulate transcription of target genes by both direct and indirect mechanisms. The signaling specificity of this system comes from two mechanisms. First, distinct members of the receptor-activated SMAD group interact with specific type I receptors. For example SMAD2 and SMAD3 associate with the TGF-β type I receptor ALK-5, whereas SMAD1 associates with bone morphogenic protein (BMP) type I receptors such as ALK-2 and ALK-3. Second, the receptor-activated SMAD/SMAD4 heterodimer regulates not only downstream effector gene expression but also the expression of a third group of SMADs, the inhibitory SMADs. These proteins (SMAD6, SMAD7), once expressed, can bind to type I receptors and prevent the association and activation of receptor-activated SMADs.

The insulin receptor (see Fig. 51-5) and the IGF-1 receptor are activated by somewhat different mechanisms, as we discuss on pages 1041–1042 for the insulin receptor and on page 996 for the IGF-1 receptor. In brief, these receptors are tetrameric; they are composed of two α and two β subunits. The α subunit contains a cysteine-rich region and functions in ligand binding. The β subunit is a single-pass transmembrane protein with a cytoplasmic tyrosine kinase domain. The α and β subunits are held together by disulfide bonds (as are the two α subunits), forming a heterotetramer. Ligand binding produces conformational changes that appear to cause allosteric interactions between the two α and β pairs, as opposed to the dimerization characteristic of the first class of RTKs (see Fig. 3-12C). Thus, insulin binding results in the autophosphorylation of tyrosine residues in the catalytic domains of the β subunits. The activated insulin receptor also phosphorylates cytoplasmic substrates such as IRS-1 (insulin-receptor substrate 1; see Fig. 51-6), which, once phosphorylated, serves as a docking site for additional signaling proteins.

CHAPTER 3  •  Signal Transduction

6 The activated GTP-Ras recruits Raf-1 and activates it.

1 Ligand binding causes receptor dimerization. Ligand

Ligand

Extracellular space

5 SOS activates Ras by causing GTP to replace GDP on Ras.

8 MEK phosphorylates and activates MAPK.

9 MAPK works as an important effector molecule by phosphorylating many cellular proteins.

7 Raf-1 phosphorylates and activates MEK.

Receptor

Plasma membrane SH2 domain

Cytosol Tyrosine kinase domain

Ras

Ras Raf-1

P

P

69

MEK

P

P

MAPK P

Cytosolic proteins

GRB2 SOS

2 The activated RTK phosphorylates itself. 3 GRB2, an SH2-containing protein, recognizes the phosphotyrosine residues.

Ra

f-1

S

SO

4 The binding of GRB2 recruits SOS.

Nucleus Modulation of transcription

Inactive transcription factor MAPK

P

P

Active transcription factor

10 MAPK translocates to the nucleus where it phosphorylates a transcription factor.

Figure 3-13  Regulation of transcription by the Ras pathway. A ligand, such as a growth factor, binds to a specific RTK, and this leads to an increase in gene transcription in a 10-step process.

Step 2: The now-activated RTK phosphorylates itself on tyrosine residues of the cytoplasmic domain (autophosphorylation). Step 3: GRB2, an SH2-containing protein, recognizes pY residues on the activated receptor. Step 4: Because GRB2 constitutively associates with the guanine nucleotide exchange factor SOS (son of sevenless), via an SH3-proline interaction, the recruitment of GRB2 automatically results in the recruitment of SOS as well. Step 5: SOS activates the small G protein Ras by catalyzing the replacement of GDP with GTP. Step 6: The activated GTP-Ras complex activates other proteins by physically recruiting them to the plasma membrane. In particular, active GTP-Ras interacts with the N-terminal portion of the cytosolic serine/threonine kinase Raf-1 (also known as MAP kinase kinase kinase or MAPKKK or MAP3K), which is the first in a series of sequentially activated protein kinases that ultimately transmits the activation signal. Step 7: Raf-1 phosphorylates and activates a protein kinase called MEK (also known as MAP kinase kinase or MAPKK). MEK is a multifunctional protein kinase that phosphorylates substrates on both tyrosine and serine/ threonine residues. Step 8: MEK phosphorylates MAPKs, cytosolic serine/ threonine kinases also called extracellular signal– regulated kinases (ERK1, ERK2). Activation of MAPK requires dual phosphorylation on neighboring serine and tyrosine residues. Raf, MEK, and MAPK typically assemble on a scaffolding protein at the inner side of the

cell membrane to facilitate interaction/phosphorylation during the activation process. Step 9: MAPK is an important effector molecule in Rasdependent signaling to the cytoskeleton. MAPK phosphorylates multiple proteins involved in actin cytoskeletal assembly and cell-matrix interactions; this phosphorylation leads to Ras-dependent changes in cell morphology and cell migration. Step 10: Once activated, MAPK disassociates from the scaffold and translocates primarily to the nucleus, where it phosphorylates a number of nuclear proteins that are transcription factors. The result is either enhancement or repression of the DNA binding and transcriptional activity of these nuclear proteins.  N3-21 Two other signal-transduction pathways (cAMP and Ca2+) can modulate the activity of some of the protein intermediates in this MAPK cascade, which suggests multiple points of integration for the various signaling systems. The Phosphatidylinositol-3-Kinase Pathway  The phosphatidylinositol-3-kinase (PI3K) is an SH2 domain–containing protein that commonly signals downstream of RTKs. PI3K is a heterodimer consisting of a p85 regulatory subunit and p110 catalytic subunit. p85 has an SH2 domain for targeting the complex to activated receptors and an SH3 domain that mediates constitutive association with p110. p110 is a lipid kinase that phosphorylates PIP2 (see p. 58) on the 3 position of the inositol ring to form PIP3. PIP2 is a relatively common lipid in the inner leaflet of the cell membrane, whereas PIP3 constitutes NH +4 ≫ Cs+ > Li+, Na+, Ca2+. Under normal physiological conditions, the permeability ratio PK/PNa is >100, and Na+ can block many K+ channels. Some K+ channels can pass Na+ current in the complete absence of K+. This characteristic is analogous to the behavior of Ca2+ channels, which can pass Na+ and K+ currents in the absence of Ca2+. Given such strong K+ selectivity and an equilibrium potential near −80 mV, the primary role of K+ channels in excitable cells is to oppose the action of excitatory Na+ and Ca2+ channels and stabilize the resting state. Whereas some K+ channels are major determinants of the resting potential, other K+ channels mediate the repolarizing phase and shape of action potentials, control firing frequency, and define the bursting behavior of rhythmic firing. Such functions are broadly important in regulating the strength and frequency of all types of muscle contraction, in terminating transmitter release at nerve terminals, in attenuating the strength of synaptic connections, and in coding the intensity of sensory stimuli. Finally, in epithelia, K+ channels also function in K+ absorption and secretion. Before understanding the molecular nature of K+ channels, electrophysiologists classified K+ currents according to their functional properties and gating behavior, grouping macroscopic K+ currents into four major types: 1. Delayed outward rectifiers 2. Transient outward rectifiers (A-type currents) 3. Ca2+-activated K+ currents 4. Inward rectifiers These four fundamental K+ currents are the macroscopic manifestation of five distinct families of genes (see Table 6-2, family No. 2): 1. Kv channels (voltage-gated K+ channels related to the Shaker family) 2. Small- and intermediate-conductance KCa channels (Ca2+-calmodulin–activated K+ channels), including SKCa and IKCa channels 3. Large-conductance KCa channels (Ca2+-activated BKCa channels and related Na+- and H+- activated K+ channels)

193

4. Kir channels (inward-rectifier K+ channels) 5. K2P channels (two-pore K+ channels) In the next three sections, we discuss the various families of K+ channels and their associated macroscopic currents.

The Kv (or Shaker-related) family of K+ channels mediates both the delayed outward-rectifier current and the transient A-type current The K+ current in the HH voltage-clamp analysis of the squid giant axon (see pp. 177–178) is an example of a delayed outward rectifier. Figure 7-18A shows that this current activates with a sigmoidal lag phase (i.e., it is delayed in time, as in Fig. 7-6C). Figure 7-18B is an I-V plot of peak currents obtained in experiments such as that presented in Figure 7-18A and shows that the outward current rises steeply at positive voltages (i.e., it is an outward rectifier). A second variety of K+ current that is also outwardly rectifying is the transient A-type K+ current. This current was first characterized in mollusk neurons, but similar currents are common in the vertebrate nervous system. A-type currents are activated and inactivated over a relatively rapid time scale. Because their voltage activation range is typically more negative than that of other K+ currents, they are activated in the negative Vm range that prevails during the afterhyperpolarizing phase of action potentials. In neurons that spike repetitively, this A-type current can be very important in determining the interval between successive spikes and thus the timing of repetitive action potentials. For example, if the A-type current is small, Vm rises relatively quickly toward the threshold, and consequently the interspike interval is short and the firing frequency is high (see Fig. 7-18C). However, if the A-type current is large, Vm rises slowly toward the threshold, and therefore the interspike interval is long and the firing frequency is low (see Fig. 7-18D). Because the nervous system often encodes sensory information as a frequency-modulated signal, these A-type currents play a critical role. The channels responsible for both the delayed outwardrectifier and the transient A-type currents belong to the Kv channel family (where v stands for voltage-gated). The prototypic protein subunit of these channels is the Shaker channel of Drosophila (see Fig. 7-12C). All channels belonging to this family contain the conserved S1 to S6 core that is characteristic of the Shaker channel (see Fig. 7-10), but may differ extensively in the length and sequence of their intracellular N-terminal and C-terminal domains. The voltagesensing element in the S4 segment underlies activation by depolarization; the S4 segment actually moves outward across the membrane with depolarizing voltage, thus increasing the probability of the channel’s being open.  N7-13 The Kv channel family has multiple subclasses (see Table 6-2, family No. 2). Individual members of this Kv channel family, whether in Drosophila or humans, exhibit profound differences in gating kinetics that are analogous to delayedrectifier (slow activation) or A-type (rapid inactivation) currents. For example, Figure 7-18E shows the macroscopic currents of four subtypes of rat brain Kv1 (or Shaker) channels heterologously expressed in frog oocytes. All of these Kv1 channel subtypes (Kv1.1 to Kv1.4) exhibit sigmoidal activation kinetics when examined on a brief time scale—

194

SECTION II  •  Physiology of Cells and Molecules

A

DELAYED ACTIVATION OF Kv CHANNELS +30 mV –60 mV The activation of the current is delayed.

1 2 Peak current

+

K current

B

OUTWARD RECTIFICATION OF Kv CHANNELS Peak K current (IK)

3

4 5 +

6

P

N

C

C

A-TYPE OUTWARD RECTIFIER: SMALL CURRENT Small A-type current

Outward The current flows only in the outward direction. That is, the channel is an “outward rectifier.”

Vm

E

A-TYPE OUTWARD RECTIFIER: LARGE CURRENT Long interspike interval Large A-type current

DIFFERENCES IN GATING KINETICS AMONG Kv-TYPE DELAYED OUTWARD RECTIFIERS Kv1.1

150 pA

Kv1.1

Kv1.2

200 pA

Kv1.2

Kv1.3

6 pA

Kv1.4

400 pA

0 F

D

25 Time (ms)

50

0

500 pA

300 pA

Kv1.3

10 pA

Kv1.4

200 pA 1

2 Time (s)

INACTIVATION OF Kv-TYPE CHANNELS 4 α subunits

4 α subunits

4 α subunits

β

The N-terminal domain ball moves in and blocks the channel.

4 α subunits

β

The β subunit moves in and blocks the channel.

3

Chapter 7    •  Electrical Excitability and Action Potentials

195

BOX 7-3  Congenital and Drug-Induced Cardiac Arrhythmias Linked to K+ Channels Congenital Long QT Syndromes As discussed in Box 7-1, congenital cardiac abnormality in some people results in lengthening of the QT interval of the electrocardiographic signal—long QT syndrome—which corresponds to a prolonged cardiac action potential. Affected children and young adults can exhibit an arrhythmic disturbance of the ventricular heartbeat that results in sudden death. As we have already seen in Box 7-1, one form of a long QT syndrome—LQT3—involves gain-of-function mutations of the cardiac Na+ channel Nav1.5 (SCN5A) that prolong Na+ channel opening. However, at least six forms of long QT syndrome—LQT1, LQT2, LQT5, LQT6, LQT7, and LQT13—are caused by loss-of-function mutations in cardiac K+ channels (see Table 6-2, family No. 2) or their accessory proteins. LQT1 is due to mutations in the KCNQ1 gene encoding KvLQT1, a 581-residue protein belonging to the Kv family of voltage-gated K+ channels. Another form of this disease, LQT2, involves mutations in the KCNH2 gene encoding HERG (for human ether-à-go-go) which is related to the gene defective in the ether-à-go-go Drosophila mutation, in which flies convulsively shake under ether anesthesia. Both KvLQT1 and HERG K+ channels participate in repolarization of the human cardiac action potential (see p. 488). KvLQT1 mediates the slowly activating delayed-rectifier component (IKs) of cardiac action potential repolarization; HERG mediates the rapidly activating repolarization component (IKr). Both LQT1 and LQT2 result from loss-offunction effects associated with decreased K+ channel expression in cardiac myocytes.

in the millisecond range (left side of Fig. 7-18E). That is, these channels display some degree of “delayed” activation. Different Kv channels exhibit different rates of activation. Thus, these currents can modulate action potential duration by either keeping it short (e.g., in nerve and skeletal muscle) when the delayed rectifier turns on quickly or keeping it long (e.g., in heart) when the delayed rectifier turns on slowly. Kv1 channels also differ markedly in their inactivation kinetics when observed over a long time scale—in the range of seconds (right side of Fig. 7-18E). Kv1.1 exhibits little time-dependent inactivation (i.e., the current is sustained throughout the stimulus). On the other hand, the Kv1.4

KvLQT1 associates with minK, a small, single-span membrane protein encoded by the KCNE1 gene. minK modulates the gating kinetics of KvLQT1, and mutations in minK cause LQT5. Three other human proteins closely related to minK are known as MiRP1, MiRP2, and MiRP3 (minK-related proteins)—the products of the genes KCNE2, KCNE3, and KCNE4, respectively. MiRP1 associates with HERG, and mutations in MiRP1 are linked to LQT6. Two other K+ channel genes also cause long QT syndromes. Mutations in Kir2.1, encoded by the gene KCNJ2, cause LQT7, whereas mutations in GIRK4, encoded by KCNJ5, cause LQT13.

Acquired Long QT Syndrome The HERG channel is notorious for its sensitivity to blockade by many classes of therapeutic drugs, including antihistamines (e.g., terfenadine), antipsychotics (e.g., sertindole), and gastrointestinal drugs (e.g., cisapride). Blockade of HERG can readily mimic the genetic condition of LQT2. The promiscuous drug sensitivity of the HERG K+ channel appears to result from particular structural features of the internal aspect of the channel pore that favor binding of many hydrophobic small molecules. People who have natural variations in ion channel genes that cause a subclinical propensity for long QT intervals or who have deficiencies in drugmetabolizing enzymes appear to be especially at risk. Many drugs have been banned or limited for therapeutic use because of the risk of HERG channel block. Today all new drugs proposed for clinical use must first undergo screening for HERG blockade in order to prevent deaths by acquired long QT syndrome.

channel completely inactivates in 10 degrees away from the center of the fovea and thus the center of gaze).

SECTION III  •  The Nervous System

To optic nerve

Nerve fiber layer

Light Ganglion cell

ipRGC

Ganglion cell layer

Amacrine cell

Inner plexiform layer

Proximal

Retina Inner nuclear layer

Outer plexiform layer Light

Vertical information flow

364

Lateral information flow Horizontal cell

Bipolar cells

Rod Outer nuclear layer

Cone

Cone

Rod

Rod

Cone

Distal

Photoreceptor outer segment Pigment epithelium

Ciliary stalk

Synaptic terminals

Folding of outer cell membrane

Inner segment

Connecting cilium

Nucleus Mitochondria Freefloating disks

Cilium

Outer segment

Disks Cone

Rod

Cytoplasmic space

Figure 15-9  Neural circuits in the primate retina. Notice that the incoming light reaches intrinsically photosensitive retinal ganglion cells (ipRGCs) immediately, but hits rod and cone photoreceptor cells only after passing through several thin, transparent layers of other neurons. The pigment epithelium absorbs the light not absorbed by the photoreceptor cells and thus minimizes reflections of stray light. The ganglion cells communicate to the thalamus by sending action potentials down their axons. However, the photoreceptor cells and other neurons communicate by graded synaptic potentials that are conducted electrotonically.

Chapter 15  •  Sensory Transduction

A CENTER OF THE RETINA (FOVEA)

B

PERIPHERY OF THE RETINA

Ganglion cells

365

membrane-bound intracellular organelles that have pinched off from the outer membrane. Cone outer segments have similarly stacked membranes, except that they are infolded and remain continuous with the outer membrane. The disk membranes contain the photopigments—rhodopsin in rods and molecules related to rhodopsin in cones. Rhodopsin moves from its synthesis site in the inner segment through the stalk and into the outer segment through small vesicles whose membranes are packed with rhodopsin to be incorporated into the disks.

Rods and cones hyperpolarize in response to light

Bipolar cells

Cone

Cone Rod

Photoreceptors

Receptive fields of ganglion cells

The receptive field of ganglion cells at the retinal periphery is much larger than that at the fovea.

Figure 15-10  Comparison of the synaptic connections and receptive fields in the fovea and periphery of the retina.

Rods and cones are elongated cells with synaptic terminals, an inner segment, and an outer segment (see Fig. 15-9). The synaptic terminals connect to the inner segment by a short axon. The inner segment contains the nucleus and metabolic machinery; it synthesizes the photopigments and has a high density of mitochondria. The inner segment also serves an optical function—its high density funnels photons into the outer segment. A thin ciliary stalk connects the inner segment to the outer segment. The outer segment is the transduction site, although it is the last part of the cell to see the light. Structurally, the outer segment is a highly modified cilium. Each rod outer segment has ~1000 tightly packed stacks of disk membranes, which are flattened,

The remarkable psychophysical experiments of Hecht and colleagues in 1942 demonstrated that five to seven photons, each acting on only a single rod, are sufficient to evoke a sensation of light in humans. Thus, the rod is performing at the edge of its physical limits because there is no light level smaller than 1 photon. To detect a single photon requires a prodigious feat of signal amplification. As Denis Baylor has pointed out, “the sensitivity of rod vision is so great that the energy needed to lift a sugar cube one centimeter, if converted to a blue-green light, would suffice to give an intense sensation of a flash to every human who ever existed.” Phototransduction involves a cascade of chemical and electrical events to detect, to amplify, and to signal a response to light. As do many other sensory receptors, photoreceptors use electrical events (receptor potentials) to carry the visual signal from the outer segment to their synapses. Chemical messengers diffusing over such a distance would simply be too slow. A surprising fact about the receptor potential of rods and cones is that it is hyperpolarizing. Light causes the cell’s Vm to become more negative than the resting potential that it maintains in the dark (Fig. 15-11A). At low light intensities, the size of the receptor potential rises linearly with light intensity; but at higher intensities, the response saturates. Hyperpolarization is an essential step in relaying the visual signal because it directly modulates the rate of transmitter release from the photoreceptor onto its postsynaptic neurons. This synapse is conventional in that it releases more transmitter—in this case glutamate—when its presynaptic terminal is depolarized and less when it is hyperpolarized. Thus, a flash of light causes a decrease in transmitter secretion. The upshot is that the vertebrate photoreceptor is most active in the dark. How is the light-induced hyperpolarization generated? Figure 15-11B shows a method to measure the current flowing across the membrane of the outer segment of a single rod. In the dark, each photoreceptor produces an ionic current that flows steadily into the outer segment and out of the inner segment. This dark current is carried mainly by inwardly directed Na+ ions in the outer segment and by outwardly directed K+ ions from the inner segment (see Fig. 15-11C). Na+ flows through a nonselective cation channel of the outer segment, which light indirectly regulates, and K+ flows through a K+ channel in the inner segment, which light does not regulate. Na+ carries ~90% of the dark current in the outer segment, and Ca2+, ~10%. In the dark, Vm is about −40 mV. Na-K pumps, primarily located within the inner segments, remove the Na+ and import K+. An Na-Ca exchanger removes Ca2+ from the outer segment.

366

SECTION III  •  The Nervous System

A

B

LIGHT-EVOKED HYPERPOLARIZATIONS

LIGHT STIMULATING A SINGLE ROD

Light flash Response to the least intense light flash –40 –45 Membrane –50 potential (mV) –55 Response to the most intense light flash

–60 –65 0 C

100

200 300 400 Time (ms)

DARK

500

600

D

LIGHT

Depolarized: high transmitter release

Hyperpolarized: low transmitter release

Rod

Rod

Inner segment

Inner segment

Nonselective cation channel K+

Na+

cGMP +

Na

cGMP

Outer segment

Na+

Outer segment

Figure 15-11  Phototransduction. A, The experiment for which results are summarized here was performed on a red-sensitive cone from a turtle. A brief flash of light causes a hyperpolarization of the photoreceptor cell. The size of the peak and the duration of the receptor potential increase with the increasing intensity of the flash. At low light intensities, the magnitude of the peak increases linearly with light intensity. At high intensities, the peak response saturates, but the plateau becomes longer. B, A single rod has been sucked into a pipette, which allows the investigators to monitor the current. The horizontal white band is the light used to stimulate the rod. C, In the absence of light, Na+ enters the outer segment of the rod through cGMPgated channels and depolarizes the cell. The electrical circuit for this dark current is completed by K+ leaving the inner segment. The dark current, which depolarizes the cell, leads to constant transmitter release. D, In the presence of light, Na+ can no longer enter the cell because cGMP levels are low, and the cGMP-gated channel closes. The photoreceptor cell thus hyperpolarizes, and transmitter release decreases. (A, Data from Baylor DA, Hodgkin AL, Lamb TD: The electrical response of turtle cones to flashes and steps of light. J Physiol 242:685–727, 1974; B, from Baylor DA, Lamb TD, Yau K-W: Responses of retinal rods to single photons. J Physiol 288:613–634, 1979.)

Chapter 15  •  Sensory Transduction

Figure 15-12  Rhodopsin, transducin, and signal transduction at the molecular

level. A, The opsin molecule is a classic seven-transmembrane receptor that couples to transducin, a G protein. When the opsin is attached to retinal (magenta structure) via amino-acid residue 296 in the seventh (i.e., most C-terminal) membrane-spanning segment of opsin, the assembly is called rhodopsin. B, The absorption of a photon by 11-cis retinal causes the molecule to isomerize to all-trans retinal. C, After rhodopsin absorbs a photon of light, it activates many transducins. The activated α subunit of transducin (Gαt) in turn activates phosphodiesterase, which hydrolyzes cGMP. The resultant decrease in [cGMP]i closes cGMP-gated channels and produces a hyperpolarization (receptor potential). GMP, 5′-guanylate monophosphate; NCKX1, the Na+/(Ca2+-K+) exchanger (SLC24A1). (A, Data from Palczewsk K, Kumasaka T, Miyano, M et al: Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289(5480):739–745, 2000. Reconstructed figure is courtesy of S. Filipek and K. Palczewski.)

Absorption of photons leads to closure of the nonselective cation channels in the outer segment. The total conductance of the cell membrane decreases. Because the K+ channels of the inner segment remain open, K+ continues to flow out of the cell, and this outward current causes the cell to hyperpolarize (see Fig. 15-11D). The number of cation channels that close depends on the number of photons that are absorbed. The range of one rod’s sensitivity is 1 to ~1000 photons. Baylor and colleagues measured the minimum amount of light required to produce a change in receptor current (see Fig. 15-11B). They found that absorption of 1 photon suppresses a surprisingly large current, equivalent to the entry of >106 Na+ ions, and thus represents an enormous amplification of energy. The single-photon response is also much larger than the background electrical noise in the rod, as it must be to produce the rod’s high sensitivity to dim light. Cones respond similarly to single photons, but they are inherently noisier and their response is only ~ 1 50 the size of that in the rod. Cone responses do not saturate, even at the brightest levels of natural light. Cones also respond faster than rods.

Rhodopsin is a G protein–coupled “receptor” for light

RHODOPSIN

Disk interior N

2 3

1

6

7

Cytosol Retinal B

5

4

Attachment site for retinal

C

RETINAL 11-cis retinal H3C CH3 H

CH3 H H

H CH3

H

H H3C

Light

C

O

All-trans retinal H3C CH3 H CH3 H

H

O

CH3 C

H CH3 C

H

H

H

H

VISUAL TRANSDUCTION

Visual pigment (rhodopsin)

Light

Disk interior

Cytosol

γ

Disk

Disk membrane

Phosphodiesterase

α

β

α

Transducin

GMP cG

Ca2+

Extracellular space

P M

Guanylyl cyclase cG

MP K+

P

Rod outer segment membrane

M cG

How can a single photon stop the flow of 1 million Na+ ions across the membrane of a rod cell? The process begins when the photon is absorbed by rhodopsin, the light receptor molecule. Rhodopsin is one of the most tightly packed proteins in the body, with a density of ~30,000 molecules per square micrometer in the disk membranes. Thus, the packing ratio is 1 protein molecule for every 60 lipid molecules! One rod contains ~109 rhodopsin molecules. This staggering density ensures an optimized capture rate for photons passing through a photoreceptor. Even so, only ~10% of the light entering the eye is used by the receptors. The rest is either absorbed by the optical components of the eye or passes between or through the receptors. Rhodopsin has two key components: retinal and the protein opsin. Retinal is the aldehyde of vitamin A, or retinol (~500 Da). Opsin is a single polypeptide (~41 kDa) with seven membranespanning segments (Fig. 15-12A). It is a member of the superfamily of GPCRs (see pp. 51–52) that includes many neurotransmitter receptors as well as the olfactory receptor molecules. To be transduced, photons are actually absorbed by retinal, which is responsible for rhodopsin’s color. The tail

A

367

NCKX1 4 Na+

Na+ Ca2+ Nonselective cation channel

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SECTION III  •  The Nervous System

of retinal can twist into a variety of geometric configurations, one of which is a kinked and unstable version called 11-cis retinal (see Fig. 15-12B). The cis form sits within a pocket of the opsin (comparable to the ligand-binding site of other GPCRs) and is covalently bound to it. However, because of its instability, the cis form can exist only in the dark. If 11-cis retinal absorbs a photon, it isomerizes within 1 ps to a straighter and more stable version called all-trans retinal. This isomerization in turn triggers a series of conformational changes in the opsin that lead to a form called metarhodopsin II, which can activate an attached molecule called transducin. Transducin carries the signal forward in the cascade and causes a reduction in Na+ conductance. Soon after isomerization, all-trans retinal and opsin separate in a process called bleaching; this separation causes the color to change from the rosy red of rhodopsin (rhodon is Greek for the color “rose”) to the pale yellow of opsin. The photoreceptor cell converts all-trans retinal to retinol (vitamin A), which then translocates to the pigment epithelium and becomes 11-cis retinal. This compound makes its way back to the outer segment, where it recombines with opsin. This cycle of rhodopsin regeneration takes a few minutes. Transducin is so named because it transduces the lightactivated signal from rhodopsin into the photoreceptor membrane’s response (see Fig. 15-12C). Transducin was the first of the large family of GTP-binding proteins (G proteins; see p. 52) to be identified, and its amino-acid sequence is very similar to that of other G proteins (see Table 3-2). When it is activated by metarhodopsin, the α subunit of transducin exchanges a bound GDP for a GTP and then diffuses within the plane of the membrane to stimulate a phosphodiesterase that hydrolyzes cGMP to 5′-guanylate monophosphate. cGMP is the diffusible second messenger that links the light-activated events of the disk membranes to the electrical events of the outer membrane. A key discovery by Fesenko and colleagues in 1985 showed that the “light-sensitive” cation channel of rods is actually a cGMP-gated cation channel (see pp. 169–172). This CNG channel was the first of its kind to be discovered (we have already discussed a similar channel in olfactory receptors). In the dark, a constitutively active guanylyl cyclase that synthesizes cGMP from GTP keeps cGMP levels high within the photoreceptor cytoplasm. This high [cGMP]i causes the cGMP-gated cation channels to spend much of their time open and accounts for the dark current (see Fig. 15-11C). Because light stimulates the phosphodiesterase and thus decreases [cGMP]i, light reduces the number of open cGMP-gated cation channels and thus reduces the dark current. The photoreceptor then hyperpolarizes, transmitter release falls, and a visual signal is passed to retinal neurons. Strong amplification occurs along the phototransduction pathway. The absorption of 1 photon activates 1 metarhodopsin molecule, which can activate ~700 transducin molecules within ~100 ms. These transducin molecules activate phosphodiesterase, which increases the rate of cGMP hydrolysis by ~100-fold. One photon leads to the hydrolysis of ~1400 cGMP molecules by the peak of the response, thus reducing [cGMP] by ~8% in the cytoplasm around the activated disk. This decrease in [cGMP]i closes ~230 of the 11,000 cGMP-gated channels that are open in the dark. As a result, the dark current falls by ~2%.

The cGMP-gated channel has additional interesting properties. It responds within milliseconds when [cGMP]i rises, and it does not desensitize in response to cGMP. The concentration-response curve is very steep at low [cGMP]i because opening requires the simultaneous binding of three cGMP molecules. Thus, the channel has switch-like behavior at physiological levels of cGMP. Ion conductance through the channel also has steep voltage dependence because Ca2+ and Mg2+ strongly block the channel (as well as permeate it) within its physiological voltage range. This open-channel block (see Fig. 7-20D) makes the normal single-channel conductance very small, among the smallest of any ion channel; the open channel normally carries a current of only 3 × 10−15 A (3 fA)! The currents of ion channels are inherently “noisy” as they flicker open and closed. However, the 11,000 channels—each with currents of 3 fA—summate to a rather noise-free dark current of 11,000 channels × 3 fA per channel = 33 pA. In contrast, if 11 channels—each with currents of 3 pA—carried the dark current of 33 pA, the 2% change in this signal (0.66 pA) would be smaller than the noise produced by the opening and closing of a single channel (3 pA). Thus, the small channels give the photoreceptor a high signal-to-noise ratio. The [cGMP]i in the photoreceptor cell represents a dynamic balance between the synthesis of cGMP by guanylyl cyclase and the breakdown of cGMP by phosphodiesterase. Ca2+, which enters through the relatively nonselective cGMPgated channel, synergistically inhibits the guanylyl cyclase and stimulates the phosphodiesterase. These Ca2+ sensitivities set up a negative-feedback system. In the dark, the incoming Ca2+ prevents runaway increases in [cGMP]i. In the light, the ensuing decrease in [Ca2+]i relieves the inhibition on guanylyl cyclase, inhibits the phosphodiesterase, increases [cGMP]i, and thus poises the system for channel reopening. When a light stimulus terminates, the activated forms of each component of the transduction cascade must be inactivated. One mechanism of this termination process appears to involve the channels themselves. As described in the preceding paragraph, closure of the cGMP-gated channels in the light leads to a fall in [Ca2+]i, which helps replenish cGMP and facilitates channel reopening. Two additional mechanisms involve the proteins rhodopsin kinase and arrestin. Rhodopsin kinase phosphorylates light-activated rhodopsin and allows it to be recognized by arrestin. Arrestin, an abundant cytosolic protein, binds to the phosphorylated light-activated rhodopsin and completely terminates its ability to activate transducin.

The eye uses a variety of mechanisms to adapt to a wide range of light levels The human eye can operate effectively over a 1010-fold range of light intensities, which is the equivalent of going from almost total darkness to bright sunlight on snow. However, moving from a bright to a dark environment, or vice versa, requires time for adaptation before the eye can respond optimally. Adaptation is mediated by several mechanisms. One mechanism mentioned above is regulation of the size of the pupil by the iris, which can change light sensitivity by ~16fold. That still leaves the vast majority of the range to account

Chapter 15  •  Sensory Transduction

Cones adapt to low light quickly… 8

…but still have a relatively high threshold. Thus, by themselves, cones do not provide effective dark-adapted vision.

7 6 Log relative 5 light threshold 4 3 0

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However, rods adapt to much dimmer light, although they do so more slowly. Figure 15-13  Effect of dark adaptation on the visual threshold. The subject was exposed to light at a level of 1600 millilumens and then switched to the dark. The graph is a plot of the time course of the subject’s relative threshold (on a log scale) for detecting a light stimulus. (Data from Hecht S, Shlaer S, Smith EL, et al: The visual functions of the complete color blind. J Gen Physiol 31:459–472, 1948.)

for. During dark adaptation, two additional mechanisms with very different time courses are evident, as we can see from a test of the detection threshold for the human eye (Fig. 15-13). The first phase of adaptation is finished within ~10 minutes and is a property of the cones; the second takes at least 30 minutes and is attributed to the rods. A fully darkadapted retina, relying on rods, can have a light threshold that is as much as 15,000 times lower than a retina relying on cones. In essence, then, the human eye has two retinas in one, a rod retina for low light levels and a cone retina for high light levels. These two systems can operate at the same time; when dark adapted, the rods can respond to the lowest light levels, but cones are available to respond when brighter stimuli appear. The rapid and slow phases of adaptation that are discussed in the preceding paragraph have both neural and photoreceptor mechanisms. The neural mechanisms are relatively fast, operate at relatively low ambient light levels, and involve multiple mechanisms within the neuronal network of the retina. The photoreceptor mechanisms involve some of the processes that are described in the previous section. Thus, in bright sunlight, rods become ineffective because most of their rhodopsin remains inactivated, or bleached. cGMP-gated channels are closed and thus Ca2+ entry is blocked, so [Ca2+]i falls to a few nanomolar as Ca2+ is removed by the Na+/(Ca2+-K+) exchanger NCKX1 (SLC24A1; see Table 5-4). After returning to darkness, the rods slowly regenerate rhodopsin and become sensitive once again. However, a component of the cGMP system also regulates photoreceptor sensitivity. In the dark, when baseline [cGMP]i is relatively high, substantial amounts of Ca2+ enter through cGMP-gated channels. The resultant high [Ca2+]i (several

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hundred nanomolars) inhibits guanylyl cyclase and stimulates phosphodiesterase, thereby preventing [cGMP]i from rising too high. Conversely, when background light levels are high, this same feedback system causes baseline [cGMP]i to remain high so that [cGMP]i can fall in response to further increases in light levels. Otherwise, the signal-transduction system would become saturated. In other words, the photoreceptor adapts to the increased back­ground light intensity and remains responsive to small changes. Additional adaptation mechanisms regulate the sensitivity of rhodopsin, guanylyl cyclase, and the cGMP-gated channel. Clearly, adaptation involves an intricate network of molecular interactions.

Color vision depends on the different spectral sensitivities of the three types of cones The human eye responds only to a small region of the electromagnetic spectrum (see Fig. 15-5), but within it, we are exquisitely sensitive to the light’s wavelength. We see assorted colors in a daytime panorama because objects absorb some wavelengths while reflecting, refracting, or transmitting others. Different sources of light may also affect the colors of a scene; the light from tungsten bulbs is reddish, whereas that of fluorescent bulbs is bluish. Research on color vision has a long history. In 1801, Thomas Young first outlined the trichromatic theory of color vision, which was championed later in the 19th century by Hermann von Helmholtz. These investigators found that they could reproduce a particular sample hue by mixing the correct intensities of three lights with the primary hues blue, green, and red. They proposed that color vision, with its wide range of distinct, perceived hues, is based on only three different pigments in the eye, each absorbing a different range of wavelengths. Microspectrophotometry of single cones in 1964 amply confirmed this scheme. Thus, although analysis of color by the human brain is sophisticated and complex, it all derives from the responses of only three types of photo­ pigments in cones. Our sensitivity to the wavelength of light depends on the retina’s state of adaptation. When it is dark adapted (also called scotopic conditions), the spectral sensitivity curve for human vision is shifted toward shorter wavelengths compared with the curve obtained after light adaptation (photopic conditions; Fig. 15-14A). The absolute sensitivity to light can also be several orders of magnitude higher under scotopic conditions (see Fig. 15-13). The primary reason for the difference in these curves is that rods are doing the transduction of dim light under dark-adapted conditions, whereas cones transduce in the light-adapted eye. As we would predict, the spectral sensitivity curve for scotopic vision is quite similar to the absorption spectrum of the rods’ rhodopsin, with a peak at 500 nm. The spectral sensitivity of the light-adapted eye depends on the photopigments in the cones. Humans have three different kinds of cones, and each expresses a photopigment with a different absorbance spectrum. The peaks of their absorbance curves fall at ~420, 530, and 560 nm, which correspond to the violet, yellow-green, and yellow-red regions of the spectrum (see Fig. 15-14B). The three cones and their pigments were historically called blue, green, and red,

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SPECTRAL SENSITIVITY UNDER DARK- AND LIGHT-ADAPTED CONDITIONS Under conditions of dark adaptation, sensitivity to light depends mainly on rods… 500

...whereas under conditions of light adaptation, sensitivity depends mainly on cones.

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Figure 15-14  Sensitivity of vision and photoreceptors at different wavelengths of light. A, The graph shows the results of a psychophysical experiment. Under dark-adapted (scotopic) conditions, the human eye is maximally sensitive at ~500 nm. Under light-adapted (photopic) conditions, the eye is maximally sensitive at ~560 nm. B, The spectral sensitivity of rods (obtained with a spectrophotometer) peaks at ~500 nm; that of the three types of cones peaks at ~420 nm for the S (blue) cone, ~530 nm for the M (green) cone, and ~560 nm for the L (red) cone; and that of melanopsin peaks at ~475 nm. Each absorbance spectrum has been normalized to its peak sensitivity. (A, Data from Knowles A: The biochemical aspects of vision. In Barlow HB, Mollon JD [eds]: The Senses. Cambridge, UK, Cambridge University Press, 1982, pp 82–101; B, rhodopsin data from Dartnell HJ, Bowmaker JK, Mollon JD: Microspectrophotometry of human photoreceptors. In Mollon JD, Sharpe LT [eds]: Colour Vision. London, Academic Press, 1983, pp 69–80; melanopsin data from Matsuyama T, Yamashita T, Imamoto Y, Shichida Y: Photochemical properties of mammalian melanopsin. Biochemistry 51:5454–5462, 2012.)

respectively. They are now more commonly called S, M, and L (for short, medium, and long wavelengths); we use this terminology here. Because the absolute sensitivity of the short-wavelength cone is only one tenth that of the other two, the spectral sensitivity of photopic human vision is dominated by the two longer-wavelength cones (compare the spectral sensitivity functions in Fig. 15-14A with the absorbance spectra of the cones in Fig. 15-14B). Single cones do not encode the wavelength of a light stimulus. If a cone responds to a photon, it generates the same response regardless of the wavelength of that photon. A glance at Figure 15-14B shows that each type of cone pigment can absorb a wide range of wavelengths. The pigment in a cone is more likely to absorb photons when their wavelength is at its peak absorbance, but light hitting the cone on the fringe of its absorbance range can still generate a large response if the light’s intensity is sufficiently high. This property of response univariance is the reason that vision in an eye with only one functioning pigment (e.g., scotopic vision using only rods) can only be monochromatic. With a single pigment system, the distinction between different colors and between differences in intensity is confounded. Two different cones (as in most New World monkeys), each with a different but overlapping range of wavelength sensitivities, remove much of the ambiguity in encoding the wavelength of light stimuli. With three overlapping pigments (as in Old World monkeys and humans), light of a single wavelength stimulates each of the three cones to different degrees, and light of any other wavelength stimulates these cones with a distinctly different pattern. Because the nervous system can compare the relative stimulation of the three cone types to decode the wavelength, it can also distinguish changes in the intensity (luminance) of the light from changes in its wavelength. Color capabilities are not constant across the retina. The use of multiple cones is not compatible with fine spatial discrimination because of wavelength-dependent differences in the eye’s ability to focus light, known as chromatic aberration, and because very small objects may stimulate only single cones. The fovea has only M and L cones, which limits its color discrimination in comparison to the peripheral portions of the retina but leaves it best adapted to discriminate fine spatial detail (Box 15-1). The four different human visual pigments have a similar structure. The presence of retinal and the mechanisms of its photoisomerization are essentially identical in each. The main difference is the primary structure of the attached protein, the opsin. M and L opsins share 96% of their amino acids. Pairwise comparisons among the other opsins show only 44% or lower sequence similarity, however. Apparently, the different amino-acid structures of the opsins affect their charge distributions in the region of the 11-cis retinal and shift its absorption spectrum to give the different pigments their specific spectral sensitivities.

The ipRGCs have unique properties and functions The ipRGC, the third retinal photoreceptor, differs from rods and cones in fundamental ways. First, instead of expressing rhodopsin or cone opsins, ipRGCs use a related but unique light-sensitive protein called melanopsin that is most

Chapter 15  •  Sensory Transduction

BOX 15-1  Inherited Defects in Color Vision

I

nherited defects in color vision are relatively common, and many are caused by mutations in visual pigment genes. For example, 8% of white males and 1% of white females have some defect in their L or M pigments caused by X-linked recessive mutations. A single abnormal pigment can lead to either dichromacy (the absence of one functional pigment) or anomalous trichromacy (a shift in the absorption spectrum of one pigment relative to normal), often with a consequent inability to distinguish certain colors. Jeremy Nathans and colleagues found that men have only one copy of the L pigment gene; but located right next to it on the X chromosome, they may have one to three copies of the M pigment gene. He proposed that homologous recombination could account for the gene duplication, loss of a gene, or production of the hybrid L-M genes that occur in red-green color blindness. Hybrid L-M pigments have spectral properties intermediate between those of the two normal pigments, probably because their opsins possess a combination of the traits of the two normal pigments. Lack of two of the three functional cone pigments leads to monochromacy. The number of people who have such true color blindness is very small, 100,000 Hz. A continuous pure tone (see p. 376) produces a wave that travels along the basilar membrane and has different amplitudes at different points along the base-apex axis (Fig. 15-24A). Increases in sound amplitude cause an increase in the rate of action potentials in auditory nerve axons—rate coding.  N15-14  The frequency of the sound determines where along the cochlea the cochlear membranes vibrate most—high frequencies at one end and low at the other— and thus which hair cells are stimulated. This selectivity is the basis for place coding in the auditory system; that is, the frequency selectivity of a hair cell depends mainly on its longitudinal position along the cochlear membranes. The cochlea is essentially a spectral analyzer that evaluates a complex sound according to its pure tonal components, with each pure tone stimulating a specific region of the cochlea.

Chapter 15  •  Sensory Transduction

N15-12  Otoacoustic Emissions

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N15-13  Auditory Range

Contributed by Philine Wangemann

Contributed by Philine Wangemann

Amplification by the outer hair cells evokes vibrations of the basilar membrane that travel through the middle ear, set the tympanic membrane in motion, and produce a sound that comes out of the ear canal. Clinically most relevant are transient otoacoustic emissions and distortion-product otoacoustic emissions. Transient otoacoustic emissions are sounds that are detected in the ear canal milliseconds after a very brief stimulus. Amplification by the outer hair cells is nonlinear, which means that the cochlea produces and emits distortion products. Distortion products in response to two pure tones at nearby frequencies (f1 and f2) relate to these stimuli by simple math, for example, 2f1 − f2 or 2f2 − f1. Transient otoacoustic emissions and distortion-product otoacoustic emissions provide useful clues for the evaluation of outer hair cell function.

The auditory frequency range of the human ear is well adapted to the perception of speech, which encompasses frequencies between 60 and 12,000 Hz. We can comfortably hear sounds with amplitudes from 0 to 120 dB SPL. Higher sound pres­ sure levels cause pain and destruction of the ear  N15-9.  Typical sound pressure levels are 20 dB SPL for whispering, 60 dB SPL for normal conversation, 80 dB SPL for loud traffic, and 120 dB SPL for a nearby train horn.

N15-14  Rate Coding Contributed by Philine Wangemann Amplitude information is transmitted by rate coding. Rate coding refers to the principle that increases in sound amplitude result in an increase the rate of action potentials. Cooperation between neurons is required to code the full range of sound pressure levels from 0 to 120 dB SPL.

Chapter 15  •  Sensory Transduction

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EFFECT OF FREQUENCY ON ENVELOPE POSITION

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Apex Helicotrema

…than its apical end.

Figure 15-24  Waves along the basilar membrane of the cochlea. A, As a wave generated by a sound of a single frequency travels along the basilar membrane, its amplitude changes. The green and yellow curves represent a sample wave at two different times. The upper and lower broken lines (i.e., the envelope) encompass all maximum amplitudes of all waves, at all points in time. Thus, a wave can never escape the envelope. The figure exaggerates the amplitudes of the traveling waves ~1 million-fold. B, For a pure tone of 10,000 Hz, the envelope is confined to a short region of the basilar membrane near the stapes. For pure tones of 4000 Hz and 200 Hz, the widest part of the envelope moves closer to the helicotrema. C, The cochlea narrows in diameter from base to apex, whereas the basilar membrane tapers in the opposite direction.

Using optical methods to study cadaver ears, Georg von Békésy found that sounds of a particular frequency generate relatively localized waves in the basilar membrane and that the envelope of these waves changes position according to the frequency of the sound (see Fig. 15-24B). Low frequencies generate their maximal amplitudes near the apex. As sound frequency increases, the envelope shifts progressively toward the basal end (i.e., near the oval and round windows). For his work, von Békésy  N15-15  received the 1961 Nobel Prize in Physiology or Medicine. Two properties of the basilar membrane underlie the low-apical to high-basal gradient of resonance: taper and stiffness (see Fig. 15-24C). If we could unwind the cochlea

and stretch it straight, we would see that it tapers from base to apex. The basilar membrane tapers in the opposite direction—wider at the apex, narrower at the base. More important, the narrow basal end is ~100-fold stiffer than its wide and floppy apical end. Thus, the basilar membrane resembles a harp. At one end—the base, near the oval and round windows—it has short, taut strings that vibrate at high frequencies. At the other end—the apex—it has longer, looser strings that vibrate at low frequencies. Although von Békésy’s experiments were illuminating, they were also paradoxical. A variety of experimental data suggested that the tuning of living hair cells is considerably sharper than the broad envelopes of von Békésy’s traveling

Chapter 15  •  Sensory Transduction

N15-15  Georg von Békésy For more information about Georg von Békésy and the work that led to his Nobel Prize, visit http://www.nobel.se/medicine/ laureates/1961/index.html (accessed December 2014).

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Figure 15-25  Peak movement of the basilar membrane. The graph illustrates the displacement of the basilar membrane in response to a pure tone as a function of distance along the base-to-apex axis. The dashed line indicates the displacement threshold for triggering of an electrical response. (Data from Ashmore JF: Mammalian hearing and the cellular mechanisms of the cochlear amplifier. In Corey DP, Roper SD [eds]: Sensory Transduction. New York, Rockefeller University Press, 1992, pp 396–412.)

waves on the basilar membrane could possibly produce.  N15-16  Recordings from primary auditory nerve cells are also very sharp, implying that this tuning must occur within the cochlea, not in the CNS. Some enhancement of tuning comes from the structure of the inner hair cells themselves. Those near the base have shorter, stiffer stereovilli, which makes them resonate to higher frequencies than possible with the longer, floppier stereovilli on cells near the apex. The blue curve in Figure 15-25 approximates von Békésy’s envelope of traveling waves for a passive basilar membrane from cadavers. It is important to note that von Békésy used unnaturally loud sounds. With reasonable sound levels, the maximum passive displacement of the basilar membrane would be slightly more than 0.1 nm. This distance is less than the pore diameter of an ion channel and also less than the threshold (0.3 to 0.4 nm) for an electrical response from a hair cell. However, measurements from the basilar membrane in living animals (the orange curve in Fig. 15-25) by very sensitive methods show that movements of the basilar membrane are much more localized and much larger than predicted by von Békésy. The maximal physiological displacement is ~20-fold greater than threshold and ~40-fold greater than that predicted by the passive von Békésy model. Moreover, the physiological displacement decays sharply on either side of the peak, >100-fold within ~0.5 mm (recall that the human basilar membrane has a total length of >30 mm). Both the extremely large physiological excursions of the basilar membrane and the exquisitely sharp tuning of the cochlea depend on the cochlear amplifier (see p. 380). Indeed, selectively damaging outer hair cells—with large doses of certain antibiotics, for example—considerably dulls the sharpness of cochlear tuning and dramatically reduces the amplification. The brain can control the tuning of hair cells. Axons that arise in the superior olivary complex in the brainstem

BOX 15-2  Cochlear Implants

T

he most common cause of human deafness is damage to the hair cells of the cochlea.  N15-18  This damage can be caused by genetic factors, a variety of drugs (e.g., some antibiotics, including quinine), chronic exposure to excessively loud sounds, and other types of disease. Even when all hair cells have been destroyed, if the auditory nerve is intact, it is often possible to restore substantial hearing with a cochlear implant. A cochlear implant  N15-19  is essentially an electronic cochlea. Most of the system resides outside the body. The user wears a headpiece with a microphone, which is connected to a small, battery-powered digital speech processor. This processor sends signals to a miniature radio transmitter next to the scalp, which transmits digitally encoded signals— no wires penetrate the skin—to a receiver/decoder that is surgically implanted in the mastoid bone behind the ear. A very thin and flexible set of wires carries the signals through a tiny hole into the basal end of the cochlea, where an array of 8 to 22 electrodes lies adjacent to the auditory nerve endings (where healthy hair cells would normally be) along the cochlea. Each electrode activates a small portion of the auditory nerve axons. The cochlear implant exploits the tonotopic arrangement of auditory nerve fibers. By stimulating near the base of the cochlea, it is possible to trigger a perception of high-frequency sounds; stimulation toward the apex evokes low-frequency sounds. The efficacy of the implant can be extraordinary. Users require training of a few months or longer, and in many cases, they achieve very good comprehension of spoken speech, even as it comes across on a telephone. As the technology and safety of cochlear implants have improved, so has their popularity. By 2010, >200,000 people were using cochlear implants worldwide, ~80,000 of them infants and children. The best candidates for cochlear implants are young children (optimally as young as 1 year) and older children or adults whose deafness was acquired after they learned some speech. Children older than ~7 years and adults whose deafness preceded any experience with speech generally do not fare as well with cochlear implants. The systems of sensory neurons in the brain, including the auditory system, need to experience normal inputs at a young age to develop properly. When the auditory system is deprived of sounds early in life, it can never develop completely normal function even if sensory inputs are restored during adulthood.

synapse mainly on the outer hair cells and, sparsely, on the afferent axons that innervate the inner hair cells.  N15-17  Stimulation of these olivocochlear efferent fibers suppresses the responsiveness of the cochlea to sound and is thought to provide auditory focus by suppressing responsiveness to unwanted sounds—allowing us to hear better in noisy environments (Box 15-2). The main efferent neurotransmitter is acetylcholine (ACh), which activates ionotropic ACh receptors (see pp. 206–207)—nonselective cation channels—and triggers an entry of Ca2+. The influx of Ca2+ activates Ca2+-activated K+ channels, causing a hyperpolarization—effectively an inhibitory postsynaptic potential—that suppresses the electromotility of outer hair cells and action potentials in afferent dendrites. Thus, the efferent axons allow the brain to control the gain of the inner ear.

Chapter 15  •  Sensory Transduction

N15-16  Sharpening of Cochlear Tuning

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N15-17  Central Processing of Auditory Patterns

Contributed by Philine Wangemann Outer hair cells express the motor protein prestin along the lateral cell wall, which is responsible for electromotility. Transduction-mediated depolarization of outer hair cells during upward movements of the basilar membrane causes prestin to contract; this shortens the hair cell body and increases the upward movement of the basilar membrane (see Fig. 15-23). Conversely, hyperpolarization during downward movements of the basilar membrane expands prestin, elongates the outer hair cells, and enlarges the downward movement of the basilar membrane. This electromotility, which amplifies and sharpens the peak of the sound-induced traveling wave, is a prerequisite for the sensitivity of hearing and the ability to sharply discriminate frequencies (see Fig. 15-25).

N15-18  Conductive Hearing Loss

Contributed by Philine Wangemann Auditory patterns are analyzed in the medial geniculate and the auditory cortex. Neurons in these areas are often highly specialized and respond only to a specific frequency and intensity pattern. Interpretation of sound elements requires cortical input beyond the auditory cortex. Central processing is clinically evaluated by auditory brainstem recordings. The coordinated firing of groups of neurons in responses to brief stimuli (clicks or tone pips) produces transient voltage fluctuations that can be detected with surface electrodes. Distinctive voltage fluctuations occur 2 to 12 ms after the stimulus and can be associated with neuronal activity in the auditory pathway including the cochlear nerve, cochlear nucleus, and superior olivary complex.

N15-19  Cochlear Implants

Contributed by Philine Wangemann

Contributed by Emile Boulpaep and Walter Boron

Conductive hearing losses are disorders that compromise the conduction of sound through the external ear, tympanic membrane, or middle ear. Pressure differences across the tympanic membrane (eardrum) can rupture it. Accumulations of fluid in the middle ear can lead to conductive hearing losses that are seen particularly often in children with middle ear infections (otitis media). With proper treatment, the hearing loss due to otitis media is usually self-limited. Otosclerosis, which stiffens the ossicular chain, is another common cause of conductive hearing loss. Treatments for conductive hearing loss encompass a palette of devices including hearing aids and middle ear implants. Hearing aids amplify the sound in the external ear canal. Prosthetic devices can replace the tympanic membrane and the ossicular chain. Middle ear implants are clamped onto the incus and enhance the vibrations of the ossicular chain.

See the following websites for more information on cochlear implants: http://www.nidcd.nih.gov/health/hearing/pages/coch.aspx http://ecs.utdallas.edu/loizou/cimplants/tutorial/

Chapter 15  •  Sensory Transduction

SOMATIC SENSORY RECEPTORS, PROPRIOCEPTION, AND PAIN

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Somatic sensation is the most widespread and diverse of the body’s sensory systems (soma means “body” in Greek). Its receptors are distributed throughout the body instead of being condensed into small and specialized sensory surfaces, as most other sensory systems are arranged. Somatosensory receptors cover the skin, subcutaneous tissue, skeletal muscles, bones and joints, major internal organs, epithelia, and cardiovascular system. These receptors also vary widely in their specificity. The body has mechanoreceptors to transduce pressure, stretch, vibration, and tissue damage; thermoreceptors to gauge temperature; and chemoreceptors to sense a variety of substances. Somatic sensation (or somesthesia) is usually considered to be a combination of at least four sensory modalities: the senses of touch, temperature, body position (proprioception), and pain (nociception).

Mechanoreceptors, which are sensitive to physical distortion such as bending or stretching, account for many of the somatic sensory receptors. They exist throughout our bodies and monitor the following: physical contact with the skin, blood pressure in the heart and vessels, stretching of the gut and bladder, and pressure on the teeth. The transduction site of these mechanoreceptors is one or more unmyelinated axon branches. Our progress in understanding the molecular nature of mechanosensory transduction has been relatively slow. Similar to the transduction process in hair cells, that in cutaneous mechanoreceptive nerve endings probably involves the gating of ion channels. Some of these channels belong to the TRP superfamily (see Table 6-2, family No. 5). Thermoreceptors respond best to changes in temperature, whereas chemoreceptors are sensitive to various kinds of chemical alterations. In the next three sections, we discuss mechanoreceptors, thermoreceptors, and chemoreceptors that are located in the skin.

A variety of sensory endings in the skin transduce mechanical, thermal, and chemical stimuli

Mechanoreceptors in the skin provide sensitivity to specific stimuli such as vibration and steady pressure

To meet a wide array of sensory demands, many kinds of specialized receptors are required. Somatic sensory receptors range from simple bare nerve endings to complex combinations of nerve, muscle, connective tissue, and supporting cells. As we have seen, the other major sensory systems have only one type of sensory receptor or a set of very similar subtypes.

Skin protects us from our environment by preventing evaporation of body fluids, invasion by microbes, abrasion, and damage from sunlight. However, skin also provides our most direct contact with the world. The two major types of mammalian skin are hairy and glabrous. Glabrous skin (or hairless skin) is found on the palms of our hands and fingertips and on the soles of our feet and pads of our toes (Fig. 15-26A).

A

B

GLABROUS (HAIRLESS) SKIN

PACINI’S CORPUSCLE Stimulus

Intact corpuscle

Papillary ridges

Epidermis Most intense stimulus

300

Dermis

Extracellular recording of 200 receptor potential 100 (µV)

Least intense stimulus

Pacini’s corpuscle is a rapidly adapting receptor.

0 Ruffini’s endings

Subcutaneous tissue

5 ms

Brief stimulus Subpapillary plexus

Free nerve endings

Sweat gland

Meissner’s Pacini’s corpuscle corpuscle

Merkel’s disk

Figure 15-26  Sensors in the skin. (Data from Mendelson M, Loewenstein WR: Mechanisms of receptor adaptation. Science 144:554–555, 1964.)

100 ms

Long stimulus

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Hairy skin makes up most of the rest and differs widely in its hairiness. Both types of skin have an outer layer, the epidermis, and an inner layer, the dermis, and sensory receptors innervate both. The receptors in the skin are sensitive to many types of stimuli and respond when the skin is vibrated, pressed, pricked, or stroked, or when its hairs are bent or pulled. These are quite different kinds of mechanical energy, yet we can feel them all and easily tell them apart. Skin also has exquisite sensitivity; for example, we can reliably feel a dot only 0.006 mm high and 0.04 mm across when it is stroked across a fingertip. The standard Braille dot is 167 times higher! The sensory endings in the skin take many shapes, and most of them are named after the 19th-century European histologists who observed them and made them popular. The largest and best-studied mechanoreceptor is Pacini’s corpuscle, which is up to 2 mm long and almost 1 mm in diameter (see Fig. 15-26B). Pacini’s corpuscle is located in the subcutaneous tissue of both glabrous and hairy skin. It has an ovoid capsule with 20 to 70 onion-like, concentric layers of connective tissue and a nerve terminal in the middle. The capsule is responsible for the rapidly adapting response of the Pacini’s corpuscle. When the capsule is compressed, energy is transferred to the nerve terminal, its membrane is deformed, and mechanosensitive channels open. Current flowing through the channels generates a depolarizing receptor potential that, if large enough, causes the axon to fire an action potential (see Fig. 15-26B, left panel). However, the capsule layers are slick, with viscous fluid between them. If the stimulus pressure is maintained, the layers slip past one another and transfer the stimulus energy away so that the underlying axon terminal is no longer deformed and the receptor potential dissipates (see Fig. 15-26B, right panel). When pressure is released, the events reverse themselves and the terminal is depolarized again. In this way, the non-neural covering of Pacini’s corpuscle specializes the corpuscle for sensing of vibrations and makes it almost unresponsive to steady pressure. Pacini’s corpuscle is most sensitive to vibrations of 200 to 300 Hz, and its threshold increases dramatically below 50 Hz and above ~500 Hz. The sensation evoked by stimulation of Pacini’s corpuscle is a poorly localized humming feeling. Werner Loewenstein and colleagues in the 1960s showed the importance of the Pacini corpuscle’s capsule to its frequency sensitivity. With fine microdissection, they were able to strip away the capsule from single corpuscles. They found that the resultant naked nerve terminal is much less sensitive to vibrating stimuli and much more sensitive to steady pressure. Clearly, the capsule modifies the sensitivity of the bare mechanoreceptive axon. The encapsulated Pacini corpuscle is an example of a rapidly adapting sensor, whereas the decapsulated nerve ending behaves like a slowly adapting sensor. Several other types of encapsulated mechanoreceptors are located in the dermis, but none has been studied as well as Pacini’s corpuscle. Meissner’s corpuscles (see Fig. 15-26A) are located in the ridges of glabrous skin and are about one tenth the size of Pacini’s corpuscles. They are rapidly adapting, although less so than Pacini’s corpuscles. Ruffini’s corpuscles resemble diminutive Pacini’s corpuscles and, like Pacini’s corpuscles, occur in the subcutaneous tissue of both

hairy and glabrous skin. Their preferred stimuli might be called “fluttering” vibrations. As relatively slowly adapting receptors, they respond best to low frequencies. Merkel’s disks are also slowly adapting receptors made from a flattened, non-neural epithelial cell that synapses on a nerve terminal. They lie at the border of the dermis and epidermis of glabrous skin. It is not clear whether it is the nerve terminal or epithelial cell that is mechanosensitive. The nerve terminals of Krause’s end bulbs appear knotted. They innervate the border areas of dry skin and mucous membranes (e.g., around the lips and external genitalia) and are probably rapidly adapting mechanoreceptors. The receptive fields of different types of skin receptors vary greatly in size. Pacini’s corpuscles have extremely broad receptive fields (Fig. 15-27A), whereas those of Meissner’s corpuscles (see Fig. 15-27B) and Merkel’s disks are very small. The last two seem to be responsible for the ability of the fingertips to make very fine tactile discriminations. Small receptive fields are an important factor in achieving high spatial resolution. Resolution varies widely, a fact easily demonstrated by measuring the skin’s two-point discrimination. Bend a paper clip into a U shape. Vary the distance between the tips and test how easily you can distinguish the touch of one tip versus two on your palm, your fingertips, your lips, your back, and your foot. To avoid bias, a colleague—rather than you—should apply the stimulus. Compare the results with standardized data (see Fig. 15-27C). The identities of somatosensory transduction molecules remain elusive. A variety of TRP channel subtypes transduce mechanical stimuli in invertebrate species (e.g., Drosophila, Caenorhabditis elegans). In mammals, rapidly adapting ion channels are associated with receptors for light touch, and several of the TRPC channels appear to be involved in sensitivity to light touch in mice. A non-TRP protein named Piezo2 is associated with rapidly adapting mechanosensory currents in mouse sensory neurons, and knocking down the expression of Piezo2 expression causes deficits in touch. Other mechanosensory channels are expressed in some sensory neurons, including TRPA1 and TRPV4, two-pore potassium channels (KCNKs), and degenerin/epithelial sodium channels (especially ASIC1 to ASIC3 and their accessory proteins), but their roles in mammalian mechanosensation are still controversial. One reason it is difficult to identify mechanosensory channels is that they often need to be associated with other cellular components in order to be sensitive to mechanical stimuli. The mechanisms by which mechanical force is transferred from cells and their membranes to mechano­ sensitive channels are unclear. Ion channels may be physically coupled to either extracellular structures (e.g., collagen fibers) or cytoskeletal components (e.g., actin, microtubules) that transfer energy from deformation of the cell to the gating mechanism of the channel. Mechani­ cally gated ion channels of sensory neurons, including those requiring Piezo2, depend on the actin cytoskeleton. Some channels may be sensitive to stress, sheer, or curvature of the lipid bilayer itself and require no other types of anchoring proteins. Other channels may respond to mechanically triggered second messengers such as DAG (acting directly on the channel) or IP3 (acting indirectly via an IP3 receptor).

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Chapter 15  •  Sensory Transduction

A

RECEPTIVE FIELD OF PACINI’S CORPUSCLES

B RECEPTIVE FIELD OF MEISSNER’S CORPUSCLES

C

TWO-POINT DISCRIMINATION ACROSS THE SKIN 4 3 Fingers 2 1 Thumb Palm Forearm Forehead Cheek Nose Upper lip

Upper arm Shoulder Breast Back Belly

Thigh

Pacini’s corpuscles

Meissner’s corpuscles

Calf

Sole Toe 0 5 10 15 20 25 30 35 40 45 50 Two-point discrimination threshold (mm) Figure 15-27  Receptive fields and spatial discrimination of skin mechanoreceptors. A, Each of the two black dots indicates an area of maximal sensitivity of a single Pacini corpuscle. Each blue-green area is the receptive field of a corpuscle (i.e., the corpuscle responds when stimulus strength increases sufficiently anywhere within the area). B, Each dot represents the entire receptive field of a single Meissner corpuscle. Note that the fields are much smaller than in A. C, The horizontal bars represent the minimum distance at which two points can be perceived as distinct at various locations over the body. Spatial discrimination depends on both receptor density and receptive-field size. (A and B, Data from Vallbo AB, Johansson RS: Properties of cutaneous mechanoreceptors in the human hand related to touch sensation. Hum Neurobiol 3:3–14, 1984; C, data from Weinstein S: Intensive and extensive aspects of tactile sensitivity as a function of body part, sex and laterality. In Kenshalo DR [ed]: The Skin Senses. Springfield, IL, Charles C Thomas, 1968.)

Two things determine the sensitivity of spatial dis­ crimination in an area of skin. The first is the size of the receptors’ receptive fields—if they are small, the two tips of your paper clip are more likely to stimulate different sets of receptors. The second parameter that determines spatial discrimination is the density of the receptors in the skin. Indeed, two-point discrimination of the fingertips is better than that of the palm, even though their receptive fields are the same size. The key to finer discrimination in the fingertips is their higher density of receptors. Crowding more receptors into each square millimeter of fingertip has a second advantage: because the CNS receives more information per stimulus, it has a better chance of detecting very small stimuli. Although we rarely think about it, hair is a sensitive part of our somatic sensory system. For some animals, hairs are a major sensory system. Rodents whisk long facial vibrissae (hairs) and feel the texture, distance, and shape of their local environment. Hairs grow from follicles embedded in the skin, and each follicle is richly innervated by free

mechanoreceptive nerve endings that either wrap around it or run parallel to it. Bending of the hair causes deformation of the follicle and surrounding tissue, which stretches, bends, or flattens the nerve endings and increases or decreases their firing frequency. Various mechanoreceptors innervate hair follicles, and they may be either slowly or rapidly adapting.

Separate thermoreceptors detect warmth and cold Neurons are sensitive to changes in temperature, as are all of life’s chemical reactions. Neuronal temperature sensitivity has two consequences: first, neurons can measure temperature; but second, to work properly, most neural circuits need to be kept at a relatively stable temperature. Neurons of the mammalian CNS are especially vulnerable to temperature changes. Whereas skin tissue temperatures can range from 20°C to 40°C without harm or discomfort, brain temperature must be near 37°C to avoid serious dysfunction. The body has complex systems to control brain (i.e., body core)

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temperature tightly (see pp. 1198–1201). Even though all neurons are sensitive to temperature, not all neurons are ther­moreceptors. Because of specific membrane mechanisms, some neurons are extremely sensitive to temperature and seem to be adapted to the job of sensing it. Although many temperature-sensitive neurons are present in the skin, they are also clustered in the hypothalamus and the spinal cord (see pp. 1198–1199). The hypothalamic temperature sensors, like their cutaneous counterparts, are important for regulation of the physiological responses that maintain stable body temperature. Perceptions of temperature apparently reflect warmth and cold receptors located in the skin. Thermoreceptors, like mechanoreceptors, are not spread uniformly across the skin. When you map the skin’s sensitivity to temperature with a small cold or warm probe, you find spots ~1 mm across that are especially sensitive to either warmth or cold, but not to both. In addition, some areas of skin in between are relatively insensitive. The spatial dissociation of the hot and cold maps shows that they are separate submodalities, with separate receptors to encode each. Recordings from single sensory fibers have confirmed this conclusion. The responses of both warmth and cold thermoreceptors adapt during long stimuli, as many sensory receptors commonly do. Most cutaneous thermoreceptors are probably free nerve endings, without obvious specialization. Their axons are small, either unmyelinated C fibers or the smallest-diameter myelinated Aδ fibers (see Table 12-1). We can perceive changes in our average skin temperature of as little as 0.01°C. Within the skin are separate types of thermoreceptors that are sensitive to a range of relatively hot or cold temperatures. Figure 15-28A shows how the steady discharge rate of both types of receptors varies with temperature. Warmth receptors begin firing above ~30°C and increase their firing rate until 44°C to 46°C, beyond which the rate falls off steeply and a sensation of pain begins, presumably mediated by nociceptive endings (see the next section). Cold receptors have a much broader temperature response. They are relatively quiet at skin temperatures of ~40°C, but their steady discharge rate increases as the temperature falls to 24°C to 28°C. Further decreases in temperature cause the steady discharge rate of the cold receptors to decrease until the temperature falls to ~10°C. Below that temperature, firing ceases and cold becomes an effective local anesthetic. In addition to the tonic response just described (i.e., the steady discharge rate), cold receptors also have a phasic response that enables them to report changes in temperature. As shown in Figure 15-28B, when the temperature suddenly shifts from 20.5°C to 15.2°C (both points are to the left of the peak in Fig. 15-28A), the firing rate transiently increases (i.e., the phasic response). However, the new steady-state level is lower, as suggested by the left pair of points in Figure 15-28A. When the temperature suddenly shifts from 35°C to 31.5°C (both points are to the right of the peak in Fig. 15-28A), the firing rate transiently increases, and the new steady-state level is higher, as suggested by the right pair of points in Figure 15-28A. The transduction of relatively warm temperatures is carried out by several types of TRPV channels (specifically TRPV1 to TRPV4—see Table 6-2, family No. 5) expressed in

A

STEADY (TONIC) RESPONSES Warmth receptor fibers

30 Mean steady firing rate 20 (impulses/s) 10

0 15

B

Cold receptor fibers

20 30 40 Skin temperature (°C)

50

TRANSIENT (PHASIC) RESPONSES OF “COLD” FIBERS 35° 31.5°

Firing rate (impulses/s) 20.5°

Temperature “step” 15.2°

Figure 15-28  Temperature sensitivity of cutaneous thermoreceptors.

A, The curves represent the mean steady firing rates of neurons from warmth receptors and cold receptors. B, These data from two experiments on cold receptors show the effects of cooling steps of similar magnitude but starting from different temperatures (20.5°C and 35°C). In both instances, the transient (phasic) responses are the same: an increase in the firing rate. When the starting temperature is 20.5°C, the final firing rate is less than the initial one. However, when the initial temperature is 35°C, the final rate is greater than the initial one. (Data from Somjen GG: Sensory Coding in the Mammalian Nervous System. New York, Appleton-Century-Crofts, 1972.)

thermoreceptors. TRPV1 is a vanilloid receptor—it is activated by the vanilloid class of compounds that includes capsaicin, the pungent ingredient that gives spicy foods their burning quality. Aptly enough, chili peppers taste “hot” because they activate some of the same ion channels that heat itself activates! TRPV1 and TRPV2 channels have painfully high temperature thresholds (~43°C and ~50°C, respectively) and thus help mediate the noxious aspects of thermoreception (see p. 387). Other TRPV channels (TRPV3 and TRPV4) are activated at more moderate temperatures and presumably provide our sensations of warmth. Yet another TRP channel, TRPM8, mediates sensations of moderate cold. TRPM8 channels begin to open at temperatures below ~27°C and are maximally activated at 8°C. In a remarkable analogy to the hot-sensitive TRPV1 channel (the capsaicin receptor), the cool-sensitive TRPM8 channel is a menthol receptor. Menthol evokes sensations of cold because it activates the same ion channel that is opened by cold temperatures.

Chapter 15  •  Sensory Transduction

Nociceptors are specialized sensory endings that transduce painful stimuli Physical energy that is informative at low and moderate levels can be destructive at higher intensity. Sensations of pain motivate us to avoid such situations. Nociceptors are the receptors mediating acutely painful feelings to warn us that body tissue is being damaged or is at risk of being damaged (as the Latin roots imply: nocere [to hurt] + recipere [to receive]). The pain-sensing system is entirely separate from the other modalities we have discussed; it has its own peripheral receptors and a complex, dispersed, chemically unique set of central circuits. Nociceptors are free nerve endings, widely distributed throughout the body. They innervate the skin, bone, muscle, most internal organs, blood vessels, and heart. Ironically, nociceptors are generally absent from the brain substance itself, although they are in the meninges. Nociceptors vary in their selectivity. Mechanical nociceptors, some of which are quite selective, respond to strong pressure—in particular, pressure from sharp objects. A subset of nociceptors expresses Mas-related G protein– coupled receptor D (MrgprD); genetic ablation of just these neurons makes mice insensitive to noxious mechanical stimuli without affecting their responses to painful heat or cold. TRPA1 channels are involved in some forms of painrelated mechanosensation, and they may transduce stimuli that trigger pain originating from viscera such as the colon and bladder. Thermal nociceptors signal either burning heat (above ~45°C, when tissues begin to be destroyed) or unhealthy cold; the heat-sensitive nociceptive neurons express the TRPV1 and TRPV2 channels, whereas the cold-sensitive nociceptors express TRPA1 and TRPM8 channels. A uniquely cold-resistant Na+ channel, Nav1.8, allows cold-sensitive nociceptors to continue firing action potentials even at temperatures low enough to silence other neurons. Chemical nociceptors, which are mechanically insensitive, respond to a variety of agents, including K+, extremes of pH, neuroactive substances such as histamine and bradykinin from the body itself, and various irritants from the environment. Some chemosensitive nociceptors may express TRP channels that respond to, among other things, plant-derived irritants such as capsaicin (TRPV1), menthol (TRPM8), and the pungent derivatives of mustard and garlic (TRPA1). Finally, polymodal nociceptors are single nerve endings that are sensitive to combinations of mechanical, thermal, and chemical stimuli. Nociceptive axons include both fast Aδ fibers and slow, unmyelinated C fibers. Aδ axons mediate sensations of sharp, intense pain; C fibers elicit more persistent feelings of dull, burning pain. The Na+ channel Nav1.7 has a particularly interesting relationship to pain. Patients with loss-of-function mutations of Nav1.7 are insensitive to noxious stimuli and experience repeated injuries because they lack protective reflexes. Several gain-of-function Nav1.7 mutations cause channel hyperexcitability and syndromes of intense chronic pain. Sensations of pain can be modulated in a variety of ways. Skin, joints, or muscles that have been damaged or inflamed are unusually sensitive to further stimuli. This phenomenon

387

Tissue damage creates an "inflammatory soup." Substance P, released from nerve endings, increases capillary permeability and contributes to inflammation.

Skin

Blood vessel

H+

Substance P

Bradykinin Serotonin Lesion K+ Nociceptive axon Axon reflex

Prostaglandins Histamine

NGF

Substance P

Mast cell

Substance P causes mast cells to release histamine, which in turn activates nociceptor endings. Figure 15-29  Hyperalgesia of inflammation.

is called hyperalgesia, and it can be manifested as a reduced threshold for pain, an increase in perceived intensity of painful stimuli, or spontaneous pain. Primary hyperalgesia occurs within the area of damaged tissue, but within ~20 minutes after an injury, tissues surrounding a damaged area may become supersensitive by a process called secondary hyperalgesia. Hyperalgesia seems to involve processes near peripheral receptors (Fig. 15-29) as well as mechanisms in the CNS. Damaged skin releases a variety of chemical substances from its many cell types, blood cells, and nerve endings. These substances—sometimes called the inflammatory soup—include neurotransmitters (e.g., glutamate, serotonin, adenosine, ATP), peptides (e.g., substance P, bradykinin), various lipids (e.g., prostaglandins, endocannabinoids), proteases, neurotrophins, cytokines, and chemokines, K+, H+, and others; they trigger the set of local responses that we know as inflammation. As a result, blood vessels become more leaky and cause tissue swelling (or edema) and redness (see Box 20-1). Nearby mast cells release the chemical histamine, which directly excites nociceptors. By a mechanism called the axon reflex, action potentials can propagate along nociceptive axons from the site of an injury into side branches of the same axon that innervate neighboring regions of skin. The spreading axon branches of the nociceptors themselves may release substances that sensitize nociceptive terminals and make them responsive to

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previously nonpainful stimuli. Such “silent” nociceptors among our small Aδ and C fibers are normally unresponsive to stimuli—even destructive ones. Only after sensitization do they become responsive to mechanical or chemical stimuli and contribute greatly to hyperalgesia. For example, the neurotrophin nerve growth factor (NGF)—part of the inflammatory soup—triggers strong hypersensitivity to heat and mechanical stimuli by modulating TRPV1 channels. Activation of TRPA1 and ASICs are also important in hyperalgesia. The cytokine tumor necrosis factor-alpha (TNF-α) potentiates the inflammatory response directly and enhances release of substances that sensitize nociceptors. Drugs that interfere with neurotrophin and cytokine actions can be effective treatments for the pain of inflammatory diseases. The cognitive sensations of pain are under remarkably potent control by the brain, more so than other sensory system. In some cases, nociceptors may fire wildly, although perceptions of pain are absent; on the other hand, pain may be crippling although nociceptors are silent. Chronic activation of nociceptors can lead to central sensitization, a chronic enhancement of central pain-processing circuits. Prolonged activity in nociceptive axons and their spinal cord synapses causes increased glutamate release, strong activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)– and NMDA (N-methyl-D-aspartate)–type glutamate receptors, and eventually a form of long-term potentiation (see pp. 329–337). Nonpainful sensory input and neural activity from various nuclei within the brain can modify pain. For example, pain evoked by activity in nociceptors (Aδ and C fibers) can be reduced by simultaneous activity in low-threshold mechanoreceptors (Aα and Aβ fibers). This phenomenon is a familiar experience—some of the discomfort of a burn, cut, or bruise can be relieved by gentle massage or rubbing (stimulating mechanoreceptors) around the injured area. In 1965, Melzack and Wall proposed that this phenomenon involves a circuit in the spinal cord that can “gate” the transmission of nociceptive information to the brain; control of the gate could be provided by other sensory information (e.g., tactile stimulation) or by descending control from the brain itself. Gate-like regulation of pain may arise from the modulation of gammaaminobutyric acid (GABA)–mediated and glycine-mediated inhibitory circuits in the spinal cord. A second mechanism for modifying the sensation of pain involves the relatively small peptides called endorphins. In the 1970s, it was discovered that a class of drugs called opioids (including morphine, heroin, and codeine) act by binding tightly and specifically to opioid receptors in the brain and, furthermore, that the brain itself manufactures “endogenous morphine-like substances,” collectively called endorphins (see p. 315).

Muscle spindles sense changes in the length of skeletal muscle fibers, whereas Golgi tendon organs gauge the muscle’s force The somatic sensory receptors described thus far provide information about the external environment. However, the body also needs detailed information about itself to know where each of its parts is in space, whether it is moving, and if so, in which direction and how fast. Proprioception

provides this sense of self and serves two main purposes. First, knowledge of the positions of our limbs as they move helps us judge the identity of external objects. It is much easier to recognize an object if you can actively palpate it than if it is placed passively into your hand so that your skin is stimulated but you are not allowed to personally guide your fingers around it. Second, proprioceptive information is essential for accurately guiding many movements, especially while they are being learned. Skeletal muscles, which mediate voluntary movement, have two mechanosensitive proprioceptors: the muscle spindles (or stretch receptors) and Golgi tendon organs (Fig. 15-30). Muscle spindles measure the length and rate of stretch of the muscles, whereas the Golgi tendon organs gauge the force generated by a muscle by measuring the tension in its tendon. Together, they provide a full description of the dynamic state of each muscle. The different sensitivities of the spindle and the tendon organ are due partly to their structures but also to their placement: spindles are located in modified muscle fibers called intrafusal muscle fibers, which are aligned in parallel with the “ordinary” forcegenerating or extrafusal skeletal muscle fibers. On the other hand, Golgi tendon organs are aligned in series with the extrafusal fibers. The Golgi tendon organ consists of bare nerve endings of group Ib axons (see Table 12-1). These endings intimately invest an encapsulated collagen matrix and usually sit at the junction between skeletal muscle fibers and the tendon. When tension develops in the muscle as a result of either passive stretch or active contraction, the collagen fibers tend to squeeze and distort the mechanosensitive nerve endings, triggering them to fire action potentials. The mammalian muscle spindle is a complex of modified skeletal muscle fibers (intrafusal fibers) combined with both afferent and efferent innervation. The spindle does not contribute significant force generation to the muscle but serves a purely sensory function. A simplified summary of the muscle spindle is that it contains two kinds of intrafusal muscle fibers (bag and chain), with two kinds of sensory endings entwined about them (the primary and secondary endings). The different viscoelastic properties of the muscle fibers make them differentially sensitive to the consequences of muscle stretch. Because the primary sensory endings of group Ia axons coil around and strongly innervate individual bag muscle fibers (in addition to chain fibers), they are very sensitive to the dynamics of muscle length (i.e., changes in its length). The secondary sensory endings of group II axons mainly innervate the chain fibers and most accurately transduce the static length of the muscle; in other words, they are slowly adapting receptors. The discharge rate of afferent neurons increases when the whole muscle—and therefore the spindle—is stretched. ENaC and ASIC2 channels may contribute to the stretch sensitivity of the sensory nerve terminals in muscle spindles. What is the function of the motor innervation of the muscle spindle? Consider what happens when the α motor neurons stimulate the force-generating extrafusal fibers and the muscle contracts. The spindle, connected in parallel to the extrafusal fibers, quickly tends to go slack, which makes it insensitive to further changes in length. To avoid this situation and to continue to maintain control over the sensitivity

Chapter 15  •  Sensory Transduction

Muscle spindle (intrafusal fibers) Extrafusal muscle fiber Muscle spindle capsule (cut open)

Golgi tendon organ (GTO) GTO capsule

Spindle afferent neuron Spindle efferent neuron

Neuromuscular synapse from α motor neuron to extrafusal fibers

Neuromuscular synapse from α motor neuron to extrafusal fibers

Collagen fibrils

GTO afferent (Ib) neuron

Tendon

389

Chain fiber Bag fiber Primary afferent (Ia) neuron γ Motor neurons to intrafusal fibers Secondary afferent (II) neuron

Figure 15-30  Golgi tendon organ and muscle spindle fibers. A muscle contains two kinds of muscle fibers, extrafusal fibers (ordinary muscle fibers that cause contraction) and intrafusal fibers (aligned in parallel with the extrafusal fibers). Some of the extrafusal fibers have Golgi tendon organs located in series between the end of the muscle fiber and the macroscopic tendon. The intrafusal fibers contain muscle spindles, which receive both afferent (sensory) and efferent (motor) innervation. The spindle (inset) contains both bag fibers, with nuclei bunched together, and chain fibers, with nuclei in a row.

of the spindle, γ motor neurons cause the intrafusal muscle fibers to contract in parallel with the extrafusal fibers. This ability of the spindle’s intrafusal fibers to change their length as necessary greatly increases the range of lengths over which the spindle can work. It also means that the sensory responses of the spindle depend not only on the length of the whole muscle in which the spindle sits but also on the contractile state of its own intrafusal muscle fibers. Presumably, the ambiguity in this code is sorted out centrally by circuits that simultaneously keep track of the spindle’s sensory output and the activity of its motor nerve supply. In addition to the muscle receptors, various mechanoreceptors are found in the connective tissues of joints, especially within the capsules and ligaments. Many resemble Ruffini, Golgi, and Pacini end organs; others are free nerve endings. They respond to changes in the angle, direction, and velocity of movement in a joint. Most are rapidly adapting,

which means that sensory information about a moving joint is rich. Nerves encoding the resting position of a joint are few. We are nevertheless quite good at judging the position of a joint, even with our eyes closed. It seems that information from joint receptors is combined with that from muscle spindles and Golgi tendon organs, and probably from cutaneous receptors as well, to estimate joint angle. Removal of one source of information can be compensated by use of the other sources. When an arthritic hip is replaced with a steel and plastic one, patients are still able to tell the angle between their thigh and their pelvis, even though all hip joint mechanoreceptors are long gone.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 15  •  Sensory Transduction

REFERENCES Books and Reviews Alper SL, Sharma AK: The SLC26 gene family of anion transporters and channels. Mol Aspects Med 34:494–515, 2013. Bowmaker JK: Visual pigments and molecular genetics of color blindness. News Physiol Sci 13:63–69, 1998. Copeland BJ, Pillsbury HC 3rd: Cochlear implantation for the treatment of deafness. Annu Rev Med 55:157–167, 2004. Corey DP: New TRP channels in hearing and mechanosensation. Neuron 39:585–588, 2003. Eatock RA, Songer JE: Vestibular hair cells and afferents: Two channels for head motion signals. Annu Rev Neurosci 34:501–534, 2011. Eijkelkamp N, Quick K, Wood JN: Transient receptor potential channels and mechanosensation. Annu Rev Neurosci 36:519– 546, 2013. Fain GL: Sensory Transduction. Sunderland, MA, Sinauer, 2003. Guinan JJ, Salt A, Cheatham MA: Progress in cochlear physiology after Békésy. Hearing Res 293:12–20, 2012. Hudspeth AJ: How hearing happens. Neuron 19:947–950, 1997. Hunt CC: Mammalian muscle spindle: Peripheral mechanisms. Physiol Rev 70:643–663, 1990. Jordt SE, McKemy DD, Julius D: Lessons from peppers and peppermint: The molecular logic of thermosensation. Curr Opin Neurobiol 13:487–492, 2003. Kazmierczak P, Müller U: Sensing sound: Molecules that orchestrate mechanotransduction by hair cells. Trends Neurosci 35: 220–229, 2011. Kinnamon SC, Margolskee RF: Mechanisms of taste transduction. Curr Opin Neurobiol 6:506–513, 1996. Kral A, Sharma A: Developmental neuroplasticity after cochlear implantation. Trends Neurosci 35:111–122, 2012. Kung C: A possible unifying principle for mechanosensation. Nature 436:647–654, 2005. Lin SY, Corey DP: TRP channels in mechanosensation. Curr Opin Neurobiol 15:350–357, 2005. Lucas RJ: Mammalian inner retinal photoreception. Curr Biol 23:R125–R133, 2013. Mattes RD: Accumulating evidence supports a taste component for free fatty acids in humans. Physiol Behav 104:624–631, 2011. Mombaerts P: Genes and ligands for odorant, vomeronasal and taste receptors. Nat Rev Neurosci 5:263–278, 2004. Nathans J: In the eye of the beholder: Visual pigments and inherited variation in human vision. Cell 78:357–360, 1994. Nobili R, Mammano F, Ashmore J: How well do we understand the cochlea? Trends Neurosci 21:159–167, 1998. Peng AW, Salles FT, Pan B, Ricci AJ: Integrating the biophysical and molecular mechanisms of auditory hair cell mechanotransduction. Nat Commun 2:523, 2011. Santos-Sacchi J: New tunes from Corti’s organ: The outer hair cell boogie rules. Curr Opin Neurobiol 13:459–468, 2003. Schnetkamp PPM: The SLC24 gene family of Na+/Ca2+–K+ exchangers: From sight and smell to memory consolidation and skin pigmentation. Mol Aspects Med 34:455–464, 2013.

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Shepherd GM: Neurogastronomy: How the Brain Creates Flavor and Why It Matters. New York, Columbia University Press, 2011, p 288. Tsunozaki M, Bautista DM: Mammalian somatosensory mechanotransduction. Curr Opin Neurobiol 19:1–8, 2009. Journal Articles Buck L, Axel R: A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell 65:175– 187, 1991. Chandrashekar J, Kuhn C, Oka Y, et al: The cells and peripheral representation of sodium taste in mice. Nature 464:297–301, 2010. Coste B, Mathur J, Schmidt M, et al: Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330:55–60, 2010. Crawford AC, Fettiplace R: Auditory nerve responses to imposed displacements of the turtle basilar membrane. Hear Res 12:199– 208, 1983. Eijkelkamp N, Linley JE, Torres JM, et al: A role for Piezo2 in EPAC1-dependent mechanical allodynia. Nat Commun 4:1682, 2013. Hecht S, Shlaer S, Pirenne MH: Energy, quanta, and vision. J Gen Physiol 25:819–840, 1942. Hudspeth AJ: How hearing happens. Neuron 19:947–950, 1997. Ishimaru Y, Inada H, Kubota M, et al: Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor. Proc Natl Acad Sci U S A 103:12569–12574, 2006. Jianga P, Josuea J, Lia X, et al: Major taste loss in carnivorous mammals. Proc Natl Acad Sci U S A 109:4956–4961, 2012. Kawaguchi H, Yamanaka A, Uchida K, et al: Activation of polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 complex by acid in mouse taste cells. J Biol Chem 285:17277–17281, 2010. Liberman MC, Gao J, He DZ, et al: Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 419:300–304, 2002. Nakamura T, Gold GH: A cyclic nucleotide–gated conductance in olfactory receptor cilia. Nature 325:442–444, 1987. Nelson G, Hoon MA, Chandrashekar J, et al: Mammalian sweet taste receptors. Cell 106:381–390, 2001. Oka Y, Butnaru M, von Buchholtz L, et al: High salt recruits aversive taste pathways. Nature 494:472–475, 2013. Taruno A, Vingtdeux V, Ohmoto M, et al: CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes. Nature 495:223–226, 2013. Yu Y, Ulbrich MH, Li M-H, et al: Molecular mechanism of the assembly of an acid-sensing receptor ion channel complex. Nat Commun 3:1252, 2012. Zimmerman K, Leffler A, Babes A, et al: Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature 447:855–858, 2007. Zhao H, Ivic L, Otaki JM, et al: Functional expression of a mammalian odorant receptor. Science 279:237–242, 1998. Zhao GQ, Zhang Y, Hoon MA, et al: The receptors for mammalian sweet and umami taste. Cell 115:255–266, 2003.

C H A P T E R 16  CIRCUITS OF THE CENTRAL NERVOUS SYSTEM Barry W. Connors

ELEMENTS OF NEURAL CIRCUITS Neural circuits process sensory information, generate motor output, and create spontaneous activity A neuron never works alone. Even in the most primitive nervous systems, all neurons participate in synaptically interconnected networks called circuits. In some hydrozoans (small jellyfish), the major neurons lack specialization and are multifunctional. They serve simultaneously as photodetectors, pattern generators for swimming rhythms, and motor neurons. Groups of these cells are repetitively interconnected by two-way electrical synapses into simple ringlike arrangements, and these networks coordinate the rhythmic contraction of the animal’s muscles during swimming. This simple neural network also has the flexibility to command defensive changes in swimming patterns when a shadow passes over the animal. Thus, neuronal circuits have profound advantages over unconnected neurons. In more complex animals, each neuron within a circuit may have very specialized properties. By the interconnection of various specialized neurons, even a simple neuronal circuit may accomplish astonishingly intricate functions. Some neural circuits may be primarily sensory (e.g., the retina) or motor (e.g., the ventral horns of the spinal cord). Many circuits combine features of both, with some neurons dedicated to providing and processing sensory input, others to commanding motor output, and many neurons (perhaps most) doing both. Neural circuits may also generate their own intrinsic signals, with no need for any sensory or central input to activate them. The brain does more than just respond reflexively to sensory input, as a moment’s introspection will amply demonstrate. Some neural functions—such as walking, running, breathing, chewing, talking, and piano playing—require precise timing, with coordination of rhythmic temporal patterns across hundreds of outputs. These basic rhythms may be generated by neurons and neural circuits called pacemakers because of their clock-like capabilities. The patterns and rhythms generated by a pacemaking circuit can always be modulated—stopped, started, or altered—by input from sensory or central pathways. Neuronal circuits that produce rhythmic motor output are sometimes called central pattern generators; we discuss these in a section below. This chapter introduces the basic principles of neural circuits in the mammalian central nervous system (CNS). We 390

describe a few examples of specific systems in detail to illuminate general principles as well as the diversity of neural solutions to life’s complex problems. However, this topic is enormous, and we have necessarily been selective and somewhat arbitrary in our presentation.

Nervous systems have several levels of organization The function of a nervous system is to generate adaptive behaviors. Because different species face unique problems, we expect brains to differ in their organization and mechanisms. Nevertheless, certain principles apply to most nervous systems. It is useful to define various levels of organization.  N16-1  We can analyze a complex behavior—reading the words on this page—in a simple way, with progressively finer detail, down to the level of ion channels, receptors, messengers, and the genes that control them. At the highest level, we recognize neural subsystems and pathways (see Chapter 10), which in this case include the sensory input from the retina (see Chapter 15) leading to the visual cortex, the central processing regions that make sense of the visual information and the motor systems that coordinate movement of the eyes and head. Many of these systems can be recognized in the gross anatomy of the brain. Each specific brain region is extensively interconnected with other regions that serve different primary functions. These regions tend to have profuse connections that send information in both directions along most sensory/central motor pathways. The advantages of this complexity are obvious; while you are interpreting visual information, for example, it can be very useful simultaneously to analyze sound and to know where your eyes are pointing and how your body is oriented. The systems of the brain can be more deeply understood by studying their organization at the cellular level. Within a local brain region, the arrangement of neurons and their synaptic connections is called a local circuit. A local circuit typically includes the set of inputs, outputs, and all the interconnected neurons that are essential to functions of the local brain region. Many regions of the brain are composed of a large number of stereotyped local circuits, almost modular in their interchangeability, that are themselves interconnected. Within the local circuits are finer arrangements of neurons and synapses sometimes called microcircuits. Microcircuits may be repeated numerous times within a local circuit, and they determine the transformations of

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N16-1  Levels of Organization of the Nervous System Contributed by Barry Connors A

BEHAVIOR

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SYSTEMS AND PATHWAYS

LOCAL CIRCUITS

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eFigure 16-1  (Data from Shepherd GM: Neurobiology, 3rd ed. New York, Oxford University Press, 1994.)

CHAPTER 16  •  Circuits of the Central Nervous System

information that occur within small areas of dendrites and the collection of synapses impinging on them. At even finer resolution, neural systems can be understood by the properties of their individual neurons (see Chapter 12), synapses, membranes, molecules (e.g., neurotransmitters and neuromodulators), and ions as well as the genes that encode and control the system’s molecular biology.

Most local circuits have three elements: input axons, interneurons, and projection (output) neurons One of the most fascinating things about the nervous system is the wide array of different local circuits that have evolved for different behavioral functions. Despite this diversity, we can define a few general components of local circuits, which we illustrate with two examples from very different parts of the CNS: the ventral horn of the spinal cord and the cerebral neocortex. Some of the functions of these circuits are described in subsequent sections; here, we examine their cellular anatomy. All local circuits have some form of input, which is usually a set of axons that originate elsewhere and terminate in synapses within the local circuit. A major input to the spinal cord (Fig. 16-1) is the afferent sensory axons in the dorsal roots. These axons carry information from somatic sensory receptors in the skin, connective tissue, and muscles (see pp. 383–389). However, local circuits in the spinal cord also have many other sources of input, including descending input from the brain and input from the spinal cord itself, both from the contralateral side and from spinal segments

Dorsal horn

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above and below. Input to the local circuits of the neocortex (Fig. 16-2) is also easily identified; relay neurons of the thalamus send axons into particular layers of the cortex to bring a range of information about sensation, motor systems, and the body’s internal state. By far, the most numerous type of input to the local circuits of the neocortex comes from the neocortex itself—from adjacent local circuits, distant areas of cortex, and the contralateral hemisphere. These two systems illustrate a basic principle: local circuits receive multiple types of input. Output is usually achieved with a subset of cells known as projection neurons, or principal neurons, which send axons to one or more targets. The most obvious spinal output comes from the α motor neurons, which send their axons out through the ventral roots to innervate skeletal muscle fibers. Output axons from the neocortex come mainly from large pyramidal neurons in layer V, which innervate many targets in the brainstem, spinal cord, and other structures, as well as from neurons in layer VI, which make their synapses back onto the cells of the thalamus. However, as was true with inputs, most local circuits have multiple types of outputs. Thus, spinal neurons innervate other regions of the spinal cord and the brain, whereas neocortical circuits make most of their connections to other neocortical circuits. Rare, indeed, is the neural circuit that has only input and output cells. Local processing is achieved by additional neurons whose axonal connections remain within the local circuit. These neurons are usually called interneurons or intrinsic neurons. Interneurons vary widely in structure and function, and a single local circuit may have many different

Dorsal root

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Figure 16-1  Local circuits in the spinal cord. A basic local circuit in the spinal cord consists of inputs (e.g., sensory axons of the dorsal roots), interneurons (both excitatory and inhibitory), and output neurons (e.g., α motor neurons that send their axons through the ventral roots).

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I II and III

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From To other Outside thalamus neocortex neocortex (input) and thalamus Figure 16-2  Local circuits in the neocortex. A basic local circuit in the neocortex consists of inputs (e.g., afferent axons from the thalamus), excitatory and inhibitory interneurons, and output neurons (e.g., pyramidal cells).

types. Both the spinal cord and neocortex have excitatory and inhibitory interneurons, interneurons that make very specific or widely divergent connections, and interneurons that either receive direct contact from input axons or process only information from other interneurons. In many parts of the brain, interneurons vastly outnumber output neurons. To take an extreme example, the cerebellum has ~1011 granule cells—a type of excitatory interneuron—which is more than the total number of all other types of neurons in the entire brain! The “principles” of local circuits outlined here have many variations. For example, a projection cell may have some of the characteristics of an interneuron, as when a branch of its output axon stays within the local circuit and makes synaptic connections. This branching is the case for the projection cells of both the neocortex (pyramidal cells) and the spinal cord (α motor neurons). On the other hand, some interneurons may entirely lack an axon and instead make their local synaptic connections through very short neurites or even dendrites. In some rare cases, the source of the input to a local circuit may not be purely synaptic but chemical (as with CO2-sensitive neurons in the medulla; see p. 714) or physical (as with temperature-sensitive neurons in the hypothalamus; see p. 1199). Although the main neurons within a generic local circuit are wired in series (see Figs. 16-1 and 16-2), local circuits, often in massive numbers, operate in parallel with one another. Furthermore, these circuits usually

demonstrate a tremendous amount of crosstalk; information from each circuit is shared mutually, and each circuit continually influences neighboring circuits. Indeed, one of the things that makes analysis of local neural circuits so exceptionally difficult is that they operate in highly interactive, simultaneously interdependent, and expansive networks.

SIMPLE, STEREOTYPED RESPONSES: SPINAL REFLEX CIRCUITS Passive stretching of a skeletal muscle causes a reflexive contraction of that same muscle and relaxation of the antagonist muscles Reflexes are among the most basic of neural functions and involve some of the simplest neuronal circuits. A motor reflex is a rapid, stereotyped motor response to a particular sensory stimulus. Although the existence of reflexes had been long appreciated, it was Sir Charles Sherrington  N10-2  who, beginning in the 1890s, first defined the anatomical and physiological bases for some simple spinal reflexes. So meticulous were Sherrington’s observations of reflexes and their timing that they offered him compelling evidence for the existence of synapses, a term he originated. Reflexes are essential, if rudimentary, elements of behavior. Because of their relative simplicity, more than a century of research has taught us a lot about their biological basis. However, reflexes are also important for understanding more complex behaviors. Intricate behaviors may sometimes be built up from sequences of simple reflexive responses. In addition, neural circuits that generate reflexes almost always mediate or participate in much more complex behaviors. Here we examine a relatively well understood example of reflex-mediating circuitry. The CNS commands the body to move about by activating motor neurons, which excite skeletal muscles (Sherrington called motor neurons the final common path). Motor neurons receive synaptic input from many sources within the brain and spinal cord, and the output of large numbers of motor neurons must be closely coordinated to achieve even uncomplicated actions such as walking. However, in some circumstances, motor neurons can be commanded directly by a simple sensory stimulus—muscle stretch—with only the minimum of neural machinery intervening between the sensory cell and motor neuron: one synapse. Understanding of this simplest of reflexes, the stretch reflex or myotatic reflex, first requires knowledge of some anatomy. Each motor neuron, with its soma in the spinal cord or brainstem, commands a group of skeletal muscle cells; a single motor neuron and the muscle cells that it synapses on are collectively called a motor unit (see pp. 241–242). Each muscle cell belongs to only one motor unit. The size of motor units varies dramatically and depends on muscle function. In small muscles that generate finely controlled movements, such as the extraocular muscles of the eye, motor units tend to be small and may contain just a few muscle fibers. Large muscles that generate strong forces, such as the gastrocnemius muscle of the leg, tend to have large motor units with as many as several thousand muscle fibers. There are two types of motor neurons (see Table 12-1): α motor neurons

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Inhibitory interneuron

Primary sensory neuron Excitatory synapse

The reflex stimulates— in the spinal cord—the motor neurons to the extensor muscle...

Ia axon from the stretch receptor Inhibitory Spinal α Motor synapse cord neuron Ventral cell bodies root

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...and inhibits the motor neurons to the flexor muscle. Patellar tendon

Flexor (semitendinosus) Extensor (quadriceps)

Knee jerk

Figure 16-3  Knee-jerk (myotatic) reflex. Tapping the patellar tendon with a percussion hammer elicits a reflexive knee jerk caused by contraction of the quadriceps muscle: the stretch reflex. Stretching the tendon pulls on the muscle spindle, exciting the primary sensory afferents, which convey their information via group Ia axons. These axons make monosynaptic connections to the α motor neurons that innervate the quadriceps, resulting in the contraction of this muscle. The Ia axons also excite inhibitory interneurons that recip­ rocally innervate the motor neurons of the antagonist muscle of the quadriceps (the flexor), resulting in relaxation of the semitendinosus muscle. Thus, the reflex relaxation of the antagonistic muscle is polysynaptic.

innervate the main force-generating muscle fibers (the extrafusal fibers), whereas γ motor neurons innervate only the fibers of the muscle spindles. The group of all motor neurons innervating a single muscle is called a motor neuron pool (see pp. 241–242). When a skeletal muscle is abruptly stretched, a rapid, reflexive contraction of the same muscle often occurs. The contraction increases muscle tension and opposes the stretch. This stretch reflex is particularly strong in physiological extensor muscles—those that resist gravity—and it is sometimes called the myotatic reflex because it is specific for the same muscle that is stretched. The most familiar version is the knee jerk, which is elicited by a light tap on the patellar tendon. The tap deflects the tendon, which then pulls on and briefly stretches the quadriceps femoris muscle. A reflexive contraction of the quadriceps quickly follows (Fig. 16-3). Stretch reflexes are also easily demonstrated in the biceps of the arm and the muscles that close the jaw. Sherrington

showed that the stretch reflex depends on the nervous system and requires sensory feedback from the muscle. For example, cutting the dorsal (sensory) roots to the lumbar spinal cord abolishes the stretch reflex in the quadriceps muscle. The basic circuit for the stretch reflex begins with the primary sensory axons from the muscle spindles (see p. 388) in the muscle itself. Increasing the length of the muscle stimulates the spindle afferents, particularly the large group Ia axons from the primary sensory endings. In the spinal cord, these group Ia sensory axons terminate monosynaptically onto the α motor neurons that innervate the same (i.e., the homonymous) muscle from which the group Ia axons originated. Thus, stretching a muscle causes rapid feedback excitation of the same muscle through the minimum possible circuit: one sensory neuron, one central synapse, and one motor neuron (Box 16-1). Monosynaptic connections account for much of the rapid component of the stretch reflex, but they are only the

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BOX 16-1  Motor System Injury

T

he motor control systems, because of their extended anatomy, are especially susceptible to damage from trauma or disease. The nature of a patient’s motor deficits often allows the neurologist to diagnose the site of neural damage with great accuracy. When injury occurs to lower parts of the motor system, such as motor neurons or their axons, deficits may be very localized. If the motor nerve to a muscle is damaged, that muscle may develop paresis (weakness) or complete paralysis (loss of motor function). When motor axons cannot trigger contractions, there can be no reflexes (areflexia). Normal muscles are slightly contracted even at rest—they have some tone. If their motor nerves are transected, muscles become flaccid (atonia) and eventually develop profound atrophy (loss of muscle mass) because of the absence of trophic influences from the nerves. Motor neurons normally receive strong excitatory influences from the upper parts of the motor system, including regions of the spinal cord, the brainstem, and the cerebral cortex. When upper regions of the motor system are injured by stroke, trauma, or demyelinating disease, for example, the signs and symptoms are distinctly different from those caused by lower damage. Complete transection of the spinal cord leads to profound paralysis below the level of the lesion. This is called paraplegia when only both legs are selectively affected, hemiplegia when one side of the body is affected, and quadriplegia when the legs, trunk, and arms are involved. For a few days after an acute injury, there is also areflexia and reduced muscle tone (hypotonia), a condition called spinal shock. The muscles are limp and cannot be controlled by the brain or by the remaining circuits of the spinal cord. Spinal shock is temporary; after days to months, it is replaced by both an exaggerated muscle tone (hypertonia) and heightened stretch reflexes (hyperreflexia) with related signs—this combination is called spasticity. The biological mechanisms of spasticity are poorly understood, although the hypertonia is the consequence of tonically overactive stretch reflex circuitry, driven by spinal neurons that have become chronically hyperexcitable.

beginning of the story. At the same time the stretched muscle is being stimulated to contract, parallel circuits are inhibiting the α motor neurons of its antagonist muscles (i.e., those muscles that move a joint in the opposite direction). Thus, as the knee-jerk reflex causes contraction of the quadriceps muscle, it simultaneously causes relaxation of its antagonists, including the semitendinosus muscle (see Fig. 16-3). To achieve inhibition, branches of the group Ia sensory axons excite specific interneurons that inhibit the α motor neurons of the antagonists. This reciprocal innervation increases the effectiveness of the stretch reflex by minimizing the antagonistic forces of the antagonist muscles.

Force applied to the Golgi tendon organ regulates muscle contractile strength Skeletal muscle contains another mechanosensory transducer in addition to the stretch receptor: the Golgi tendon organ (see p. 388). Tendon organs are aligned in series with the muscle; they are exquisitely sensitive to the tension

within a tendon and thus respond to the force generated by the muscle rather than to muscle length. Tendon organs may respond during passive muscle stretch, but they are stimulated particularly well during active contractions of a muscle. The group Ib sensory axons of the tendon organs excite both excitatory and inhibitory interneurons within the spinal cord (Fig. 16-4). In some cases, this interneuron circuitry inhibits the muscle in which tension has increased and excites the antagonistic muscle; therefore, activity in the tendon organs can yield effects that are almost the opposite of the stretch reflex. Under other circumstances, particularly during rapid movements such as locomotion, sensory input from Golgi tendon organs actually excites the motor neurons activating the same muscle. The reflex effects of Golgi tendon organ activity vary because the interneurons receiving input from Ib axons also receive input from other sensory endings in the muscle and skin, and from axons descending from the brain. In general, reflexes mediated by the Golgi tendon organs serve to control the force within muscles and the stability of particular joints.

Noxious stimuli can evoke complex reflexive movements Sensations from the skin and connective tissue can also evoke strong spinal reflexes. Imagine walking on a beach and stepping on a sharp piece of shell. Your response is swift and coordinated and does not require thoughtful reflection: you rapidly withdraw the wounded foot by activating the leg flexors and inhibiting the extensors. To keep from falling, you also extend your opposite leg by activating its extensors and inhibiting its flexors (Fig. 16-5). This response is an example of a flexion-withdrawal reflex. The original stimulus for the reflex came from fast pain afferent neurons in the skin, primarily the group Aδ axons. This bilateral flexor reflex response is coordinated by sets of inhibitory and excitatory interneurons within the spinal gray matter. Note that this coordination requires circuitry not only on the side of the cord ipsilateral to the wounded side but also on the contralateral side. That is, while you withdraw the foot that hurts, you must also extend the opposite leg to support your body weight. Flexor reflexes can be activated by most of the various sensory afferents that detect noxious stimuli. Motor output spreads widely up and down the spinal cord, as it must to orchestrate so much of the body’s musculature into an effective response. A remarkable feature of flexor reflexes is their specificity. Touching a hot surface, for example, elicits reflexive withdrawal of the hand in the direction opposite the side of the stimulus, and the strength of the reflex is related to the intensity of the stimulus. Unlike simple stretch reflexes, flexor reflexes coordinate the movement of entire limbs and even pairs of limbs. Such coordination requires precise and widespread wiring of the spinal interneurons.

Spinal reflexes are strongly influenced by control centers within the brain Axons descend from numerous centers within the brainstem and the cerebral cortex and synapse primarily on the spinal interneurons, with some direct input to the motor neurons.

CHAPTER 16  •  Circuits of the Central Nervous System

Dorsal root ganglion

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Inhibitory interneuron

Primary sensory neuron

Ib axon from Golgi tendon organ The reflex inhibits—in the spinal cord—the motor neurons to the extensor muscle...

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α Motor neuron axons ...and stimulates the motor neurons to the flexor muscle. Flexor (semitendinosus) Extensor (quadriceps)

Patellar tendon Golgi tendon organ Figure 16-4  Golgi tendon organ reflex. Contraction of the quadriceps muscle can elicit a reflexive relaxation of this muscle and contraction of the antagonistic semitendinosus muscle. Contraction of the muscle pulls on the tendon; this squeezes and excites the sensory endings of the Golgi tendon organ, which convey their information via group Ib axons. These axons synapse on both inhibitory and excitatory interneurons in the spinal cord. The inhibitory interneurons innervate α motor neurons to the quadriceps, relaxing this muscle. The excitatory interneurons innervate α motor neurons to the antagonistic semitendinosus muscle, contracting it. Thus, both limbs of the reflex are polysynaptic.

This descending control is essential for all conscious (and much unconscious) command of movement, a topic beyond the scope of this chapter. Less obvious is that the descending pathways can alter the strength of reflexes. For example, to heighten an anxious patient’s stretch reflexes, a neurologist will sometimes ask the patient to perform the Jendrassik maneuver. The patient clasps his or her hands together and pulls; while the patient is distracted with that task, the examiner tests the stretch reflexes of the leg. Another example of the brain’s modulation of a stretch reflex occurs when you catch a falling ball. If a ball were to fall unexpectedly from the sky and hit your outstretched hand, the force applied to your arm would cause a rapid stretch reflex—contraction in the stretched muscles and reciprocal inhibition in the antagonist muscles. The result would be that your hand would slap the ball back up into the air. However, if you anticipate catching the falling ball, for a short period around the time of impact (about ±60 ms), both your stretched muscles and the antagonist muscles contract! This maneuver stiffens your arm just when you need to squeeze that ball to avoid dropping it. Stretch reflexes of the leg also vary dramatically during each step as we walk, thereby facilitating movement of the legs. Like stretch reflexes, flexor reflexes can also be strongly affected by descending pathways. With mental effort,

painful stimuli can be tolerated and withdrawal reflexes sup­pressed. On the other hand, anticipation of a painful stimulus may heighten the vigor of a withdrawal reflex when the stimulus actually arrives. Most of the brain’s influence on spinal circuitry is achieved by control of the many spinal interneurons. Spinal reflexes are frequently studied in isolation from one another, and textbooks often describe them this way. However, under realistic conditions, many reflex systems operate simultaneously, and motor output from the spinal cord depends on interactions among them as well as on the state of controlling influences descending from the brain. It is now well accepted that reflexes do not simply correct for external perturbations of the body; in addition, they play a key role in the control of all movements. The neurons involved in reflexes are the same neurons that generate other behaviors. Think again of the flexor response to the sharp shell—the pricked foot is withdrawn while the opposite leg extends. Now imagine that a crab pinches that opposite foot—you respond with the opposite pattern of withdrawal and extension. Repeat this a few times, crabs pinching you left and right, and you have achieved the basic pattern necessary for walking! Indeed, rhythmic locomotor patterns use components of these same spinal reflex circuits, as discussed next.

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Cutaneous afferent fiber from the nociceptor (Aδ) Extensor (quadriceps) In the leg that feels the pain, the reflex inhibits— in the spinal cord—the motor neurons to the extensor muscle… …and stimulates the motor neurons to the flexor muscle.

Flexor (semitendinosus) In the opposite leg, the reflex stimulates—in the spinal cord—the motor neurons to the extensor muscle… Extensor (quadriceps)

Stimulated leg withdraws Cutaneous receptor for sharp pain

…and inhibits the motor neurons to the flexor muscle.

Opposite leg supports

Figure 16-5  Flexion-withdrawal reflex. A painful stimulus to the right foot elicits a reflexive flexion of the right knee and an extension of the left knee. The noxious stimulus activates nociceptor afferents, which convey their information via group Aδ axons. These axons synapse on both inhibitory and excitatory interneurons. The inhibitory interneurons that project to the right side of the spinal cord innervate α motor neurons to the quadriceps and relax this muscle. The excitatory interneurons that project to the right side of the spinal cord innervate α motor neurons to the antagonistic semitendinosus muscle and contract it. The net effect is a coordinated flexion of the right knee. Similarly, the inhibitory interneurons that project to the left side of the spinal cord innervate α motor neurons to the left semitendinosus muscle and relax this muscle. The excitatory interneurons that project to the left side of the spinal cord innervate α motor neurons to the left quadriceps and contract it. The net effect is a coordinated extension of the left knee.

RHYTHMIC ACTIVITY: CENTRAL PATTERN GENERATORS Central pattern generators in the spinal cord can create a complex motor program even without sensory feedback A common feature of motor control is the motor program, a set of structured muscle commands that are determined by the nervous system before a movement begins and that can be sent to the muscles with the appropriate timing so that a sequence of movements occurs without any need for sensory feedback. The best evidence for the existence of motor programs is that the brain or spinal cord can command a variety of voluntary and automatic movements, such as walking and breathing (see pp. 706–709), even in the complete absence

of sensory feedback from the periphery. The existence of motor programs certainly does not mean that sensory information is unimportant; on the contrary, motor behavior without sensory feedback is always different from that with normal feedback. The neural circuits responsible for various motor programs have been defined in a wide range of species. Although the details vary endlessly, certain broad principles emerge, even when vertebrates and invertebrates are compared. Here we focus on central pattern generators, wellstudied circuits that underlie many of the rhythmic motor activities that are central to animal behavior. Rhythmic behavior includes walking, running, swimming, breathing, chewing, certain eye movements, shivering, and even scratching. The central pattern generators driving each of these activities share certain basic properties. At their core is a set of cyclic, coordinated timing signals that are

CHAPTER 16  •  Circuits of the Central Nervous System

A

ALTERNATING CONTRACTIONS IN A SINGLE LIMB

B

LH LF RH RF

When the extensors contract…

the flexors relax…

…and the foot is planted.

LH

RH

LF

RF

Extensors (LH)

Flexors (LH)

Foot planted Foot lifted

LH LF RH RF LH LF RH RF LH LF RH RF

397

STEPPING PATTERN OF A CAT DURING VARIOUS GAITS WALK

Foot planted

Foot lifted

TROT

PACE

GALLOP

Time

Figure 16-6  Rhythmic patterns during locomotion. A, The experimental tracings are electromyograms (EMGs)—extracellular recordings of the electrical activity of muscles—from the extensor and flexor muscles of the left hind limb of a walking cat. The pink bars indicate that the foot is lifted; the purple bars indicate that the foot is planted. B, The walk, trot, pace, and gallop not only represent different patterns and frequencies of planting and lifting for a single leg but also different patterns of coordination among the legs. LF, left front; LH, left hind; RF, right front; RH, right hind. (Data from Pearson K: The control of walking. Sci Am 2:72–86, 1976.)

generated by a cluster of interconnected neurons. These basic signals are used to command as many as several hundred muscles, each precisely contracting or relaxing during a particular phase of the cycle; for example, with each walking step, the knee must first be flexed and then extended. Figure 16-6A shows how the extensor and flexor muscles of the left hind limb of a cat contract rhythmically—and out of phase with one another—while the animal walks. Rhythms must also be coordinated with other rhythms; for humans to walk, one leg must move forward while the other thrusts backward, then vice versa, and the arms must swing in time with the legs, but with the opposite phase. For four-footed animals, the rhythms are even more complicated and must be able to accommodate changes in gait (see Fig. 16-6B). For coordination to be achieved among the various limbs, sets of central pattern generators must be interconnected. The motor patterns must also have great flexibility so that they can be altered on a moment’s notice—consider the adjustments necessary when one foot strikes an obstacle while walking or the changing motor patterns necessary to go from walking, to trotting, to running, to jumping. Finally, reliable methods must be available for regulating the speed of the patterns and for turning them on and off. The central pattern generators for some rhythmic functions, such as breathing, are in the brainstem (see p. 706). Surprisingly, those responsible for locomotion reside in the spinal cord itself. Even with the spinal cord transected so that the lumbar segments are isolated from all higher centers, cats on a treadmill can generate well-coordinated stepping

movements. Furthermore, stimulation of sensory afferents or descending tracts can induce the spinal pattern generators in four-footed animals to switch rapidly from walking, to trotting, to galloping patterns by altering not only the frequency of motor commands but also their pattern and coordination. During walking and trotting and pacing, the hind legs alternate their movements, but during galloping, they both flex and extend simultaneously (compare the different leg patterns in Fig. 16-6B). Grillner and colleagues showed that each limb has at least one central pattern generator. If one leg is prevented from stepping, the other continues stepping normally. Under most circumstances, the various spinal pattern generators are coupled to one another, although the nature of the coupling must change to explain, for example, the switch from trotting to galloping patterns.

Pacemaker cells and synaptic interconnections both contribute to central pattern generation How do neural circuits generate rhythmic patterns of activity? There is no single answer, and different circuits use different mechanisms. The simplest pattern generators are single neurons whose membrane characteristics endow them with pacemaker properties that are analogous to those of cardiac muscle cells (see p. 489) and smooth muscle cells (see p. 244). Even when experimentally isolated from other neurons, pacemaker neurons may be able to generate rhythmic activity by relying only on their intrinsic membrane conductances (see Fig. 12-4). It is easy to imagine how

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SECTION III  •  The Nervous System

Excitatory interneuron

Flexor motor neuron

Inhibitory interneurons

Tonic descending or afferent input

Excitatory interneuron

Extensor motor neuron

Figure 16-7  Half-center model for alternating rhythm generation in flexor and extensor motor neurons. Stimulating the upper excitatory interneuron has two effects. First, the stimulated excitatory interneuron excites the motor neuron to the flexor muscle. Second, the stimulated excitatory interneuron excites an inhibitory interneuron, which inhibits the lower pathway. Stimulating the lower excitatory interneuron has the opposite effects. Thus, when one motor neuron is active, the opposite one is inhibited.

intrinsic pacemaker neurons might act as the primary rhythmic driving force for sets of motor neurons that in turn command cyclic behavior. Among vertebrates, however, pacemaker neurons may contribute to some central pattern generators, but they do not appear to be solely responsible for generating rhythms. Instead, pacemakers are embedded within interconnected circuits, and it is the combination of intrinsic pacemaker properties and synaptic interconnections that generates rhythms. Neural circuits without pacemaker neurons can also generate rhythmic output. In 1911, T. Graham Brown proposed a pattern-generating circuit for locomotion. The essence of Brown’s half-center model is a set of excitatory and inhibitory interneurons arranged to inhibit one another reciprocally (Fig. 16-7). The half-centers are the two halves of the circuit, each commanding one of a pair of antagonist muscles. For the circuit to work, a tonic (i.e., nonrhythmic) drive must be applied to the excitatory interneurons; this drive could come from axons originating outside the circuit (e.g., from neurons in the brain) or from the intrinsic excitability of the neurons themselves. Furthermore, some built-in mechanism must limit the duration of the inhibitory activity so that excitability can cyclically switch from one half-center to the other. Note that feedback from the muscles is not needed for the rhythms to proceed indefinitely. In fact, studies of >50 vertebrate and invertebrate motor circuits have confirmed that rhythm generation can continue in the absence of sensory information.

Central pattern generators in the spinal cord take advantage of sensory feedback, interconnections among spinal segments, and interactions with brainstem control centers The half-center model can produce rhythmic, alternating neural activity, but it is clearly too simplistic to account for most features of locomotor pattern generation. Analysis of vertebrate pattern generators is a daunting task, made difficult by the complexity of the circuits and the behaviors they control. In one of the most detailed investigations, Grillner and colleagues studied a simple model of vertebrate locomotion circuits: the spinal cord of the sea lamprey. Lampreys

are among the simplest fish, and they swim with undulating motions of their body by using precisely coordinated waves of contractions of body muscles. At each spinal segment, muscle activity alternates—one side contracts as the other relaxes. As in mammals, the rhythmic pattern is generated within the spinal cord, and neurons in the brainstem control the initiation and speed of the patterns. The basic patterngenerating circuit for the lamprey spinal cord is repeated in each of the animal’s 100 or so spinal segments. The lamprey pattern-generating circuit improves on the half-center model in three ways. The first is sensory feedback. The lamprey has two kinds of stretch receptor neurons in the lateral margin of the spinal cord itself. These neurons sense stretching of the cord and body, which occurs as the animal bends during swimming. One type of stretch receptor excites the pattern generator interneurons on that same side and facilitates contraction, whereas the other type inhibits the pattern generator on the contralateral side and suppresses contraction. Because stretching occurs on the side of the cord that is currently relaxed, the effect of both stretch receptors is to terminate activity on the contracted side of the body and to initiate contraction on the relaxed side. The second improvement of the lamprey circuit over the half-center model is the interconnection of spinal segments, which ensures the smooth progression of contractions down the length of the body, so that swimming can be efficient. Specifically, each segment must command its muscles to contract slightly later than the one anterior to it, with a lag of ~1% of a full activity cycle for normal forward swimming. Under some circumstances, the animal can also reverse the sequence of intersegment coordination to allow it to swim backward! A third improvement over the half-center model is the reciprocal communication between the lamprey spinal pattern generators and control centers in the brainstem. Not only does the brainstem use numerous pathways and transmitters to modulate the generators, but the spinal generators also inform the brainstem of their activity. The features outlined for swimming lampreys are relevant to walking cats and humans. All use spinal pattern generators to produce rhythms. All use sensory feedback to modulate locomotor rhythms (in mammals, feedback from muscle,

CHAPTER 16  •  Circuits of the Central Nervous System

joint, and cutaneous receptors is all-important). All coordinate the spinal pattern generators across segments, and all maintain reciprocal communication between spinal generators and brainstem control centers.

SPATIAL REPRESENTATIONS: SENSORY AND MOTOR MAPS IN THE BRAIN We have already seen that the spinal cord can receive sensory input, integrate it, and produce motor output that is totally independent of the brain. The brain also receives this sensory information and uses it to control the motor activity of the spinal reflexes and central pattern generators. How does the brain organize this sensory input and motor output? In many cases, it organizes these functions spatially with neural maps. In everyday life, we use maps to represent spatial locations. You may use endless ways to construct a map, depending on which features of an area you want to highlight and what sort of transformation you make as you take measurements from the source (the thing being mapped) and place them on the target (the map). Maps of the earth may emphasize topography, the road system, political boundaries, distributions of air temperature and wind direction, population density, or vegetation. A map is a model of a part of the world—and a very limited model at that. The brain also builds maps, most of which represent very selected aspects of our sensory information about the environment or the motor systems controlling our body. These maps can represent spatial qualities of various sensory modalities (e.g., a place in the visual field) or nonspatial qualities (e.g., smell).

The nervous system contains maps of sensory and motor information Almost all sensory receptors are laid out in planar sheets. In some cases, these receptor sheets are straightforward spatial maps of the sensory environment that they encode. For example, the somatic sensory receptors of the skin literally form a map of the body surface. Similarly, a tiny version of the visual scene is projected onto the mosaic of retinal photoreceptors. The topographies of other sensory receptor sheets represent qualities other than spatial features of the sensory stimuli. For example, the position of a hair cell along the basilar membrane in the cochlea determines the range of sound frequencies to which it will respond. Thus, the sheet of hair cells is a frequency map of sound rather than a map of the location of sounds in space. Olfactory and taste receptors also do not encode stimulus position; instead, because the receptor specificity varies topographically, the receptor sheets may be chemical maps of the types of stimuli. The most interesting thing about sensory receptor maps is that they often project onto many different regions of the CNS. In fact, each sensory surface may be mapped and remapped many times within the brain, the characteristics of each map being unique. In some cases, the brain constructs maps of stimulus features even when these features are not mapped at the level of the receptors themselves. Sound localization is a good example of this property (see the next section). Some neural maps may also combine the features of other neural

399

maps, for example, overlaying visual information with auditory information.

The cerebral cortex has multiple visuotopic maps Some of the best examples of brain maps are those of the visual fields. Figure 16-8A shows the basic anatomical pathway extending from the retina to the lateral geniculate nucleus of the thalamus and on to the primary visual cortex (area V1). Note that area V1 actually maps the visual thalamus, which in turn maps the retina, the first visuotopic map in the brain. Thus, the V1 map is sometimes referred to as a retinotopic map. Figure 16-8B shows how the visual fields are mapped onto cortical area V1. The first thing to notice is that the left half of the visual field is represented on the right cortex and the upper half of the visual field is represented on the lower portions of the cortex. This orientation is strictly determined by the system’s anatomy. For example, all the retinal axons from the left-most halves of both eyes (which are stimulated by light from the right visual hemifield) project to the left half of the brain. Compare the red and blue pathways in Figure 16-8A. During development, each axon must therefore make an unerring decision about which side of the brain to innervate when it reaches the optic chiasm! The second thing to notice is that scaling of the visual fields onto the visual cortex—often called the magnification factor—is not constant. In particular, the central region of the visual fields—the fovea—is greatly magnified on the cortical surface. Behavioral importance ultimately determines mapping in the brain. Primates require vision of particularly high resolution in the center of their gaze; photoreceptors and ganglion cells are thus packed as densely as possible into the central retinal region (see p. 363). About half of the primary visual cortex is devoted to input from the relatively small fovea and the retinal area just surrounding it. Understanding a visual scene requires us to analyze many of its features simultaneously. An object may have shape, color, motion, location, and context, and the brain can usually organize these features to present a seamless interpretation, or image. The details of this process are only now being worked out, but it appears that the task is accomplished with the help of numerous visual areas within the cerebral cortex. Studies of monkey cortex by a variety of electrophysiological and anatomical methods have identified >25 areas that are mainly visual in function, most of which are in the vicinity of area V1. According to recent estimates, humans devote almost half of their neocortex primarily to the processing of visual information. Several features of a visual scene, such as motion, form, and color, are processed in parallel and, to some extent, in separate stages of processing. The neural mechanisms by which these separate features are somehow melded into one image or concept of an object remain unknown, but they depend on strong and reciprocal interconnections between the visual maps in various areas of the brain. The apparently simple topography of a sensory map looks much more complex and discontinuous when it is examined in detail. Many cortical areas can be described as maps on maps. Such an arrangement is especially striking in the visual system. For example, within area V1 of Old World monkeys and humans, the visuotopic maps of the two eyes remain

400

SECTION III  •  The Nervous System

A

CORONAL SECTION SHOWING PROJECTION OF THE RETINA TO THE PRIMARY VISUAL CORTEX Visual fields Left

Right

B

Fixation point

SAGITTAL SECTIONS SHOWING VISUOTOPY OF THE PRIMARY VISUAL CORTEX Foveal visual field

Left visual field

Right visual field

Right eye Retina Optic chiasm Right optic tract Right LGN Optic radiation Fovea

Fovea

Primary visual cortex (area V1) Figure 16-8  Visual fields and visual maps. A, The right sides of both retinas (which sense the left visual hemifield) project to the right lateral geniculate nucleus (LGN), which in turn projects to the right primary visual cortex (area V1). B, The upper parts of the visual fields project to lower parts of the contralateral visual cortex, and vice versa. Although the fovea represents only a small part of the visual field, its representation is greatly magnified in the primary visual cortex, which reflects the large number of retinal ganglion cells that are devoted to the fovea.

segregated. In layer IV of the primary visual cortex, this segregation is accomplished by having visual input derived from the left eye alternate every 0.25 to 0.5 mm with visual input from the right. Thus, two sets of information, one from the left eye and one from the right eye, remain separated but adjacent. Viewed edge on, these left-right alternations look like columns (Fig. 16-9A); hence their name: ocular dominance columns, which were identified by David Hubel and Torsten Wiesel, who shared half of the 1981 Nobel Prize in Physiology or Medicine.   N16-2  Viewed from the surface of the brain, this alternating left-right array of inputs looks like bands or zebra stripes (see Fig. 16-9B). Superimposed on the zebra-stripe ocular dominance pattern in layer IV of the primary visual cortex, but quite distinct from these zebra stripes, layers II and III have structures called blobs. These blobs are visible when the cortex is stained for the mitochondrial enzyme cytochrome oxidase. Viewed edge on, these blobs look like round pegs (see Fig. 16-9). Viewed from the surface of the brain (see Fig. 16-9), the blobs appear as a polka-dot pattern of small dots that are ~0.2 mm in diameter. Adjacent to the primary visual cortex (V1) is the secondary visual cortex (V2), which has, instead of blobs, a

series of thick and thin stripes that are separated by pale interstripes. Some other higher-order visual areas also have striped patterns. Whereas ocular dominance columns demarcate the left and right eyes, blobs and stripes seem to demarcate clusters of neurons that process and channel different types of visual information between areas V1 and V2 and pass them on to other visual regions of the cortex. For example, neurons within the blobs of area V1 seem to be especially attuned to information about color and project to neurons in the thin stripes of V2. Other neurons throughout area V1 are very sensitive to motion but are insensitive to color. They channel their information mainly to neurons of the thick stripes in V2.

Maps of somatic sensory information magnify some parts of the body more than others One of the most famous depictions of a neural map came from studies of the human somatosensory cortex by Penfield and colleagues. Penfield stimulated small sites on the cortical surface in locally anesthetized but conscious patients during neurosurgical procedures; from their verbal descriptions of the position of their sensations, he drew a

CHAPTER 16  •  Circuits of the Central Nervous System

N16-2  David Hubel and Torsten Wiesel David H. Hubel and Torsten N. Wiesel shared the 1981 Nobel Prize in Physiology or Medicine with Roger W. Sperry. Hubel and Wiesel were cited “for their discoveries concerning information processing in the visual system.” For more informa­tion visit, http://nobelprize.org/nobel_prizes/medicine/laureates/ 1981/.

400.e1

CHAPTER 16  •  Circuits of the Central Nervous System

A

B

LAYERS OF AREA V1

Cytochrome oxidase blobs

401

SPLIT-OPEN VIEW

Cytochrome oxidase blobs

I

II, III

II, III

Righ t eye

Left eye

Righ t eye

IV

IV

V, VI

V, VI

Left eye

Ocular dominance columns

Right eye

Left eye

Ocular dominance columns

Figure 16-9  Ocular dominance columns and blobs in the primary visual cortex (area V1). A, Ocular dominance columns are shown as alternating black (right eye) and gray (left eye) structures in layer IV. The alternating light and dark bands are visible in an autoradiograph taken 2 weeks after injecting one eye with 3H-labeled proline and fucose. The 3H label moved from the optic nerve to neurons in the lateral geniculate nucleus and then to the axon terminals in the V1 cortex that are represented in this figure. The blobs are shown as tealcolored pegs in layers II and III. They represent the regular distribution of cytochrome oxidase–rich neurons and are organized in pillar-shaped clusters. B, Cutting the brain parallel to its surface, but between layers III and IV, reveals a polka-dot pattern of blobs in layer II/III and zebra-like stripes in layer IV. (Data from Hubel D: Eye, Brain and Vision. New York, WH Freeman, 1988.)

homunculus, a little person representing the somatotopy— mapping of the body surface—of the primary somatic sensory cortex (Fig. 16-10A). The basic features of Penfield’s map have been confirmed with other methods, including recording from neurons while the body surface is stimulated and modern brain-imaging methods, such as positron emission tomography and functional magnetic resonance imaging. The human somatotopic map resembles a trapeze artist hanging upside down—the legs are hooked over the top of the postcentral gyrus and dangle into the medial cortex between the hemispheres, and the trunk, upper limbs, and head are draped over the lateral aspect of the postcentral gyrus. Two interesting features should be noticed about the somatotopic map in Figure 16-10A. First, mapping of the body surface is not always continuous. For example, the representation of the hand separates those of the head and face. Second, the map is not scaled like the human body. Instead, it looks like a cartoon character: the mouth, tongue, and fingers are very large, whereas the trunk, arms, and legs are tiny. As was the case for mapping of the visual fields onto the visual cortex, it is clear in Penfield’s map that the magnification factor for the body surface is not a constant but varies for different parts of the body. Fingertips are magnified on the cortex much more than the tips of the toes. The relative size of cortex that is devoted to each body part is correlated with the density of sensory input received from that part, and 1 mm2 of fingertip skin has many more sensory endings than a similar patch on the buttocks. Size on the map is also related to the importance of the sensory input from that part

of the body; information from the tip of the tongue is more useful than that from the elbow. The mouth representation is probably large because tactile sensations are important in the production of speech, and the lips and tongue are one of the last lines of defense in deciding whether a morsel is a potential piece of food or poison. The importance of each body part differs among species, and indeed, some species have body parts that others do not. For example, the sensory nerves from the facial whisker follicles of rodents have a huge representation on the cortex, whereas the digits of the paws receive relatively little. Rodent behavior explains this paradox. Most are nocturnal, and to navigate they actively sweep their whiskers about as they move. By touching their local environment, they can sense shapes, textures, and movement with remarkable acuity. For a rat or mouse, seeing things with its eyes is often less important than “seeing” things with its whiskers. As we have already seen for the visual system, other sensory systems usually map their information numerous times. Maps may be carried through many anatomical levels. The somatotopic maps in the cortex begin with the primary somatic sensory axons (see Table 12-1) that enter the spinal cord or the brainstem, each at the spinal segment appropriate to the site of the information that it carries. The sensory axons synapse on second-order neurons, and these cells project their axons into various nuclei of the thalamus and form synapses. Thalamic relay neurons in turn send their axons into the neocortex. The topographical order of the body surface (i.e., somatotopy) is maintained at each anatomical stage, and somatotopic maps are located within the

402 A

SECTION III  •  The Nervous System

SOMATOSENSORY

Arm Head Elbow Neck Trunk Forearm Hip Hand Genitals Fingers Leg Thumb

Eye Nose Face

runs through the postcentral gyrus of the cerebral cortex, shown as a blue band on the image of the brain. B, The plane of section runs through the precentral gyrus of the cerebral cortex, shown as a violet band on the image of the brain. (Data from Penfield W, Rasmussen T: The Cerebral Cortex of Man. New York, Macmillan, 1952.)

Foot Toes

Upper lips Lips Lower lips Teeth

spinal cord, the brainstem, and the thalamus as well as in the somatosensory cortex. Within the cortex, the somatic sensory system has several maps of the body, each unique and each concerned with different types of somatotopic information. Multiple maps are the rule in the brain.

The cerebral cortex has a motor map that is adjacent to and well aligned with the somatosensory map

Gums Jaw Tongue Midline

Pharynx Intra-abdominal

B

Figure 16-10  Somatosensory and motor maps. A, The plane of section

MOTOR

Lateral

Hand Wrist Little Ring Elbow Middle Shoulder Trunk Index

Thumb Neck

Hip

Eyebrow

Ankle

Eyelid and eyeball Face

Toes

Lips Jaw Tongue Swallowing Midline Lateral

Neural maps are not limited to sensory systems; they also appear regularly in brain structures that are considered to have primarily motor functions. Studies done in the 1860s by Fritsch and Hitzig showed that stimulation of particular parts of the cerebral cortex evokes specific muscle contractions in dogs. Penfield and colleagues generated maps of the primary motor cortex in humans (see Fig. 16-10B) by microstimulating and observing the evoked movements. They noted an orderly relationship between the site of cortical stimulation and the body part that moved. Penfield’s motor maps look remarkably like his somatosensory maps, which lie in the adjacent cortical gyrus (see Fig. 16-10A). Note that the sensory and motor maps are adjacent and similar in basic layout (legs represented medially and head laterally), and both have a striking magnification of the head and hand regions. Not surprisingly, there are myriad axonal interconnections between the primary motor and primary somatosensory areas. However, functional magnetic resonance imaging of the human motor cortex shows that the motor map for hand movements is not nearly as simple and somatotopic as Penfield’s drawings might imply. Movements of individual fingers or the wrist that are initiated by the individual activate specific and widely distributed regions of motor cortex, but these regions also overlap one another. Rather than following an obvious somatotopic progression, it instead appears that neurons in the arm area of the motor cortex form distributed and cooperative networks that control collections of arm muscles. Other regions of the motor cortex also have a distributed organization when they are examined on a fine scale, although Penfield’s somatotopic maps still suffice to describe the gross organization of the motor cortex. In other parts of the brain, motor and sensory functions may even occupy the same tissue, and precise alignment of the motor and sensory maps is usually the case. For example, a paired midbrain structure called the superior colliculus receives direct retinotopic connections from the retina as well as input from the visual cortex. Accordingly, a spot of light in the visual field activates a particular patch of neurons in the colliculus. The same patch of collicular neurons can also command, through other brainstem connections, eye and head movements that bring the image of the light spot into the center of the visual field so that it is imaged onto the

CHAPTER 16  •  Circuits of the Central Nervous System

A

VISUAL MAP

l Nasa al r o p Tem

erio Infe r rior

Superior colliculus of a cat

S up

fovea. The motor map for orientation of the eyes is in precise register with the visual response map. In addition, the superior colliculus has maps of both auditory and somatosensory information superimposed on its visual and motor maps; the four aligned maps work in concert to represent points in polysensory space and help control an animal’s orienting responses to prominent stimuli (Fig. 16-11).

403

Sensory and motor maps are fuzzy and plastic B

l Nasa ral o p Tem

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Superior Inferior

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SOMATOSENSORY MAP

l

Vent ra

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We have described a sample of the sensory and motor maps in the brain, but we are left to wonder just why neural maps are so ubiquitous, elaborate, and varied. What is the advantage of mapping neural functions in an orderly way? You could imagine other arrangements: spatial information might be widely scattered about on a neural structure, much as the bytes of one large digital file may be scattered across the array of memory elements in a computer. Various explanations may be proposed for the phenomenon of orderly mapping in the nervous system, although most remain speculations. Maps may be the most efficient way of generating nearest-neighbor relationships between neurons that must be interconnected for proper function. For example, the collicular neurons that participate in sensing stimuli 10 degrees up and 20 degrees to the left and other collicular neurons that command eye movements toward that point undoubtedly need to be strongly interconnected. Orderly collicular mapping enforces togetherness for those cells and minimizes the length of axons necessary to interconnect them. In addition, if brain structures are arranged topographically, neighboring neurons will be most likely to become activated synchronously. Neighboring neurons are very likely to be interconnected in structures such as the cortex, and their synchronous activity serves to reinforce the strength of their interconnections because of the inherent rules governing synaptic plasticity (see pp. 328–333). An additional advantage of mapping is that it may simplify establishment of the proper connections between neurons during development. For example, it is easier for an axon from neuron A to find neuron B if distances are short. Maps may thus make it easier to establish interconnections precisely among the neurons that represent the three sensory maps and one motor map in the superior colliculus. Another advantage of maps may be to facilitate the effectiveness of inhibitory connections. Perception of the edge of a stimulus (edge detection) is heightened by lateral connections that suppress the activity of neurons representing the space slightly away from the edge. If sensory areas are mapped, it is a simple matter to arrange the inhibitory connections onto nearby neurons and thereby construct an edge-detector circuit. It is worth clarifying several general points about neural maps. “The map is not the territory,” as the philosopher Alfred Korzybski pointed out. In other words, all maps, including neural maps, are abstract representations. They are also distorted by the shortcomings of particular experimental measurements. A problem with neural maps is that different experimenters, using different methods, may sometimes generate quite different maps of the same part of the brain. As more and better-refined methods become available, our understanding of these maps is evolving. Moreover,

Here we show all three maps superimposed. Rostral D

SUPERIMPOSITION

Lateral

Medial

Caudal Figure 16-11  Polysensory space in the superior colliculus. A, The representation of visual space projected onto the right superior colliculus of a cat. Note that visual space is divided into nasal versus temporal space and superior versus inferior space. B and C, Comparable auditory and somatosensory maps, respectively. D, Superimposition of the preceding three maps. Note the approximate correspondence among the visual (red), auditory (green), and somatosensory (blue) maps. The motor map for orienting the eyes (not shown) is in almost perfect register with the visual map in A. (Data from Stein BE, Wallace MT, Meredith MA: Neural mechanisms mediating attention and orientation to multisensory cues. In Gazzaniga M [ed]: The Cognitive Neurosciences. Cambridge, MA, MIT Press, 1995.)

404 A

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SOMATOSENSORY REORGANIZATION

Normal organization

Owl monkey

Median (input) nerve cut

Dorsum

3b (S1 proper)

D4

Body

Dorsum H

Face

I

P3 I P2 P1 T

P2 D2

I

P3

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Deprived

P3 H

D1

Portion—corresponding to the palm—deprived by the median nerve section…

...now corresponds to the dorsal (hairy) skin of the first three digits.

MOTOR-SYSTEM REORGANIZATION un

k

Hind limb

Hind limb

FL N

Jaw

Jaw

Normal organization

Facial nerve cut

Weeks

elim

b

Ey

For

Ey

e

elim

b

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For

iss

e

br

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Vi

b elim For

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M1

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Ey

iss

e

a

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Tr

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k

Hind limb

Tr

k

M1

Tr

Rat cortex

un

B

I

D4

P3

H P4

D5

I

D3

P1 T

D1

D1

D4

Dorsum

H P4

D5

I

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D3

D5 D4 D3 D2

P4

Dorsum

H P4

D5

Reorganization

Jaw

Reorganization

Figure 16-12  Plasticity of maps. A, The first panel on the right, labeled “Normal organization,” shows the somatotopic organization of the right hand in the left somatosensory cortex of the monkey brain. The colors correspond to different regions of the hand (viewed from the palm side, except for portions labeled “Dorsum”). The second panel shows (in gray) the territory that is deprived of input by sectioning the median nerve. The third panel shows that the cortical map is greatly changed several months after nerve section. The nerve was not allowed to regrow, but the previously deprived cortical region now responds to the dorsal skin of D3, D2, and D1. Notice that responses to regions P1, P2, and T have disappeared; region I has encroached; and regions H and P3 have suddenly appeared at a second location. B, The first panel on the right, labeled “Normal organization,” shows the somatotopic organization of the left motor cortex (M1) of the rat brain. The colors correspond to the muscles that control different regions of the body. The second panel shows (in gray) the territory that normally provides motor output to the facial nerve, which has been severed. The third panel shows that, after several weeks, the deprived cortical territory is now remapped. Notice that the deprived territory that once evoked whisker movements now evokes eye, eyelid, and forelimb movements. FL, additional representation of forelimb; N, neck area. (A, Data from Kaas JH: The reorganization of sensory and motor maps in adult mammals. In Gazzaniga M [ed]: The Cognitive Neurosciences. Cambridge, MA, MIT Press, 1995; B, data from Sanes J, Suner S, Donoghue JP: Dynamic organization of primary motor cortex output to target muscles in adult rats: Long-term patterns of reorganization following motor or mixed peripheral nerve lesions. Exp Brain Res 79:479–491, 1990.)

the brain itself muddies its maps. Maps of sensory space onto a brain area are not point-to-point representations. On the contrary, a point in sensory space (e.g., a spot of light) activates a relatively large group of neurons in a sensory region of the brain. However, such activation of many neurons is not due to errors of connectivity; the spatial dissemination of activity is part of the mechanism used to encode and to process information. The strength of activation is most intense within the center of the activated neuronal group, but the population of more weakly activated neurons may encompass a large portion of an entire brain. This diversity in strength of activation means that a point in sensory space is unlikely to be encoded by the activity of a single neuron;

instead it is represented by the distributed activity in a large population of neurons. Such a distributed code has computational advantages, and some redundancy also guards against errors, damage, and loss of information. Finally, maps may change with time. All sensory and motor maps are clearly dynamic and can be reorganized rapidly and substantially as a function of development, behavioral state, training, or damage to the brain or periphery. Such changes are referred to as plasticity. Figure 16-12 illustrates two examples of dramatic changes in neocortical mapping, one sensory and one motor, after damage to peripheral nerves. In both cases, severing a peripheral nerve causes the part of the map that normally relates to the body

CHAPTER 16  •  Circuits of the Central Nervous System

part served by this severed nerve to become remapped to another body part. Although the mechanisms of these reorganizations are only partially known, they probably reflect the same types of processes that underlie our ability to learn sensorimotor skills with practice and to adjust and improve after neural damage from trauma or stroke.

Neural circuits are very good at resolving time intervals, in some cases down to microseconds or less. One of the most demanding tasks of timing is performed by the auditory system as it localizes the source of certain sounds. Sound localization is an important skill, whether you are prey, predator, or pedestrian. Vertebrates use several different strategies for localization of sound, depending on the species, the frequency of the sound, and whether the task is to localize the source in the horizontal (left-right) or vertical (up-down) plane. In this subchapter, we briefly review general strategies of sound localization and then explain the mechanism by which a brainstem circuit measures the relative timing of low-frequency sounds so that the source of the sounds can be localized with precision. Sound localization along the vertical plane (the degree of elevation) depends, in humans at least, on the distinctive shape of the external ear, the pinna. Much of the sound that we hear enters the auditory canal directly, and its energy is transferred to the cochlea. However, some sound reflects off the curves and folds of the pinna and tragus before it enters the canal and thus takes slightly longer to reach the cochlea. Notice what happens when the vertical direction of the sound changes. Because of the arcing shape of the pinna, the reflected path of sounds coming from above is shorter than that of sounds from below (Fig. 16-13). The two sets of sounds (the direct and, slightly delayed, the reflected) combine to create sounds that are slightly different on entering the auditory canal. Because of the interference patterns created by the direct and reflected sounds, the combined sound has spectral properties that are characteristic of the elevation of the sound source. This mechanism of vertical sound localization works well even with one ear at a time, although its precise neural mechanisms are not clear. For humans, accurate determination of the direction of a sound along the horizontal plane (the azimuth) requires two working ears. Sounds must first be processed by the cochlea in each ear and then compared by neurons within the CNS to estimate horizontal direction. But what exactly is compared? For sounds that are relatively high in frequency (~2 to 20 kHz), the important measure is the interaural (i.e., ear-to-ear) intensity difference. Stated simply, the ear facing the sound hears it as louder than the ear facing away because the head casts a “sound shadow” (Fig. 16-14A). If the sound is directly to the right or left of the listener, this difference is maximal; if the sound is straight ahead, no difference is heard; and if the sound comes from an oblique direction, intensity differences are intermediate. Note that this system can be fooled. A sound source straight ahead gives the

Pinna

Path 1 direct sound Path 1 reflected sound

TEMPORAL REPRESENTATIONS: TIME-MEASURING CIRCUITS To localize sound, the brain compares the timing and intensity of input to the ears

405

Tragus Auditory canal

Path 2 direct sound Path 2 reflected sound

Path 3 reflected sound Path 3 direct sound

Figure 16-13  Detection of sound in the vertical plane. The detection of sound in the vertical plane requires only one ear. Regardless of the source of a sound, the sound reaches the auditory canal by both direct and reflected pathways. The brain localizes the source of the sound in the vertical plane by detecting differences in the combined sounds from the direct and reflected pathways.

same intensity difference (i.e., none) as a sound source directly behind. The interaural intensity difference is not helpful at lower frequencies. Sounds below ~2 kHz have a wavelength that is longer than the width of the head itself. Longer sound waves are diffracted around the head, and differences in interaural intensity no longer occur. At low frequencies, the nervous system uses another strategy—it measures interaural delay (see Fig. 16-14B). Consider a 200-Hz sound coming directly from the right. Its peak-to-peak distance (i.e., the wavelength) is ~172 cm, which is considerably more than the 20-cm width of the head. Each sound wave peak will reach the right ear ~0.6 ms before it reaches the left ear. If the sound comes from a 45-degree angle ahead, the interaural delay is ~0.3 ms; if it comes from straight ahead (or directly behind), the delay is 0 ms. Delays of small fractions of a millisecond are well within the capabilities of certain brainstem auditory neurons to detect. Sounds need not be continuous for the interaural delay to be detected. Sound onset or offset, clicks, or any abrupt changes in the sound give opportunities for interaural time comparisons. Obviously, measurement of interaural delay is subject to the same front-back ambiguity as interaural intensity, and indeed, it is sometimes difficult to distinguish whether a sound is in front of or behind your head.

The brain measures interaural timing by a combination of neural delay lines and coincidence detectors How does the auditory system measure interaural timing? Surprisingly, to detect very small time differences, the nervous system uses a precise arrangement of neurons in space. Figure 16-15A summarizes the neuroanatomy of the

406 A

SECTION III  •  The Nervous System

HIGH-FREQUENCY SOUND

Sound shadow

B

The CNS detects a difference in intensity between the two ears.

Sound waves

LOW-FREQUENCY SOUND

The CNS detects the sound delay between the two ears.

Sound waves 0.6 ms 172 cm (200 Hz) Figure 16-14  Sound detection in a horizontal plane. A, Two ears are necessary for the detection of sound in a horizontal plane. For frequencies between 2 kHz and 20 kHz, the CNS detects the ear-to-ear intensity difference. In this example, the sound comes from the right. The left ear hears a weaker sound because it is in the shadow of the head. B, For frequencies 0 ∆V>0 ∆P>0

Compliance = ∆V ∆P Figure 17-9  Compliance: changes in pressure with vessels of different compliances.

we assume that the entire body is at the level of the heart, we do not need to add a hydrostatic pressure component to the various intravascular pressures. Thus, the mean pressure in the aorta is 95 mm Hg, and—because it takes a driving pressure of ~5 mm Hg to pump blood into the end of the large arteries—the mean pressure at the end of the large arteries in the foot and head is 90 mm Hg. Similarly, the mean pressure in the large veins draining the foot and head is 5 mm Hg, and—because it takes a driving pressure of ~3 mm Hg to pump blood to the right atrium—the mean pressure in the right atrium is 2 mm Hg. When a 180-cm tall person is standing (see Fig. 17-8B), we must add a 130-cm column of blood (the Δh between the heart and large vessels in the foot) to the pressure pre­vailing in the large arteries and veins of the foot. Because a water column of 130 cm is equivalent to 95 mm Hg, the mean pressure for a large artery in the foot will be 90 + 95 = 185 mm Hg, and the mean pressure for a large vein in the foot will be 5 + 95 = 100 mm Hg. On the other hand, we must subtract a 50-cm column of blood from the pressure prevailing in the head. Because a water column of 50 cm is equivalent to 37 mm Hg, the mean pressure for a large artery in the head will be 90 − 37 = 53 mm Hg, and the mean pressure for a large vein in the head will be 5 − 37 = −32 mm Hg. Of course, this “negative” value really means that the pressure in a large vein in the head is 32 mm Hg lower than the reference pressure at the level of the heart. In this example, we have simplified things somewhat by ignoring the valves that interrupt the blood column. In reality, the veins of the limbs have a series of one-way valves that allow blood to flow only toward the heart. These valves act like a series of relay stations, so that the contraction of skeletal muscle around the veins pushes blood from one valve to another (see p. 516). Thus, veins in the foot do not “see” the full hydrostatic column of 95 mm Hg when the leg muscles pump blood away from the foot veins. Although the absolute arterial and venous pressures are much higher in the foot than in the head, the ΔP that drives blood flow is the same in the vascular beds of the foot and head. Thus, in the horizontal position, the ΔP across the vascular beds in the foot or head is 90 − 5 = 85 mm Hg. In the upright position, the ΔP for the foot is 185 − 100 = 85 mm Hg, and for the head, 53 − (−32) = 85 mm Hg. Thus,

gravity does not affect the driving pressure that governs flow. On the other hand, in “dependent” areas of the body (i.e., vessels “below” the heart in a gravitational sense), the hydrostatic pressure does tend to increase the transmural pressure (intravascular versus extravascular “tissue” pressure) and thus the diameter of distensible vessels. Because various anatomical barriers separate different tissue compartments, it is assumed that gravity does not appreciably affect this tissue pressure.

Low compliance of a vessel causes the transmural pressure to increase when the vessel blood volume is increased Until now, we have considered blood vessels to be rigid tubes, which, by definition, have fixed volumes. If we were to try to inject a volume of fluid into a truly rigid tube with closed ends, we could in principle increase the pressure to infinity without increasing the volume of the tube (Fig. 17-9A). At the other extreme, if the wall of the tube were to offer no resistance to deformation (i.e., infinite compliance), we could inject an infinite volume of fluid without increasing the pressure at all (see Fig. 17-9B). Blood vessels lie between these two extremes; they are distensible but have a finite compliance (see p. 454). Thus, if we were to inject a volume of blood into the vessel, the volume of the vessel would increase by the same amount (ΔV), and the intravascular pressure would also increase (see Fig. 17-9C). The ΔP accompanying a given ΔV is greater if the compliance of the vessel is lower. The relationship between ΔP and ΔV is a static property of the vessel wall and holds whether or not there is flow in the vessel. Thus, if we were to infuse blood into a patient’s blood vessels, the intravascular pressure would rise throughout the circulation, even if the heart were stopped.

The viscous resistance of blood causes an axial pressure difference when there is flow As we saw in Ohm’s law of hydrodynamics (see Equation 17-1), during steady flow down the axis of a tube (see Fig. 17-2), the driving pressure (ΔP) is proportional to both flow and resistance. Viewed differently, if we want to achieve a constant flow, then the greater the resistance, the greater the

420

SECTION IV  •  The Cardiovascular System

ΔP that we must apply along the axis of flow. Of the four sources of pressure in the circulatory system, this ΔP due to viscous resistance is the only one that appears in Poiseuille’s law (see Equation 17-9).

P = 100

The inertia of the blood and vessels causes pressure to decrease when the velocity of blood flow increases For the most part, we have been assuming that the flow of blood as well as its mean linear velocity is steady. However, as we have already noted, blood flow in the circulation is not steady; the heart imparts its energy in a pulsatile manner, with each heartbeat. Therefore, v in the aorta increases and reaches a maximum during systole and falls off during diastole. As we shall shortly see, these changes in velocity lead to compensatory changes in intravascular pressure. The tradeoff between velocity and pressure reflects the conversion between two forms of energy. Although we generally state that fluids flow from a higher to a lower pressure, it is more accurate to say that fluids flow from a higher to a lower total energy. This energy is made up of both the pressure or potential energy and the kinetic energy (KE = 1 2 mv 2). The impact of the interconversion between these two forms of energy is manifested by the familiar Bernoulli effect. As fluid flows along a horizontal tube with a narrow central region, which has half the diameter of the two ends, the pressure in the central region is actually lower than the pressure at the distal end of the tube (Fig. 17-10). How can the fluid paradoxically flow against the pressure gradient from the lower-pressure central to the higherpressure distal region of the tube? We saw above that flow is the product of cross-sectional area and velocity (see Equation 17-8). Because the flow is the same in both portions of the tube, but the cross-sectional area in the center is lower by a factor of 4, the velocity in the central region must be 4-fold higher (see table at bottom of Fig. 17-10). Although the blood in the central region has a lower potential energy (pressure = 60) than the blood at the distal end of the tube (pressure = 80), it has a 16-fold higher kinetic energy. Thus, the total energy of the fluid in the center exceeds that in the distal region, so that the fluid does indeed flow down the energy gradient. This example illustrates an interconversion between potential energy (pressure) and kinetic energy (velocity) in space because velocity changes along the length of a tube even though flow is constant. We will see on pages 511–513 that during ejection of blood from the left ventricle into the aorta, the flow and velocity of blood change with time at any point within the aorta. These changes in velocity contribute to the changes in pressure inside the aorta. The Bernoulli effect has important practical implications for measurement of blood pressure with an open-tipped catheter. The pressure recorded with the open tip facing the flow is higher than the actual pressure by an amount corresponding to the kinetic energy of the oncoming fluid (Fig. 17-11). Conversely, the pressure recorded with the open tip facing away from the flow is lower than the actual pressure by an equal amount. The measured pressure is correct only when the opening is on the side of the catheter, perpendicular to the flow of blood.

P = 90

P = 80

v

P = 100

P = 80 P = 60

v

A

A/ 4

Cross-sectional area (A)

1

1 4

1

P

100

60

80

Velocity (v )

2

8

2

Kinetic energy (ρv 2 /2)

2

32

2

Total energy (E)

102

92

82

Figure 17-10  Bernoulli effect. For the top tube, which has a uniform radius, velocity (v) is uniform and transmural pressure (P) falls linearly with the length, which we artificially compress to fit in the available space. The bottom tube has the same upstream and downstream pressures but a constriction in the middle that is short enough so as not to increase overall resistance or overall fall in P. The constriction has crosssectional area that is only one fourth that of the two ends. Thus, velocity in the narrow portion must be 4-fold higher than it is at the ends. Although the total energy of fluid falls linearly along the tube, pressure is lower in the middle than at the distal end.

HOW TO MEASURE BLOOD PRESSURE, BLOOD FLOW, AND CARDIAC VOLUMES Blood pressure can be measured directly by puncturing the vessel One can record blood pressure anywhere along the circulation—inside a heart chamber, inside an artery, within a capillary, or within a vein. Clinicians are generally concerned with the intravascular pressure at a particular site (e.g., in a systemic artery) in reference to the barometric pressure outside the body and not with pressure differences between two sites. The most direct approach for measurement of pressure is to introduce a needle or a catheter into a vessel and position

CHAPTER 17  •  Organization of the Cardiovascular System

97 mm Hg Facing upstream

Blood

93 mm Hg Facing downstream

95 mm Hg Side pressure

Figure 17-11  Effects of kinetic energy on the measurement of blood pressure with catheters.

Strain gauge on flexible diaphragm

Brachial artery

Catheter

Membrane Amplifier

Recorder

Figure 17-12  Direct method for determining blood pressure.

the open tip at a particular site. In the first measurements of blood pressure ever performed, Stephen Hales in 1733 found that a column of blood from a presumably agitated horse rose to fill a brass pipe to a height of 3 m. It was Poiseuille who measured blood pressure for the first time by connecting a mercury-filled U-tube to arteries through a tube containing a solution of saturated NaHCO3. In modern times, a saline-filled transmission or conduit system connects the blood vessel to a pressure transducer. In the most primitive form of this system, a catheter was connected to a closed chamber, one wall of which was a deformable diaphragm. Nowadays, the pressure transducer is a stiff diaphragm bonded to a strain gauge that converts mechanical strain into a change in electrical resistance, capacitance, or inductance (Fig. 17-12). The opposite face of the diaphragm is open to the atmosphere, so that the blood pressure is referenced to barometric pressure. The overall performance of the system depends largely on the properties of the catheter and the strain gauge. The presence of air bubbles and a long or

421

narrow catheter can decrease the displacement, velocity, and acceleration of the fluid in the catheter. Together, these properties determine overall performance characteristics such as sensitivity, linearity, damping of the pressure wave, and frequency response. To avoid problems with fluid transmission in the catheter, some high-fidelity devices employ a solidstate pressure transducer at the catheter tip. In catheterizations of the right heart, the clinician begins by sliding a fluid-filled catheter into an antecubital vein and, while continuously recording pressure, advances the catheter tip into the superior vena cava, through the right atrium and the right ventricle, and past the pulmonary valve into the pulmonary artery. Eventually, the tip reaches and snugly fits into a smaller branch of the pulmonary artery, recording the pulmonary wedge pressure (see p. 519). The wedge pressure effectively measures the pressure downstream from the catheter tip, that is, the left atrial pressure. In catheterizations of the left heart, the clinician slides a catheter into the brachial artery or femoral artery, obtaining the systemic arterial blood pressure. From there, the catheter is advanced into the aorta, the left ventricle, and finally the left atrium. Clinical measurements of venous pressure are typically made by inserting a catheter into the jugular vein. Because of the low pressures, these venous measurements require very sensitive pressure transducers or water manometers. In the research laboratory, one can measure capillary pressure in exposed capillary beds by inserting a micropipette that is pressurized just enough (with a known pressure) to keep fluid from entering or leaving the pipette.

Blood pressure can be measured indirectly by use of a sphygmomanometer In clinical practice, one may measure arterial pressure indirectly by use of a manual sphygmomanometer (Fig. 17-13). An inextensible cuff containing an inflatable bag is wrapped around the arm (or occasionally, the thigh). Inflation of the bag by means of a rubber squeeze bulb to a pressure level above the expected systolic pressure occludes the underlying brachial artery and halts blood flow downstream. The pressure in the cuff, measured by means of a mercury or aneroid manometer, is then allowed to slowly decline (see Fig. 17-13, diagonal red line). The physician can use either of two methods to monitor the blood flow downstream of the slowly deflating cuff. In the palpatory method, the physician detects the pulse as an indicator of flow by feeling the radial artery at the wrist. In the auscultatory method, the physician detects flow by using a stethoscope to detect the changing character of Korotkoff sounds over the brachial artery in the antecubital space. The palpatory method permits determination of the systolic pressure; that is, the pressure in the cuff below which it is just possible to detect a radial pulse. Because of limited sensitivity of the finger, palpation probably slightly underestimates systolic pressure. The auscultatory method permits the detection of both systolic and diastolic pressure. The sounds heard during the slow deflation of the cuff can be divided into five phases (see Fig. 17-13). During phase I, there is a sharp tapping sound, indicating that a spurt of blood is escaping under the cuff when cuff pressure is just

422

SECTION IV  •  The Cardiovascular System

The systolic pressure corresponds to the first tapping sound. 110

Time

130

90 70

110

50

Cuff pressure (mm Hg) 90

30 10

Mercury reservoir Inflation bulb

Sphygmomanometer cuff

14 mm Hg

20 mm Hg 6 mm Hg 5 mm Hg

Arterial 70 pressure

Cuff pressure

10

The diastolic pressure corresponds to the muffling of the sounds.

Relative intensity 5 of sounds

0

Silence

Phase I

II

V

e nc le Si g flin uf ng pi

IV

um

s

ur

m

ur

M

g in pp s Ta ne to

III

M

Palpation of radial artery

Th

Auscultation of brachial artery

40 mm Hg

Figure 17-13  Sphygmomanometry. The clinician inflates the cuff to a pressure that is higher than the anticipated systolic pressure and then slowly releases the pressure in the cuff.

below systolic pressure. The pressure at which these taps are first heard closely represents systolic pressure. In phase II, the sound becomes a blowing or swishing murmur. During phase III, the sound becomes a louder thumping. In phase IV, as the cuff pressure falls toward the diastolic level, the sound becomes muffled and softer. Finally, in phase V, the sound disappears. Although some debate persists about whether the point of muffling or the point of silence is the correct diastolic pressure, most favor the point of muffling as being more consistent. Actual diastolic pressure may be somewhat overestimated by the point of muffling but underestimated by the point of silence. Practical problems arise when a sphygmomanometer is used with children or obese adults or when it is used to obtain a measurement on a thigh. Ideally, one would like to use a pressure cuff wide enough to ensure that the pressure inside the cuff is the same as that in the tissue surrounding the artery. In 1967, the American Heart Association recommended that the pneumatic bag within the cuff be 20% wider than the diameter of the limb, extend at least halfway around the limb, and be centered over the artery. More recent studies indicate that accuracy and reliability improve when the pneumatic bag completely encircles the limb, as long as the width of the pneumatic bag is at least the limb diameter.

Blood flow can be measured directly by electromagnetic and ultrasound flowmeters The spectrum of blood flow measurements in the circulation ranges from determinations of total blood flow (cardiac

output) to assessment of flow within an organ or a particular tissue within an organ. Moreover, one can average blood flow measurements over time or record continuously. Examples of continuous recording include recordings of the phasic blood flow that occurs during the cardiac cycle or any other periodic event (e.g., breathing). We discuss both invasive and noninvasive approaches. Invasive Methods  Invasive approaches require direct access to the vessel under study and are thus useful only in research laboratories. The earliest measurements of blood flow involved collecting venous outflow into a graduated cylinder and timing the collection with a stopwatch. This direct approach was limited to short time intervals to minimize blood loss and the resulting changes in hemodynamics. Blood loss could be avoided by ingenious but now antiquated devices that returned the blood to the circulation, in either a manual or a semiautomated fashion. The most frequently used modern instruments for measurement of blood flow in the research laboratory are electromagnetic flowmeters based on the electromagnetic induction principle (Fig. 17-14). The vessel is placed in a magnetic field. According to Faraday’s induction law, moving any conductor (including an electrolyte solution, such as blood) at right angles to lines of the magnetic field generates a voltage difference between two points along an axis perpendicular to both the axis of the movement and the axis of the magnetic field. The induced voltage is



E = BvD

(17-14)

CHAPTER 17  •  Organization of the Cardiovascular System

sections include discussions of two indirect methods that clinicians use to measure mean blood flow.

The movement of an electrical conductor (i.e., blood in a vessel) through a magnetic field induces a voltage between two points (e1 and e2) along an axis that is mutually perpendicular to both the axis of the magnetic field and the axis of blood flow. Axis of magnetic field

Cardiac output can be measured indirectly by the Fick method, which is based on the conservation of mass The Fick method requires that a substance be removed from or added to the blood during its flow through an organ. The rate at which X passes a checkpoint in the circulation ( Q ) is simply the product of the rate at which blood volume passes the checkpoint (F) and the concentration of X in that blood:

e1 +

N

S

Q = F ⋅[ X ] moles liters moles Units: = ⋅ s s liter

Voltmeter

D –

e2 Magnet

v

423

Blood vessel Axis of blood flow Figure 17-14  Electromagnetic flowmeter.

where B is the density of magnetic flux, v is the average linear velocity, and D is the diameter of the moving column of blood. Ultrasound flowmeters employ a pair of probes placed at two sites along a vessel. One probe emits an ultrasound signal, and the other records it. The linear velocity of blood in the vessel either induces a change in the frequency of the ultrasound signal (Doppler effect) or alters the transit time of the ultrasound signal. Both the electromagnetic and ultrasound methods measure linear velocity, not flow per se. Noninvasive Methods  The electromagnetic or ultrasonic

flowmeters require the surgical isolation of a vessel. How­ ever, ultrasonic methods are also widely used transcutaneously on surface vessels in humans. This method is based on recording of the backscattering of the ultrasound signal from moving red blood cells. To the extent that the red blood cells move, the reflected sound has a frequency different from that of the emitted sound (Doppler effect). This frequency difference may thus be calibrated to measure flow. Plethysmographic methods are noninvasive approaches for measurement of changes in the volume of a limb or even of a whole person (see p. 617). Inflation of a pressure cuff enough to occlude veins but not arteries allows blood to continue to flow into (but not out of) a limb or an organ, so that the volume increases with time. The record of this rise in volume, as recorded by the plethysmograph, is a measure of blood flow. With the exception of transcutaneous ultrasonography, the direct methods discussed for measurement of blood flow are largely confined to research laboratories. The next two

(17-15)

The Fick principle is a restatement of the law of conservation of mass. The amount of X per unit time that passes a downstream checkpoint (Q B) minus the amount of X that passes an upstream checkpoint (Q A) must equal the amount of X added or subtracted per unit time (Q added subtracted) between these two checkpoints (Fig. 17-15A): Q added subtracted = Q B − Q A



(17-16)

Q added subtracted is positive for the addition of X. If the volume flow is identical at both checkpoints, combining Equation 17-15 and Equation 17-16 yields the Fick equation: Q added subtracted = F ([ X]B − [ X]A )



(17-17)

We can calculate flow from the amount of X added or subtracted and the concentrations of X at the two checkpoints: F=



Q added subtracted [ X]B − [ X]A

=

moles min moles liter

=

liters min

(17-18)

It is easiest to apply the Fick principle to the blood flow through the lungs, which is the cardiac output (see Fig. 17-15B). The quantity added to the bloodstream is the O2 uptake (Q O2) by the lungs, which we obtain by measuring the subject’s O2 consumption. This value is typically 250 mL of O2 gas per minute. The upstream checkpoint is the pulmonary artery (point A), where the O2 content ([O2]A) is typically 15 mL of O2 per deciliter of blood. The sample for this checkpoint must reflect the O2 content of mixed venous blood, obtained by means of a catheter within the right atrium or the right ventricle or pulmonary artery. The downstream checkpoint is a pulmonary vein (point B), where the O2 content ([O2]B) is typically 20 mL O2 per deciliter of blood. We can obtain the sample for this checkpoint from any systemic artery. Using these particular values, we calculate a cardiac output of 5 L/min:



250 mL O2 min Q O2 = [O2 ]B − [O2 ]A (20 − 15) mL O2 dL blood (17-19) = 5 L blood min

F=

424

SECTION IV  •  The Cardiovascular System

A

THE FICK PRINCIPLE ˙ added/subtracted Q

˙B Q

˙A Q

Checkpoint A B

Checkpoint B

MEASUREMENT OF CARDIAC OUTPUT 250 mL O2 / min

[O2]A =15 mL O2 / dL blood Pulmonary artery

˙A Q

Lung [O2]B = 20 mL O2 / dL blood Pulmonary vein

˙B Q

˙O Q 2

Left heart

Right heart

˙A Q Vena cava

˙O Q 2

˙B Q

250 mL O2 / min

Aorta

Tissue Figure 17-15  Fick method for determining cardiac output.

Cardiac output can be measured indirectly by dilution methods The dye dilution method, inaugurated by G.N. Stewart in 1897 and extended by W.F. Hamilton in 1932, is a variation of the Fick procedure. One injects a known quantity of a substance (X) into a systemic vein (e.g., antecubital vein) at site A while simultaneously monitoring the concentration downstream at site B (Fig. 17-16A). It is important that the substance not leave the vascular circuit and that it be easy to follow the concentration, by either successive sampling or continuous monitoring. If we inject a single known amount (QX) of the indicator, an observer downstream at checkpoint B will see a rising concentration of X, which, after reaching its peak, falls off exponentially. Concentration measurements provide the interval (Δt) between the time the dye makes its first appearance at site B and the time the dye finally disappears there. If site B is in the pulmonary artery, then the entire amount QX that we injected into the peripheral vein must pass site B during the interval Δt, carried by the entire cardiac output. We can deduce the average concentration [X] during the interval Δt from the concentration-versus-time curve in Figure 17-16B. From the conservation of mass, we know that

Q X = V ⋅[ X ]

Units: moles = liters ⋅

moles liter

(17-20)

Because the volume of blood (V) that flowed through the pulmonary artery during the interval Δt is, by definition, the product of cardiac output and the time interval (CO · Δt),

QX = CO ⋅ ∆t ⋅[ X] liters moles Units: moles = ⋅s⋅ s liter

(17-21)

Note that the product Δt · [ X ] is the area under the concentration-versus-time curve in Figure 17-16B. Solving for CO, we have

CO =

QX Q = X [ X] ⋅ ∆t Area

(17-22)

In practice, cardiologists monitor [X] in the brachial artery. Obviously, only a fraction of the cardiac output passes through a brachial artery; however, this fraction is the same as the fraction of QX that passes through the brachial artery. If we were to re-derive Equation 17-22 for the brachial artery,

425

CHAPTER 17  •  Organization of the Cardiovascular System

A

PRINCIPLE OF DYE DILUTION

˙X Q Checkpoint A Right heart

Left heart

Brachial artery

Systemic vein

˙A Q

˙ B = >0 Q Checkpoint B Photocell

Pulmonary artery

Dye stream

Lamp Recorder B

C

CONCENTRATION PROFILE AT PULMONARY ARTERY 25

[X]

20

D

CONCENTRATION PROFILE AT BRACHIAL ARTERY (WITHOUT RECIRCULATION)

CONCENTRATION PROFILE AT BRACHIAL ARTERY (WITH RECIRCULATION)

10

10 Mixing in total cardiac output

[X] mg/liter

Amount injected 2

[X]

2

0 3 ∆t

Time (s)

Recirculation

Dispersion

30

0 3

Time (s)

30

2

0 3

Time (s)

30

∆t Figure 17-16  Dye dilution method for determining blood flow. In B, C, and D, the areas underneath the three red curves—as well as the three green areas—are all the same.

we would end up multiplying both the CO and QX terms by this same fraction. Therefore, even though only a small portion of both cardiac output and injected dye passes through any single systemic artery, we can still use Equa­ tion 17-22 to compute cardiac output with data from that artery. Compared with the [X] profile in the pulmonary artery, the [X] profile in the brachial artery is not as tall and is more spread out, so that [X] is smaller and Δt is longer. However, the product [X] · Δt in the brachial artery—or any other systemic artery—is the same as that in the pulmonary artery. Indocyanine green dye (Cardiogreen) is the most common dye employed. Because the liver removes this dye from the

circulation, it is possible to repeat the injections, after a sufficient wait, without progressive accumulation of dye in the plasma. Imagine that after we inject 5 mg of the dye, [X] under the curve is 2 mg/L and Δt is 0.5 min. Thus,

CO =

5 mg 2 mg L × 0.5 min

= 5 L min

(17-23)

A practical problem is that after we inject a marker into a systemic vein, blood moves more quickly through some pulmonary beds than others, so that the marker arrives at checkpoint B at different times. This process, known as dispersion, is the main cause of the flattening of the [X] profile

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SECTION IV  •  The Cardiovascular System

in the brachial artery (see Fig. 17-16C) versus the pulmonary artery (see Fig. 17-16B). If we injected the dye into the left atrium and monitored it in the systemic veins, the dispersion would be far worse because of longer and more varied path lengths in the systemic circulation compared with the pulmonary circulation. In fact, the concentration curve would be so flattened that it would be difficult to resolve the area underneath the [X] profile. A second practical problem with a closed circulatory system is that before the initial [X] wave has waned, recirculation causes the injected indicator to appear for a second time in front of the sensor at checkpoint B (see Fig. 17-16D). Extrapolation of the exponential decay of the first wave can correct for this problem. The thermodilution technique is a convenient alternative approach to the dye technique. In this method, one injects a bolus of cold saline and an indwelling thermistor is used to follow the dilution of these “negative calories” as a change of temperature at the downstream site. In the thermodilution technique, a temperature-versus-time profile replaces the concentration-versus-time profile. During cardiac catheterization, the cardiologist injects a bolus of cold saline into the right atrium and records the temperature change in the pulmonary artery. The distance between upstream injection and downstream recording site is kept short to avoid heat exchange in the pulmonary capillary bed. The advantages of this method are that (1) the injection of cold saline can be repeated without harm, (2) a single venous (versus venous and arterial) puncture allows access to both the upstream and the downstream sites, (3) less dispersion occurs because no capillary beds are involved, and (4) less recirculation occurs because of adequate temperature equilibration in the pulmonary and systemic capillary beds. A potential drawback is incomplete mixing, which may result from the proximity between injection and detection sites.

Regional blood flow can be measured indirectly by “clearance” methods The methods used to measure regional blood flow are often called clearance methods, although the term here has a meaning somewhat different from its meaning in kidney physiology. Clearance methods are another application of the Fick principle, using the rate of uptake or elimination of a substance by an organ together with a determination of the difference in concentration of the indicator between the arterial inflow and venous outflow (i.e., the a-v difference). By analogy with Equation 17-18, we can compute the blood flow through an organ (F) from the rate at which the organ removes the test substance X from the blood (Q X ) and the concentrations of the substance in arterial blood ([X]a) and venous blood ([X]v):

F=

moles min Q X = = liters min (17-24) [ X]a − [ X]v moles liter

One can determine hepatic blood flow with the use of BSP (bromsulphthalein), a dye that the liver almost completely clears and excretes into the bile (see p. 951). Here, Q X is the rate of removal of BSP from the blood, estimated as the rate at which BSP appears in the bile. [X]a is the

BOX 17-2  Thallium Scanning for Assessment of Coronary Blood Flow

T

hallium is an ion that acts as a potassium analog and enters cells through the same channels or transporters as K+ does. Active cardiac muscle takes up injected 201Tl, provided there is adequate blood flow. Therefore, the rate of uptake of the 201Tl isotope by the heart is a useful qualitative measure of coronary blood flow. Complete 201Tl myocardial imaging is possible by two-dimensional scanning of the emitted gamma rays or by computed tomography for a threedimensional image. Thus, in those portions of myocardial tissue supplied by stenotic coronary vessels, the uptake is slower, and these areas appear as defects on a thallium scan. Thallium scans are used to detect coronary artery disease during exercise stress tests.

concentration of BSP in a systemic artery, and [X]v is the concentration of BSP in the hepatic vein. In a similar manner, one can determine renal blood flow with the use of PAH (para-aminohippurate). The kidneys almost completely remove this compound from the blood and secrete it into the urine (see pp. 749–750). It is possible to determine coronary blood flow or regional blood flow through skeletal muscle from the tissue clearance of rapidly diffusing inert gases, such as the radioisotopes 133Xe and 85Kr. Finally, one can use the rate of disappearance of nitrous oxide (N2O), a gas that is historically important as the first anesthetic, to compute cerebral blood flow. A similar although qualitative approach is thallium scanning to assess coronary blood flow. Here one measures the uptake of an isotope by the heart muscle, rather than its clearance (Box 17-2).

Ventricular dimensions, ventricular volumes, and volume changes can be measured by angiography and echocardiography Clinicians can use a variety of approaches to examine the cardiac chambers. Gated radionuclide imaging employs N17-6  compounds of the gamma-emitting isotope 99mTc,  which has a half-life of 6 hours. After 99mTc is injected, a gamma camera provides imaging of the cardiac chambers. Electrocardiogram (ECG) gating (i.e., synchronization to a particular spot on the ECG) allows the apparatus to snap a picture at a specific part of the cardiac cycle and to sum these pictures over many cycles. Because this method does not provide a high-resolution image, it yields only a relative ventricular volume. From the difference between the count at the maximally filled state (end-diastolic volume) and at its minimally filled state (end-systolic volume), the cardiologist can estimate the fraction of ventricular blood that is ejected during systole—the ejection fraction—which is an important measure of cardiac function. Angiography can accurately provide the linear dimensions of the ventricle, allowing the cardiologist to calculate absolute ventricular volumes. A catheter is threaded into either the left or the right ventricle, and saline containing a

CHAPTER 17  •  Organization of the Cardiovascular System

N17-6 

99m

Tc Scanning

Contributed by Emile Boulpaep Several compounds labeled with 99mTc—for instance, technetium Tc 99m sestamibi and technetium Tc 99m tetrofosmin— have been introduced for imaging myocardial perfusion. The 99m Tc label emits gamma radiation at 140 keV by an isomeric transition (indicated by the m in 99m); it has a half-life of 6 hours. Following injection, the initial distribution of these agents in the myocardium is proportional to the relative distribution of myocardial blood flow. The radiochemical enters cardiac myocytes passively in such a way that about 30% to 40% of the chemical is extracted by the myocardium. Extraction may be enhanced by administering nitrates prior to injection. Because the radiochemical leaves the myocyte rather slowly (over several hours), one can perform the imaging with the gamma camera over a time period of hours. Note that absolute measurements of myocardial blood flow would require positron-emission tomography (PET), which can quantitate counts per unit volume of tissue. It is possible to use 99mTc-labeled compounds not only for assessing myocardial perfusion but also for assessing myocardial function. In single-photon emission computed tomography (SPECT), the computer acquires imaging data synchronized with the R wave of the ECG (see Fig. 21-7). This gated imaging allows one to display end-diastolic and endsystolic images along various axes of the heart. These enddiastolic and end-systolic dimensions can then be compared to assess ejection fraction, stroke volume, regional wall motion, and regional wall thickening.

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CHAPTER 17  •  Organization of the Cardiovascular System

contrast substance (i.e., a chemical opaque to x-rays) is injected into the ventricle. This approach provides a twodimensional projection of the ventricular volume as a function of time. In magnetic resonance imaging, the physician obtains a nuclear magnetic resonance (NMR) image of the protons in the water of the heart muscle and blood. However, because standard NMR requires long data-acquisition times, it does not provide good time resolution. Echocardiography, which exploits ultrasonic waves to visualize the heart and great vessels, can be used in two modes. In M-mode echocardiography (M is for motion), the technician places a single transducer in a fixed position on the chest wall and obtains a one-dimensional view of heart components. As shown in the upper portion of Figure 17-17A, the ultrasonic beam transects the anterior wall of the right ventricle, the right ventricle, the septum, the left ventricle, the leaflets of the mitral valve, and the posterior wall of the left ventricle. The lower portion of Figure 17-17A shows the positions of the borders between these structures (x-axis) during a single cardiac cycle (y-axis) and thus how the size of the left ventricle—along the axis of the beam— changes with time. Of course, the technician can obtain other views by changing the orientation of the beam. In two-dimensional echocardiography, the probe auto­ matically and rapidly pivots, scanning the heart in a single anatomical slice or plane (see Fig. 17-17A, area between the two broken lines) and providing a true cross section. This approach is therefore superior to angiography, which provides only a two-dimensional projection. Because cardiac output is the product of heart rate and stroke volume, one can calculate cardiac output from echocardiographic measurements of ventricular end-diastolic and end-systolic volume. A problem common to angiography and M-mode echocardiography is that it is impossible to compute ventricular volume from a single dimension because the ventricle is not a simple sphere. As is shown in Figure 17-17B, the left ventricle is often assumed to be a prolate ellipse, with a long axis L and two short axes D1 and D2. To simplify the calculation and to allow ventricular volume to be computed from a single measurement, it is sometimes assumed that D1 and D2 are identical and that D1 is half of L. Unfortunately, use of this algorithm and just a single dimension, as provided by M-mode echocardiography,  N17-7  often yields grossly erroneous volumes. Use of two-dimensional echocardiography to sum information from several parallel slices through the ventricle, or from planes that are at a known angle to one another, can yield more accurate volumes. In addition to ultrasound methods and angiography, the technique of magnetic resonance angiography, an application of magnetic resonance tomography, is used to obtain twodimensional images of slices of ventricular volumes or of blood vessels. In contrast to standard echocardiography, Doppler echocardiography provides information on the velocity, direction, and character of blood flow, just as police radar monitors traffic. In Doppler echocardiography (as with police radar), most information is obtained with the beam parallel to the flow of blood. In the simplest application of Doppler flow measurements, one can continuously monitor the velocity of flowing blood in a blood vessel or part of the heart. On such a record, the x-axis represents time, and the y-axis

A

427

PRINCIPLE OF ECHOCARDIOGRAPHY Right ventricle

Aorta

Transducer can rotate to produce views of other axes.

Mitral valves

Left ventricle

Sound waves Reflected waves 0

Time (s)

0.5

1.0

B

Motion of boundaries

ASSUMED VENTRICULAR GEOMETRY

L D1 D2

Figure 17-17  M-mode and two-dimensional echocardiography. In A, the tracing on the bottom shows the result of an M-mode echocardiogram (i.e., transducer in a single position) during one cardiac cycle. The waves represent motion (M) of heart boundaries transected by a stationary ultrasonic beam. In two-dimensional echocardiography (upper panel), the probe rapidly rotates between the two extremes (broken lines), producing an image of a slice through the heart at one instant in time.

represents the spectrum of velocities of the moving red blood cells (i.e., different cells can be moving at different velocities). Flow toward the transducer appears above baseline, whereas flow away from the transducer appears below baseline. The intensity of the record at a single point on the y-axis (encoded by a gray scale or false color) represents the strength of the returning signal, which depends on the number of red blood cells moving at that velocity. Thus, Doppler echocardiography is able to distinguish the

428

SECTION IV  •  The Cardiovascular System

B

A

Figure 17-18  The colors, which encode the velocity of blood flow, are superimposed on a two-dimensional echocardiogram, which is shown in a gray scale. A, Blood moves through the mitral valve and into the left ventricle during diastole. Because blood is flowing toward the transducer, its velocity is encoded as red. B, Blood moves out of the ventricle and toward the aortic valve during systole. Because blood is flowing away from the transducer, its velocity is encoded as blue. (From Feigenbaum H: Echocardiography. In Braunwald E [ed]: Heart Disease: A Textbook of Cardiovascular Medicine, 5th ed. Philadelphia, WB Saunders, 1997.)

character of flow: laminar versus turbulent. Alternatively, at one instant in time, the Doppler technician can scan a region of a vessel or the heart, obtaining a two-dimensional, color-encoded map of blood velocities. If we overlay such two-dimensional Doppler data on a two-dimensional echocardiogram, which shows the position of the vessel or cardiac structures, the result is a color flow Doppler echocardiogram (Fig. 17-18).

Finally, a magnetic resonance scanner can also be used in two-dimensional phase-contrast mapping to yield quantitative measurements of blood flow velocity.

REFERENCES The reference list is available at www.StudentConsult.com.

CHAPTER 17  •  Organization of the Cardiovascular System

N17-7  Ventricular Volume from M-Mode Echocardiography Contributed by Emile Boulpaep As shown in Figure 17-17B, the left ventricle is often assumed to be a prolate ellipse, with a long axis L and two short axes D1 and D2. To simplify the calculation, and to allow ventricular volume to be computed from a single measurement, it is sometimes assumed that D1 and D2 are identical, and that D1 is half of L. Unfortunately, use of this algorithm and just a single dimension, as provided by M-mode echocardiography, often yields grossly erroneous volumes. One can obtain a more accurate estimate of ventricular volume by including an independent measurement of a second dimension, as is done in two-dimensional echocardiography. For example, one could obtain the long axis (L) in addition to the short axes (D1 and D2, which are assumed to be the same in the simple calculation). However, the ventricle often does not resemble a prolate ellipse, certainly not in pathological states. Thus, cardiologists have used more complex geometric models (e.g., bullet shape).

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428.e2

SECTION IV  •  The Cardiovascular System

REFERENCES Books and Reviews Badeer HS: Hemodynamics for medical students. Adv Physiol Educ 25:44–52, 2001. Caro CG, Pedley TJ, Schroter RC, Seed WA: The Mechanics of the Circulation. Oxford, UK, Oxford University Press, 1978. Lassen NA, Henriksen O, Sejrsen P: Indicator methods for measurement of organ and tissue blood flow. In Handbook of Physiology, Section 2: The Cardiovascular System, vol 3. Bethesda, MD, American Physiological Society, 1979, pp 21–63. Levine RA, Gillam LD, Weyman AE: Echocardiography in cardiac research. In Fozzard HA, Haber E, Jennings RB, et al (eds): The Heart and Cardiovascular System. New York, Raven Press, 1986, pp 369–452. Maeda N, Shiga T: Velocity of oxygen transfer and erythrocyte rheology. News Physiol Sci 9:22–27, 1994. Rowland T, Obert P: Doppler echocardiography for the estimation of cardiac output with exercise. Sports Med 32:973–986, 2002. Journal Articles Coulter NA Jr, Pappenheimer JR: Development of turbulence in flowing blood. Am J Physiol 159:401–408, 1949.

Cournand A, Ranges HA: Catheterization of the right auricle. Proc Soc Exp Biol Med 46:462–466, 1941. Fähraeus R, Lindqvist T: The viscosity of the blood in narrow capillary tubes. Am J Physiol 96:562–568, 1931. Hamilton WF, Moore JW, Kinsman JM, Spurling RG: Studies on the circulation. IV. Further analysis of the injection method and of changes in hemodynamics under physiological and pathological conditions. Am J Physiol 99:534–551, 1932. Haynes RH: Physical basis of the dependence of blood viscosity on tube radius. Am J Physiol 198:1193–1200, 1960. Poiseuille JLM: Recherches expérimentales sur le mouvement des liquides dans les tubes de très petits diamètres. Mem Savant Etrangers Paris 9:433–544, 1846. Reynolds O: An experimental investigation of the circumstances which determine whether the motion of water shall be direct or sinusoid, and of the law of resistance in parallel channels. Philos Trans R Soc Lond B Biol Sci 174:935–982, 1883. Thury A, van Langenhove G, Carlier SG, et al: High shear stress after successful balloon angioplasty is associated with restenosis and target lesion revascularization. Am Heart J 144:136–143, 2002.

C H A P T E R 18  BLOOD Emile L. Boulpaep

Blood is a complex fluid consisting of plasma—extracellular fluid rich in proteins—and of formed elements—red blood cells (RBCs), white blood cells (WBCs), and platelets. Total blood volume is ~70 mL/kg body weight in the adult woman and ~80 mL/kg body weight in the adult man (see Table 5-1).

BLOOD COMPOSITION Whole blood is a suspension of cellular elements in plasma If you spin down a sample of blood containing an anticoagulant for ~5 minutes at 10,000 g, the bottom fraction contains formed elements—RBCs (or erythrocytes), WBCs (leukocytes, which include granulocytes, lymphocytes, and monocytes), and platelets (thrombocytes); the top fraction is blood plasma (Fig. 18-1). The RBCs having the highest density are at the bottom of the tube, whereas most of the WBCs and platelets form a whitish gray layer—the buffy coat—between the RBCs and plasma. Only a small amount of WBCs, platelets, and plasma is trapped in the bottom column of RBCs. The hematocrit (see p. 102) is the fraction of the total column occupied by RBCs. The normal hematocrit is ~40% for adult women and ~45% for adult men. The hematocrit in the newborn is ~55% and falls to ~35% at 2 months of age, from which time it rises during development to reach adult values at puberty. The hematocrit is a measure of concentration of RBCs, not of total body red cell mass. Expansion of plasma volume in a pregnant woman reduces the hematocrit, whereas her total red cell volume also increases but less than plasma volume (see p. 1142). Immediately after hemorrhage, the hematocrit may be normal despite the loss of blood volume (see pp. 585–586). Total RBC volume is ~28 mL/kg body weight in the adult woman and ~36 mL/kg body weight in the adult man. Plasma is a pale-white watery solution of electrolytes, plasma proteins, carbohydrates, and lipids. Pink-colored plasma suggests the presence of hemoglobin caused by hemolysis (lysis of RBCs) and release of hemoglobin into the plasma. A brown-green color may reflect elevated bilirubin levels (see Box 46-1). Plasma can also be cloudy in cryoglobulinemias (see pp. 438–439). The electrolyte composition of plasma differs only slightly from that of interstitial

fluid on account of the volume occupied by proteins and their electrical charge (see Table 5-2). Plasma proteins at a normal concentration of ~7.0 g/dL account for a colloid osmotic pressure or oncotic pressure of ~25 mm Hg (see p. 470). Principal plasma proteins are albumin, fibrinogen, globulins, and other coagulation factors. The molecular weights of plasma proteins range up to 970 kDa (Table 18-1). The plasma concentration of albumin ranges from 3.5 to 5.5 g/dL, which provides the body with a total plasma albumin pool of ~135 g. Albumin is synthesized by the liver at a rate of ~120 mg/kg body weight per day and, due to catabolism, has a half-life in the circulation of ~20 days. Urinary losses of albumin are normally negligible (40 mm thick in certain inflammatory disorders). This rate of fall is called the erythrocyte sedimentation rate (ESR). Although it is nonspecific because so many different conditions can cause it to increase, the ESR is still widely used by clinicians to assess the presence and severity of inflammation. It is a simple technique, easily performed in a physician’s office. As an example of its utility, a patient with an inflammatory process that naturally waxes and wanes, such as lupus erythematosus, may present with nonspecific complaints such as fatigue, weakness, and achiness. An elevated ESR would suggest that these complaints are due to the reactivation of the disease and not just to a poor night’s sleep or depression.

FUNCTION

Transthyretin

62

Binds T3 and T4 Binds vitamin A

Albumin

69

Oncotic pressure Binds steroids, T3, bilirubin, bile salts, fatty acids

α1-antitrypsin (α1AT)

54

Protease inhibitor Deficiency causes emphysema

α2-macroglobulin

725

Broad-spectrum protease inhibitor Synthesized by liver

Haptoglobin

100

Binds hemoglobin

β-lipoprotein = low-density lipoprotein (LDL)

380

Binds lipid

80

Binds iron

Transferrin

Hematocrit = Height of RBCs Total height

MOLECULAR WEIGHT (kDa)

Complement C3

185

Third component of complement system

Fibrinogen

340

Clotting protein Precursor of fibrin

Immunoglobulin A (IgA)

160

Mucosal immunity Synthesized by plasma cells in exocrine glands

Immunoglobulin D (IgD)

170

Synthesized by B lymphocytes

Immunoglobulin E (IgE)

190

Synthesized by B lymphocytes Binds to mast cells or basophils

Immunoglobulin G (IgG)

150

Humoral immunity Synthesized by plasma cells

Immunoglobulin M (IgM)

970

Humoral immunity Synthesized by B lymphocytes

*The proteins are listed in the approximate order of decreasing electrophoretic mobility.

mobility (Fig. 18-2A): albumin, α1-globulins, α2-globulins, β-globulins, fibrinogen, and γ-globulins. The three most abundant peaks are albumin, fibrinogen, and γ-globulins. The γ-globulins include the immunoglobulins or antibodies, which can be separated into IgA, IgD, IgE, IgG, and IgM. Immunoglobulins are synthesized by B lymphocytes and plasma cells. Clinical laboratories most often perform electrophoresis of blood proteins on serum instead of plasma (see Fig. 18-2B). Table 18-1 shows the major protein components

Chapter 18  •  Blood

A

PLASMA PROTEINS Alb

2

1

Gc

Bone marrow is the source of most blood cells

12

AT3

Hpt

-Lp



Pl CRP C1q

1Ac Tf C3 Fibr C4 C5 -LP IgM C1Inh Hpx C1s IgA IgD(E) Cer 2M

1At

IaTl 1Ag

Pre A

Alb

IgG FB

Fibrinogen

12



C1r B

SERUM PROTEINS Alb

1

2

Gc

AT3

Hpt

-Lp

Pl C1q

CRP

1Ac 2M

1At IaTl 1Ag

Pre A

-LP C1Inh C1s Cer

Tf

C4 C5

IgA

Alb

Notice absence of fibrinogen.

C3

Hpx

IgM IgD(E) IgG

C1r

431

FB

Figure 18-2  Electrophoretic pattern of human plasma and serum proteins. Normal concentration ranges are as follows: total protein, 6 to 8 g/dL; albumin, 3.1 to 5.4 g/dL; α1-globulins, 0.1 to 0.4 g/dL; α2-globulins, 0.4 to 1.1 g/dL; β-globulins, 0.5 to 1.2 g/dL; γ-globulins, 0.7 to 1.7 g/dL.

that are readily resolved by electrophoresis. Proteins present in plasma at low concentrations are identified by immunological techniques, such as radioimmunoassay (see p. 976) or enzyme-linked immunosorbent assay. Not listed in Table 18-1 are several important carrier proteins present in plasma: ceruloplasmin (see p. 970), transcobalamin (see p. 937), corticosteroid-binding globulin (CBG; see p. 1021), insulin-like growth factor (IGF)–binding proteins (see p. 996), sex hormone–binding globulin (SHBG or TeBG; see pp. 1119–1120), thyroid-binding globulin (see pp. 1008– 1009), and vitamin D–binding protein (see p. 1064). The liver synthesizes most of the globulins and coagulation factors.  N18-1

If you spread a drop of anticoagulated blood thinly on a glass slide, you can detect under the microscope the cellular elements of blood. In such a peripheral blood smear, the following mature cell types are easily recognized: erythrocytes; granulocytes divided in neutrophils, eosinophils, and basophils; lymphocytes; monocytes; and platelets (Fig. 18-3). Hematopoiesis is the process of generation of all the cell types present in blood. Because of the diversity of cell types generated, hematopoiesis serves multiple roles ranging from the carriage of gases to immune responses and hemostasis. Pluripotent long-term hematopoietic stem cells (LT-HSCs) constitute a population of adult stem cells found in bone marrow that are multipotent and able to self-renew. The short-term hematopoietic stem cells (ST-HSCs) give rise to committed stem cells or progenitors, which after proliferation are able to differentiate into lineages that in turn give rise to burst-forming units (BFUs) or colony-forming units (CFUs), each of which ultimately will produce one or a limited number of mature cell types: erythrocytes, the megakaryocytes that give rise to platelets, eosinophils, basophils, neutrophils, monocytes-macrophages/dendritic cells, and B or T lymphocytes and natural killer cells (Fig. 18-4). Soluble factors known as cytokines guide the development of each lineage. The research of Donald Metcalf demonstrated the importance of a family of hematopoietic cytokines that stimulate colony formation by progenitor cells, the colonystimulating factors. The main colony-stimulating factors are granulocyte-macrophage colony-stimulating factor (GM-CSF; see p. 70), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin-3 (IL-3) and IL-5 (see p. 70), thrombopoietin (TPO), and erythropoietin (EPO; see pp. 431–433). GM-CSF is a glycoprotein that stimulates proliferation of a common myeloid progenitor and promotes the production of neutrophils, eosinophils, and monocytes-macrophages. Recombinant GM-CSF (sargramostim [Leukine]) is used clinically after bone marrow transplantation and in certain acute leukemias. G-CSF and M-CSF are glycoproteins that guide the ultimate development of granulocytes and monocytesmacrophages/dendritic cells, respectively. Recombinant GCSF (filgrastim [Neupogen]) is used therapeutically in neutropenia (e.g., after chemotherapy). M-CSF is also required for osteoclast development (see p. 1057 and Fig. 52-4). IL-3 (also known as multi-CSF) has a broad effect on multiple lineages. The liver and the kidney constitutively produce this glycoprotein. IL-5 (colony-stimulating factor, eosinophil), a homodimeric glycoprotein, sustains the terminal differentiation of eosinophilic precursors. TPO binds to a TPO receptor called c-Mpl, which is the cellular homolog of the viral oncogene v-mpl (murine myeloproliferative leukemia virus). On stimulation by TPO, the Mpl receptor induces an increase in the number and size of megakaryocytes—the cells that produce platelets— which thereby greatly augments the number of circulating platelets. N18-2  which is homologous to TPO, is proEPO,  duced by the kidney and to a lesser extent by the liver. This cytokine supports erythropoiesis or red cell development

Chapter 18  •  Blood

431.e1

N18-1  Plasma Proteins Contributed by Emile Boulpaep Protein

Conventional Units

International Units

Protein, total   Electrophoresis

6.4–8.3 g/dL Albumin: 3.5–5.0 g/dL α1-globulin: 0.1–0.3 g/dL α2-globulin: 0.6–1.0 g/dL β-globulin: 0.7–1.1 g/dL γ-globulin: 0.8–1.6 g/dL

64.0–83.0 g/L 35–50 g/L 1–3 g/L 6–10 g/L 7–11 g/L 8–16 g/L M: 2.5–11.7 U/L F: 0.3–9.2 U/L 0.17–0.68 µkat/L 0.12–0.60 µkat/L 34–48 g/L Adult (>20 yr) 0.43–1.70 µkat/L 0.46–2.23 µkat/L 1.2 mM

1

CO2 40 mm Hg

2A

3A

CO2 40 mm Hg

CO2 1.2 mM

Equilibrate

H2O

H2O

HCO–3 H+ + 0.000,068 mM 14 mM

HCO–3 PPV > PA 0 cm H2O

50

0

–3 cm H2O

+7 cm H 2O

5

10 15 Distance (cm)

0 cm H2O

+10 cm H2O

20 Apex

0 cm H2O

+5 cm H2O

C—LUNG ZONES Zone 1 conditions occur only when PA is high (e.g., positive-pressure ventilation) or when PPA is low (e.g., hemorrhage).

+20 cm H2O

+10 cm H2O

Zone 2 +15 cm H2O at midpoint

Zone 3 Zone 4

PIP leads to partial collapse of extraalveolar vessels.

Zone 4 PPA > PPV > PA

Lungs normally have Zones 2 through 4. Smaller regional lung volume leads to less mechanical tethering. Figure 31-9  Physiological nonuniformity of pulmonary perfusion.

0 cm H2O

Chapter 31  •  Ventilation and Perfusion of the Lungs

pressures. We define the first three zones based on how alveolar blood vessels are affected by the relative values of three different pressures: alveolar pressure (PA), the pressure inside pulmonary arterioles (PPA), and the pressure inside pulmonary venules (PPV). In the fourth zone, we instead focus on how extra-alveolar vessels are affected by intrapleural pressure (PIP). Zone 1: PA > PPA > PPV  These conditions prevail at the apex of the lung under certain circumstances. The defining characteristic of a zone 1 alveolar vessel is that PPA and PPV are so low that they have fallen below PA. At the level of the left atrium (the reference point for the pressure measurements), the mean PPA is ~15 mm Hg (see Table 31-1), which—because mercury is 13.6-fold more dense than water—corresponds to ~20 cm H2O (see Fig. 31-9C, lower illustration for zone 3). Similarly, mean PPV is ~8 mm Hg, or ~10 cm H2O. As we move upward closer to the apex of an upright lung, the actual pressures in the lumens of pulmonary arterioles and venules fall by 1 cm H2O for each 1 cm of vertical ascent. In the hypothetical case in which alveoli at the lung apex are 20 cm above the level of the left atrium, the mean PPA of these alveoli would be 0 cm H2O (see Fig. 31-9C, zone 1). The corresponding PPV would be about −10 cm H2O. The pressure inside the pulmonary capillary (Pc) would be intermediate, perhaps −5 cm H2O. In principle, blood would still flow through this capillary—the driving pressure would be ~10 cm H2O— were it not for the pressure inside the surrounding alveoli, which is 0 cm H2O between breaths. Therefore, because PA is much higher than Pc, the negative PTM (see p. 414) would tend to crush the capillary and reduce blood flow. Fortunately, zone 1 conditions do not exist for normal people at rest. However, they can arise if there is either a sufficient decrease in PPA (e.g., in hemorrhage) or a sufficient increase in PA (e.g., in positive-pressure ventilation). Zone 2: PPA > PA > PPV  These conditions normally prevail from the apex to the mid-lung. The defining characteristic of zone 2 is that mean PPA and PPV are high enough so that they sandwich PA (see Fig. 31-9C, zone 2). Thus, at the arteriolar end, the positive PTM causes the alveolar vessel to dilate. Further down the capillary, though, luminal pressure gradually falls below PA, so that the negative PTM squeezes the vessel, raising resistance and thus reducing flow. As we move downward in zone 2, the crushing force decreases because the hydrostatic pressures in the arteriole, capillary, and venule all rise in parallel by 1 cm H2O for each 1 cm of descent (see Fig. 31-9C, upper → lower illustrations for zone 2). Simultaneously, resistance decreases. The conversion of a closed vessel (or one that is open but not conducting) to a conducting one by increased PPA and PPV is an example of recruitment. Zone 3: PPA > PPV > PA  These conditions prevail in the middle to lower lung. The defining characteristic of zone 3 is that mean PPA and PPV are so high that they both exceed PA (see Fig. 31-9C, zone 3). Thus, PTM is positive along the entire length of the alveolar vessel, tending to dilate it. As we move downward in zone 3, the hydrostatic pressures in the arteriole, capillary, and venule all continue to rise by 1 cm

689

H2O for each 1 cm of descent. Because PA between breaths does not vary with height in the lung, the gradually increasing pressure of the alveolar vessel produces a greater and greater PTM, causing the vessel to dilate more and more—an example of distention (see Fig. 31-9C, upper → lower illustrations for zone 3). This distention causes a gradual decrease in resistance of the capillaries as we move downward in zone 3. Hence, although the driving force (PPA − PPV) remains constant, perfusion increases toward the base of the lung. The arrangement in which a variable PTM controls flow is known as a Starling resistor. Keep in mind that the driving force (PPA − PPV) is constant in all of the zones. Zone 4: PPA > PPV > PA  These conditions prevail at the extreme base of the lungs. In zone 4, the alveolar vessels behave as in zone 3; they dilate more as we descend toward the base of the lung. However, the extra-alveolar vessels behave differently. At the base of the lung, PIP is least negative (see Fig. 31-5C). Thus, as we approach the extreme base of the lung, the distending forces acting on the extra-alveolar blood vessels fade, and the resistance of these extra-alveolar vessels increases (see Fig. 31-9C, zone 4). Recall that we saw a similar effect—at the level of the whole lung (see Fig. 31-7B, blue curve)—where resistance of the extra-alveolar vessels increased as lung volume fell (i.e., as PIP became less negative). Because these extra-alveolar vessels feed or drain  begins to fall from its peak as we the alveolar vessels, Q approach the extreme base of the lungs (see Fig. 31-9B). These lung zones are physiological, not anatomical. The boundaries between the zones are neither fixed nor sharp. For example, the boundaries can move downward with positive-pressure ventilation (which increases PA) and can move upward with exercise (which increases PPA). In our discussion of lung zones, we have tacitly assumed that PA is always zero and that the values of PPA and PPV are stable and depend only on height in the lung. In real life, of course, things are more complicated. During the respiratory cycle, PA becomes negative during inspiration (promoting dilation of alveolar vessels) but positive during expiration. During the cardiac cycle, the pressure inside the arterioles and pulmonary capillaries is greatest during systole (promoting dilation of the vessel) and lowest during diastole. Thus, we would expect blood flow through an alveolar vessel to be greatest when inspiration coincides with systole.

MATCHING VENTILATION AND PERFUSION The greater the ventilation-perfusion ratio, the higher the PO2 and the lower the PCO2 in the alveolar air In Figure 31-4 we saw that, all other factors being equal, alveolar ventilation determines alveolar PO2 and PCO2. The greater the ventilation, the more closely PA O2 and PA CO2 approach their respective values in inspired air. However, in Figure 31-4 we were really focusing on total alveolar ventilation and how this influences the average, or idealized, alveolar PO2 and PCO2. In fact, we have already learned that both ventilation and perfusion vary among alveoli. In any group of alveoli, the greater the local ventilation, the more closely the composition of local alveolar air approaches that of the inspired air. Similarly, because blood flow removes O2

690

A

SECTION V  •  The Respiratory System

· · DEPENDENCE OF VA/Q ON HEIGHT IN LUNG Base Apex 4 3

· VA · 2 Q

· · VA/Q

· Q

200

· VA

THE O2-CO2 DIAGRAM

Mixedvenous blood: · · VA/Q = 0

5

4 3 Rib number

·

0

·

Low VA/Q Arterial blood

20

0

2

60

40 PCO2 (mm Hg)

100

1 0

· · VA or Q Unit volume (arbitrary units)

B

40

60

High · V

A /Q

100 120 PO2 (mm Hg)

80

·

Inspired air: · · VA/Q = ∞

140

160

 ratio and alveolar gas composition. (Data from West JB: Ventilation/ Figure 31-10  Regional differences in V A /Q Blood Flow and Gas Exchange. Oxford, UK, Blackwell, 1985.)

TABLE 31-3  Effect of Regional Differences in V A /Q on the Composition of Alveolar Air and Pulmonary-Capillary Blood LOCATION Apex Base Overall

FRACTION OF TOTAL LUNG VOLUME

V A /Q

PO2 (mm Hg)

PCO2 (mm Hg)

pH

Q (L/min)

7%

3.3

132

28

7.55

0.07

13% 100%

0.6 0.84*

89

42

7.38

1.3

100

40

7.40

5.0

*Because the transport of both O2 and CO2 is perfusion limited, we assume that end-capillary values of PO2 and PCO2 are the same as their respective alveolar  ratio for the values. If the overall alveolar ventilation for the two lungs is 4.2 L/min, and if the cardiac output (i.e., perfusion) is 5 L/min, then the overall V A /Q two lungs is (4.2 L/min)/(5 L/min) = 0.84. Modified from West JB: Ventilation/Blood Flow and Gas Exchange. Oxford, UK, Blackwell, 1989.

from the alveolar air and adds CO2, the greater the perfusion, the more closely the composition of local alveolar air approaches that of mixed-venous blood. Thus, the local  ) determines the local  A /Q ventilation-perfusion ratio (V PA O2 and PA CO2. You might view the alveoli as a sports venue where ventilation and perfusion are engaged in a continuous struggle over control of the composition of alveolar air. To the extent that ventilation gains the upper hand, PA O2 rises and PA CO2 falls. To the extent that perfusion holds sway, these parameters change in the opposite direction. As a physical analog of this struggle over control of alveo A ) from a lar PO2 , consider water flowing (analogous to V  through a faucet into a sink (alveoli); the water exits (Q) drain with an adjustable opening. If the drain opening is in midposition and we begin flowing water moderately fast, then the water level (PA O2) will gradually increase and reach  A ) will cause a steady state. Increasing the inflow of water ( V the water level (PA O2 ) to rise until the product of pressure head and drain conductance is high enough to drive water down the drain as fast as the water flows in. If we increase  then the drain opening and thus the outflow of water (Q), the water level (PA O2 ) will fall until the decrease in the pressure head matches the increase in drain conductance, so that once again water inflow and outflow are balanced. Just as a high faucet-drain ratio will raise the water level, a high  ratio will increase alveolar PO .  A /Q V 2

Because of the action of gravity, the regional V A /Q ratio in an upright subject is greater at the apex of the lung than at the base We have already seen that when a subject is upright in a gravitational field, ventilation falls from the base to the apex of the lung (see Fig. 31-5B) and that perfusion also falls, but more steeply (see Fig. 31-9B). Thus, it is not surprising  itself varies with height in the lung  A /Q that the ratio V  is lowest near the base, where Q  exceeds  A /Q (Fig. 31-10A). V  A. The ratio gradually increases to 1 at about the level of V the third rib and further increases toward the apex, where  falls more precipitously than V  A. Q  at the apex and  A /Q Table 31-3 shows how differences in V base of the lungs influence the regional composition of alveolar air. At the apex (the most rostral 7% of lung volume in  is highest, alveolar PO and PCO  A /Q this example), where V 2 2 most closely approach their values in inspired air. Because both O2 transport and CO2 transport across the blood-gas barrier are perfusion limited (see pp. 671–673), O2 and CO2 have completely equilibrated between the alveolar air and the blood by the end of the pulmonary capillaries. Thus, blood leaving the apex has the same high PO2 and low PCO2 as the alveolar air. Of course, the relatively low PCO2 produces a respiratory alkalosis (see p. 634) in the blood leaving the apex. The situation is just the opposite near the base of the lung (the most caudal 13% of lung volume in this example).

Chapter 31  •  Ventilation and Perfusion of the Lungs

A

ALVEOLAR DEAD-SPACE VENTILATION WITHOUT COMPENSATION B

3 Perfusion of other lung increases, causing · · V/Q.

1 Because perfusion to this lung stops, while ventilation continues, · · V/Q ∞. PO2 PCO2

691

COMPENSATION: BRONCHIOLAR CONSTRICTION 1 PO2, PCO2, and pH around smooth muscle causes bronchiolar constriction, diverting airflow to “normal” airways. 2 In response to blood flow, alveolar type II pneumocytes produce less surfactant, causing compliance and ventilation (alveoli shrink!).

PO2 = 149 PCO2 = 0

2 The alveolar gas assumes the composition of inspired air.  mismatch and compensatory response—alveolar dead-space ventilation. Figure 31-11  Extreme V A /Q

 here is lowest, alveolar PO and PCO tend more  A /Q Because V 2 2 toward their values in mixed-venous blood. What impact do these different regions of the lung, each with its own  ratio, have on the composition of systemic arterial  A /Q V blood? Each region makes a contribution that is proportional to its blood flow (see the rightmost column in Table 31-3). Because the apex is poorly perfused, it makes only a small contribution to the overall composition of arterial blood. On the other hand, pulmonary tissue at the base of the lungs, which receives ~26% of total cardiac output, makes a major contribution. As a result, the average composition of blood exiting the lung more closely reflects the composition of the blood that had equilibrated with the air in the base of the lung. The O2-CO2 diagram introduced as Figure 29-11 is a  ratios through A /Q helpful tool for depicting how different V out the lung produce different blood-gas compositions. The curve in Figure 31-10B represents all possible combinations of PO2 and PCO2 in the alveolar air or end-pulmonarycapillary blood. The H2O-saturated inspired air (PO2 = 149, PCO2 = ~0 mm Hg) represents the rightmost extreme of the  ratio of inspired air is ∞,  A /Q diagram. By definition, the V because it does not come into contact with pulmonarycapillary blood. The mixed-venous blood (PO2 = 40, PCO2 = 46 mm Hg) represents the other extreme. By definition, the  ratio of mixed-venous blood is zero, because it has not  A /Q V yet come into contact with alveolar air. With the end points of the diagram established, we can now predict—with the help of the alveolar gas equation (see Equation 31-17), the Bohr effect (see p. 652), and the Haldane effect (see p. 657)— all possible combinations of PO2 and PCO2 throughout the lung. As shown in Figure 31-10B, the base, midportion, and apex of the lungs correspond to points along the O2-CO2 diagram between mixed-venous blood at one extreme and inspired air at the other.

The ventilation of unperfused alveoli (local V A /Q = ∞) triggers compensatory bronchoconstriction and a fall in surfactant production The effects of gravity on ventilation and perfusion cause  to vary widely, even in idealized lungs (see  A /Q regional V Fig. 31-10A). However, microscopic or local physiological and pathological variations in ventilation and perfusion can  , the extremes of  A /Q cause even greater mismatches of V which are alveolar dead-space ventilation (this section) and shunt (next section). Alveolar Dead-Space Ventilation  At one end of the spec mismatches is the elimination of blood  A /Q trum of V flow to a group of alveoli. For example, if we ligated the pulmonary artery feeding one lung, the affected alveoli would receive no perfusion even though ventilation would initially continue normally (Fig. 31-11A). Above, we saw that such alveolar dead space together with the anatomical dead space constitute the physiological dead space (see Equation 31-8). The ventilation of the unperfused alveoli is called alveolar dead-space ventilation because it does not contribute to gas exchange. Thus, these alveoli behave like conducting airways.  N31-12 A natural cause of alveolar dead-space ventilation is a pulmonary embolism, which obstructs blood flow to a group of alveoli. Because one task of the lung is to filter small emboli from the blood (see p. 600), the lung must deal with small regions of alveolar dead-space ventilation on a recurring basis. At the instant the blood flow ceases, the alveoli supplied by the affected vessel(s) contain normal alveolar air. However, each cycle of inspiration and expiration replaces some stale alveolar air with fresh, inspired air. Because no exchange of O2 and CO2 occurs between these unperfused alveoli and pulmonary-capillary blood, the alveolar gas

Chapter 31  •  Ventilation and Perfusion of the Lungs

N31-12  Notes on the Differences Between Anatomical and Physiological Dead Space Contributed by Emile Boulpaep and Walter Boron There is a fundamental difference between the anatomical and alveolar dead space. The conducting airways are in series with and upstream from (proximal to) the alveoli. The conducting airways have the composition of inspired air only after an inspiration; after an expiration, they have the composition of alveolar air. On the other hand, unperfused alveoli are in parallel with normal alveoli and have the composition of inspired air, regardless of the position in the respiratory cycle.

691.e1

692

SECTION V  •  The Respiratory System

A

SHUNT WITHOUT COMPENSATION

B COMPENSATION: VASOCONSTRICTION

1 Because ventilation to this lung stops, while perfusion continues, · · V/Q 0.

3 Ventilation of other lung increases, · · causing V/Q.

In response to local alveolar hypoxia, the arterioles feeding the alveoli constrict: hypoxic vasoconstriction.

PO2 PCO2

2 The alveolar gas assumes the composition of mixed-venous blood.  mismatch and compensatory response—shunt. Figure 31-12  Extreme V A /Q

gradually achieves the composition of moist inspired air, with alveolar PO2 rising to ~149 mm Hg and PCO2 falling to ~0 mm Hg (see Fig. 31-11A, step 2). By definition, alveolar  ratio of ∞, as described by the  A /Q dead space has a V “Inspired air” point on the x-axis of an O2-CO2 diagram (see Fig. 31-10B). Redirection of Blood Flow  Blocking blood flow to one group of alveoli diverts blood to other “normal” alveoli, which then become somewhat hyperperfused. Thus, the  in alveoli downstream  A /Q blockage not only increases V  in other regions.  A /Q from the blockage, but also decreases V Redirection of blood flow thus accentuates the nonuniformity of ventilation. Regulation of Local Ventilation  Because alveolar deadspace ventilation causes alveolar PCO2 to fall to ~0 mm Hg in downstream alveoli, it leads to a respiratory alkalosis (see p. 634) in the surrounding interstitial fluid. These local changes trigger a compensatory bronchiolar constriction in the adjacent tissues (see Fig. 31-11B), so that over a period of seconds to minutes, airflow partially diverts away from the unperfused alveoli and toward normal alveoli, to which blood flow is also being diverted. This compensation makes  shift  A /Q teleological sense, because it tends to correct the V in both the unperfused and normal alveoli. The precise mechanism of bronchiolar constriction is unknown, although bronchiolar smooth muscle may contract—at least in part— in response to a high extracellular pH.  N31-13 In addition to a local respiratory alkalosis, the elimination of perfusion has a second consequence. Downstream from the blockage, alveolar type II pneumocytes become starved for various nutrients, including the lipids they need to make surfactant. (These cells never become starved for O2!) As a result of the decreased blood flow, surfactant production falls over a period of hours to days. The result is a local decrease in compliance, further reducing local ventilation.

These compensatory responses—bronchiolar constriction (i.e., increased resistance, a property of conducting airways) and reduced surfactant production (i.e., decreased compliance, a property of alveoli)—work well only if the alveolar dead space is relatively small, so that an ample volume of healthy tissue remains into which the airflow can divert.

The perfusion of unventilated alveoli (local V A /Q = 0) triggers a compensatory hypoxic vasoconstriction Shunt  Alveolar dead-space ventilation is at one end of the  mismatches. At the opposite end is  A /Q spectrum of V shunt—the flow of blood past unventilated alveoli. For example, if we ligate a mainstem bronchus, then inspired air cannot refresh alveoli distal to the obstruction (see Fig. 31-12A). As a result, mixed-venous blood perfusing the unventilated alveoli “shunts” from the right heart to the left heart, without benefit of ventilation. When the low-O2 shunted blood mixes with high-O2 unshunted blood (which is ventilated), the result is that the mixture has a lower-thannormal PO2 , causing hypoxia in the systemic arteries. It is possible to calculate the extent of the shunt from the degree of hypoxia.  N31-14 Natural causes of airway obstruction include aspiration of a foreign body or the presence of a tumor in the lumen of a conducting airway. The collapse of alveoli (atelectasis) also produces a right-to-left shunt, a pathological example of which is pneumothorax (see p. 608). Atelectasis also occurs naturally in dependent regions of the lungs, where PIP is not so negative (see Fig. 31-5C) and surfactant levels gradually decline. Sighing or yawning stimulates surfactant release (see p. 615) and can reverse physiological atelectasis. Imagine that an infant aspirates a peanut. Initially, the air trapped distal to the obstruction has the composition of normal alveolar air. However, pulmonary-capillary blood gradually extracts O2 from the trapped air and adds CO2.

Chapter 31  •  Ventilation and Perfusion of the Lungs

692.e1

N31-13  Bronchiolar Constriction during Alveolar Dead-Space Ventilation Contributed by Emile Boulpaep and Walter Boron The precise mechanism of bronchiolar constriction in response to alveolar dead-space ventilation is unknown. However, it is intriguing to speculate that, at least in part, the mechanism may parallel that for the autoregulation of blood flow in the brain. The vascular smooth-muscle cells (VSMCs) of the penetrating cerebral arterioles constrict in response to respiratory alkalosis— which is why one feels dizzy after hyperventilating. This

constriction of the VSMCs occurs when one imposes an alkalosis in the complete absence of CO2 /HCO3−. Furthermore, the alkalosis-induced vasoconstriction is due entirely to a pH decrease on the outside of the VSMC. In other words, these cells have some sort of an extracellular pH sensor. A pH increase on the inside of the cell actually has the opposite effect: vasodilation. During extracellular acidosis, the vessels dilate.

N31-14  The Shunt Equation Contributed by Emile Boulpaep and Walter Boron   mismatch and arises when A shunt is one extreme of a V/Q blood perfuses unventilated alveoli. Alveoli may be unventilated because they are downstream from an obstructed conducting airway. Regardless of the mechanism that prevents airflow to these alveoli, the resulting right-to-left shunt causes mixedvenous blood to remain relatively unoxygenated and to go directly to the left heart, where it mixes with oxygenated “arterial” blood. This process is known as venous admixture. Imagine that 80% of the blood flow to the lungs goes to alveoli that are appropriately ventilated but that 20% goes to alveoli that are downstream from completely obstructed conduct . The shunt ing airways. The total perfusion of the lungs is Q T  , 20% in this example, perfusion of the unventilated alveoli is Q S and the shunted blood has an O2 content (units: mL O2/dL) identi −Q  ) cal to that of mixed-venous blood (Cv ). The difference (Q T S is the perfusion to the normally ventilated alveoli, 80% in our example, and this unshunted blood has an O2 content appropriate for the end of a pulmonary capillary (Cc′). The blood emerging from the lungs is a mixture of shunted and unshunted blood so that the O2 emerging from the lung is partially O2 carried by the shunted blood and partially O2 carried by the unshunted blood: Total O2 leaving lungs = Shunted O2 + Unshunted O2 (NE 31-35)  How much O2 per minute emerges from the lungs in the systemic arterial blood? This amount is the product of the O2 content of this arterial blood (Ca) and the total blood flow out the  ): lungs (Q T

. Total O2 leaving lungs = Ca × QT

(NE 31-36) 

Similarly, the O2 contributed by the shunted blood is the product of the O2 content and the flow of shunted blood:

O2 contributed by shunted blood = mL O2 /min

Cv

. × QS

mL O2 100 mL blood

mL blood min

(NE 31-37) 

Finally, the amount of O2 contributed per minute by the un­ shunted blood is . . O2 contributed by unshunted blood = Cc' × (Q T – QS)

(NE 31-38) 

Inserting the expressions for each of the terms in Equations NE 31-36 through NE 31-38 into Equation NE 31-35, we have

. . . . Ca × QT = Cv × QS + Ca × QT – QS

(

TotalO2 leaving lungs

)

O2 carried by shunted blood

(

O2 carried by unshunted blood

)

(NE 31-39) 

Rearranging this equation and solving for the fraction of total  /Q  ), we have blood flow that is represented by the shunt (Q S T

. QS Cc' − Ca . = QT Cc' − Cv

(NE 31-40) 

This expression is known as the shunt equation.  /Q  in our What does Equation NE 31-40 predict for Q S T example? We will assume that the O2 content of mixed-venous blood is 15 mL O2/dL blood, whereas that for blood at the end of the pulmonary capillaries is 20 mL O2/dL blood. These values are similar to those given in Table 29-3. If our hypothetical subject—who is affected by a 20% shunt—has systemic arterial blood with an O2 content of 19 mL O2/dL blood, then the shunt equation predicts

. QS Cc' − Ca 20−19 = =20% . = QT Cc' − Cv 20−15

(NE 31-41) 

Thus, the shunt equation predicts that the shunt is 20% of the total blood flow, which is reasonable, inasmuch as we started the example by assuming that 80% of the blood flowed through properly ventilated alveoli.

Chapter 31  •  Ventilation and Perfusion of the Lungs

Eventually, the PO2 and PCO2 of the trapped air drift to their values in mixed-venous blood. If the shunt is small, so that it does not materially affect the PO2 or PCO2 of the systemic arterial blood, then the alveoli will have a PO2 of 40 mm Hg and a PCO2 of 46 mm Hg. By definition, shunted alveoli have  of zero and are represented by the “Mixed-venous  A /Q aV blood” point on an O2-CO2 diagram (see Fig. 31-10B). Redirection of Airflow  Blocking airflow to one group of alveoli simultaneously diverts air to normal parts of the lung, which then become somewhat hyperventilated. Thus, shunt  in unventilated alveoli, but also  A /Q not only decreases V  in other regions. The net effect is a widening  A /Q increases V  ratios.  A /Q of the nonuniformity of V Asthma  Although less dramatic than complete airway

.  A /Q obstruction, an incomplete occlusion also decreases V An example is asthma, in which hyperreactivity of airway smooth muscle increases local airway resistance and decreases ventilation of alveoli distal to the pathology. Normal Anatomical Shunts  The thebesian veins drain some of the venous blood from the heart muscle, particularly the left ventricle, directly into the corresponding cardiac chamber. Thus, delivery of deoxygenated blood from thebesian veins into the left ventricle (10 times a day) to the scrutiny of the renaltubule epithelium. If it were not for such a high turnover of the ECF, only small volumes of blood would be “cleared” per unit time (see p. 731) of certain solutes and water. Such a low clearance would have two harmful consequences for the renal excretion of solutes that renal tubules cannot adequately secrete. First, in the face of a sudden increase in the plasma level of a toxic material—originating either from metabolism or from food or fluid intake—the excretion of the material would be delayed. A high blood flow and a high GFR allow the kidneys to eliminate harmful materials rapidly by filtration. A second consequence of low clearance would be that steady-state plasma levels would be very high for waste materials that depend on filtration for excretion. The following example by Robert Pitts, a major contributor to renal physiology, illustrates the importance of this concept. Consider two individuals consuming a diet that contains 70 g/day of protein, one with normal renal function (e.g., GFR of

180 L/day) and the other a renal patient with sharply reduced glomerular filtration (e.g., GFR of 18 L/day). Each individual produces 12 g/day of nitrogen in the form of urea (urea nitrogen) derived from dietary protein and must excrete this into the urine. However, these two individuals achieve urea balance at very different blood urea levels. We make the simplifying assumption that the tubules neither absorb nor secrete urea, so that only filtered urea can be excreted, and all filtered urea is excreted. The normal individual can excrete 12 g/day of urea nitrogen from 180 L of blood plasma having a [blood urea nitrogen] of 12 g/180 L, or 6.7 mg/dL. In the patient with end-stage renal disease (ESRD), whose GFR may be only 10% of normal, excreting 12 g/day of urea nitrogen requires that each of the 18 L of filtered blood plasma have a blood urea nitrogen level that is 10 times higher, or 67 mg/dL. Thus, excreting the same amount of urea nitrogen—to maintain a steady state—requires a much higher plasma blood urea nitrogen concentration in the ESRD patient than in the normal individual.

The clearance of inulin is a measure of GFR The ideal glomerular marker for measuring GFR would be a substance X that has the same concentration in the glomerular filtrate as in plasma and that also is not reabsorbed, secreted, synthesized, broken down, or accumulated by the tubules (Table 34-1). In Equation 33-4, we saw that Input into Bowman’s space



Output into urine

 PX ⋅GFR = U X ⋅ V mg mL

mL min

(34-1) 

mg mL mL min

PX is the concentration of the solute in plasma, GFR is the sum of volume flow of filtrate from the plasma into all Bowman’s spaces, UX is the urine concentration of the solute,  is the urine flow. Rearranging this equation, we have and V



U X × V PX mL (mg/mL) × (mL/min) = min (mg/mL)

GFR =

(34-2) 

Note that Equation 34-2 has the same form as the clearance equation (see Equation 33-3) and is identical to Equation 739

740

SECTION VI  •  The Urinary System

33-5. Thus, the plasma clearance of a glomerular marker is the GFR.  N34-1 Inulin is an exogenous starch-like fructose polymer that is extracted from the Jerusalem artichoke and has a molecular weight of 5000 Da. Inulin is freely filtered at the glomerulus, but neither reabsorbed nor secreted by the renal tubules (Fig. 34-1A). Inulin also fulfills the additional requirements listed in Table 34-1 for an ideal glomerular marker. Assuming that GFR does not change, three tests demonstrate that inulin clearance is an accurate marker of GFR. First, as shown in Figure 34-1B, the rate of inulin excretion TABLE 34-1  Criteria for Use of a Substance to Measure GFR 1. Substance must be freely filterable in the glomeruli. 2. Substance must be neither reabsorbed nor secreted by the renal tubules. 3. Substance must not be synthesized, broken down, or accumulated by the kidney. 4. Substance must be physiologically inert (not toxic and without effect on renal function).

A HANDLING OF INULIN

Efferent arteriole

Afferent arteriole

Glomerular capillary

 ) is directly proportional to the plasma inulin con(U In ⋅ V centration (PIn), as implied by Equation 34-2. The slope in Figure 34-1B is the inulin clearance. Second, inulin clearance is independent of the plasma inulin concentration (see Fig. 34-1C). This conclusion was already implicit in Figure 34-1B, in which the slope (i.e., inulin clearance) does not vary with PIn. Third, inulin clearance is independent of urine flow (see Fig. 34-1D). Given a particular PIn, after the renal corpuscles filter the inulin, the total amount of inulin in the urine does not change. Thus, diluting this glomerular marker in a large amount of urine, or concentrating it in a small volume, does  ). If the not affect the total amount of inulin excreted (U In ⋅ V urine flow is high, the urine inulin concentration will be  ) is fixed, proportionally low, and vice versa. Because (U In ⋅ V  (U In ⋅ V)/PIn is also fixed. Two lines of evidence provide direct proof that inulin clearance represents GFR. First, by collecting filtrate from single glomeruli, Richards and coworkers showed in 1941 that the concentration of inulin in Bowman’s space of the mammalian kidney is the same as that in plasma. Thus, inulin is freely filtered. Second, by perfusing single tubules with known amounts of labeled inulin, Marsh and B

DEPENDENCE OF INULIN EXCRETION ON PLASMA [INULIN] 2500 The slope is the 2000 clearance of inulin. . 1500 UIn · V (mg/min) 1000 500 0

Bowman’s space Amount of inulin filtered is PIn · GFR.

C Peritubular capillary

4

8 12 PIn (mg/mL)

16

20

16

20

DEPENDENCE OF INULIN CLEARANCE ON PLASMA [INULIN] 250

.

200

UIn · V/PIn 150 (mL/min) 100

Because inulin is not reabsorbed…

50 0

…and not secreted… D …the amount excreted . in the urine (UIn · V) is the same as the amount filtered.

0

Renal vein

0

4

8 12 PIn (mg/mL)

DEPENDENCE OF INULIN CLEARANCE ON URINE FLOW 250 200

.

UIn · V/PIn 150 (mL/min) 100 50 0

Figure 34-1  Clearance of inulin.

0

1

2

3

.4 5 6 V (mL/min)

7

8

9

10

Chapter 34  •  Glomerular Filtration and Renal Blood Flow

N34-1  Units of Clearance Contributed by Erich Windhager and Gerhard Giebisch Clearance values are conventionally given in milliliters of total plasma per minute, even though plasma consists of 93% “water” and 7% protein, with only the “plasma water”—that is, the protein-free plasma solution, including all solutes small enough to undergo filtration—undergoing glomerular filtration. As pointed out in Chapter 5 (see Table 5-2) the concentrations of plasma solutes can be expressed in millimoles per liter of total plasma, or millimoles per liter of protein-free plasma (i.e., plasma water). Customarily, clinical laboratories report values in millimoles (or milligrams) per deciliter of plasma, not plasma water. When we say that the GFR is 125 mL/min, we mean that each minute the kidney filters all ions and small solutes contained in 125 mL of plasma. However, because the glomerular capillary blood retains the proteins, only 0.93 × 125 mL = 116 mL of plasma water appear in Bowman’s capsule. Nevertheless, GFR is defined in terms of volume of blood plasma filtered per minute rather than in terms of the volume of protein-free plasma solution that actually arrives in Bowman’s space (i.e., the filtrate).

740.e1

Chapter 34  •  Glomerular Filtration and Renal Blood Flow

Frasier showed that the renal tubules neither secrete nor reabsorb inulin. Although the inulin clearance is the most reliable method for measuring GFR, it is not practical for clinical use. One must administer inulin intravenously to achieve reasonably constant plasma inulin levels. Another deterrent is that the chemical analysis for determining inulin levels in plasma and urine is sufficiently demanding to render inulin unsuitable for routine use in a clinical laboratory. The normal value for GFR in a 70-kg man is ~125 mL/ min. Population studies show that GFR is proportional to body surface area. Because the surface area of an average 70-kg man is 1.73 m2, the normal GFR in men is often reported as 125 mL/min per 1.73 m2 of body surface area. In women, this figure is 110 mL/min per 1.73 m2. Age is a second variable. GFR is very low in the newborn, owing to incomplete development of functioning glomerular units. Beginning at ~2 years of age, GFR normalizes for body surface area and gradually falls off with age as a consequence of progressive loss of functioning nephrons.

The clearance of creatinine is a useful clinical index of GFR Because inulin is not a convenient marker for routine clinical testing, nephrologists use other compounds that have clearances similar to those of inulin. The most commonly used compound in human studies is 125I-iothalamate. However, even 125I-iothalamate must be infused intravenously and is generally used only in clinical research studies rather than in routine patient care. The problems of intravenous infusion of a GFR marker can be completely avoided by using an endogenous substance with inulin-like properties. Creatinine is such a substance, and creatinine clearance (CCr) is commonly used to estimate GFR in humans. Tubules, to a variable degree, secrete creatinine, which, by itself, would lead to a ~20% overestimation of GFR in humans. Moreover, when GFR falls to low levels with chronic kidney disease, the overestimation of GFR by CCr becomes more appreciable. In clinical practice, determining CCr is an easy and reliable means of assessing the GFR, and such determination avoids the need to inject anything into the patient. One merely obtains samples of venous blood and urine, analyzes them for creatinine concentration, and makes a simple calculation (see Equation 34-3 below). Although CCr may overestimate the absolute level of GFR, assessing changes in CCr is extremely useful for monitoring relative changes in GFR in patients. The source of plasma creatinine is the normal metabolism of creatine phosphate in muscle. In men, this metabolism generates creatinine at the rate of 20 to 25 mg/kg body weight per day (i.e., ~1.5 g/day in a 70-kg man). In women, the value is 15 to 20 mg/kg body weight per day (i.e., ~1.2  g/day in a 70-kg woman), owing to a lower muscle mass. In the steady state, the rate of urinary creatinine excretion equals this rate of metabolic production. Because metabolic production of creatinine largely depends on muscle mass, the daily excretion of creatinine depends strongly not only on gender but also on age, because elderly patients tend to have lower muscle mass. For a CCr measurement, the patient generally collects urine over an entire 24-hour period, and

741

· PCr · CCr = UCr · V = Constant 20

200

15 Plasma creatinine concentration 10 (mg/dL)

150 Blood urea 100 nitrogen (mg/dL) 50

5 0

0

0 25 50 75 100 125 GFR (mL/min)

Figure 34-2  Dependence of plasma creatinine and blood urea nitrogen on the GFR. In the steady state, the amount of creatinine appearing in the urine per day (UCr ⋅ V ) equals the production rate. Because all filtered creatinine (PCr · CCr) appears in the urine, (PCr · CCr) equals (UCr ⋅ V ), which is constant. Thus, PCr must increase as CCr (i.e., GFR) decreases, and vice versa. If we assume that the kidney handles urea in the same way that it handles inulin, then a plot of blood urea nitrogen versus GFR will have the same shape as that of creatinine concentration versus GFR.

the plasma sample is obtained by venipuncture at one time during the day based on the assumption that creatinine production and excretion are in a steady state. Frequently, clinicians make a further simplification, using the endogenous plasma concentration of creatinine (PCr), normally 1 mg/dL, as an instant index of GFR. This use rests on the inverse relationship between PCr and CCr:

CCr =

 U Cr ⋅ V ≈ GFR PCr

(34-3) 

In the steady state, when metabolic production in muscle  ) of creatinine, and equals the urinary excretion rate (U Cr ⋅ V both remain fairly constant, this equation predicts that a plot of PCr versus CCr (i.e., PCr versus GFR) is a rectangular hyperbola (Fig. 34-2). For example, in a healthy person whose GFR is 100 mL/min, plasma creatinine concentration is ~1 mg/dL. The product of GFR (100 mL/min) and PCr (1 mg/dL) is thus 1 mg/min, which is the rate both of creatinine production and of creatinine excretion. If GFR suddenly drops to 50 mL/min (Fig. 34-3, top), the kidneys will initially filter and excrete less creatinine (see Fig. 34-3, middle), although the production rate is unchanged. As a result, the plasma creatinine level will rise to a new steady state, which is reached at a PCr of 2 mg/dL (see Fig. 34-3, bottom). At this point, the product of the reduced GFR (50 mL/min) and the elevated PCr (2 mg/dL) will again equal 1 mg/min, the rate of endogenous production of creatinine. Similarly, if GFR were to fall to one fourth of normal, PCr would rise to 4 mg/dL. This concept is reflected in the rightrectangular hyperbola of Figure 34-2.  N34-2

Molecular size and electrical charge determine the filterability of solutes across the glomerular filtration barrier The glomerular filtration barrier consists of four elements (see p. 726): (1) the glycocalyx overlying the endothelial cells, (2) endothelial cells, (3) the glomerular basement membrane, and (4) epithelial podocytes. Layers 1, 3, and 4 are

Chapter 34  •  Glomerular Filtration and Renal Blood Flow

741.e1

N34-2  Calculating Estimated Glomerular Filtration Rate Contributed by Gerhard Giebisch, Peter Aronson, Walter Boron, and Emile Boulpaep Clinicians can use the plasma creatinine concentration (PCr) to calculate CCr—that is, the estimated GFR (eGFR)—without the necessity of collecting urine. Researches have derived empirical equations for calculating eGFR based on patient data, including not only PCr, but also parameters that include patient age, weight, eGFR



(

mL/ min⋅1.73 m2

)

= 175 ( PCr )

−1.154

⋅ ( Age)

mg/dL

−0.203

gender, and race. In using these equations, we recognize that daily creatinine excretion depends on muscle mass, which in turn depends on age, weight, sex, and race. An example is the Modification of Diet in Renal Disease (MDRD) Study equation:

⋅ (0.742[if female]) ⋅ (1.212[if African American])

Improving upon the MDRD equation was the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) calculator for eGFR (http://www.qxmd.com/calculate-online/nephrology/ ckd-epi-egfr):

Thus, the MDRD calculation takes into account PCr, age, sex, and—in the United States—whether or not the person is African American. Because MDRD is normalized to body surface area, it does not include body weight. eGFR



(

mL/ min ⋅1.73m 2

)

P  = 141⋅ smaller of  Cr  or 1  k 

a

(NE 34-1) 

Years

P  ⋅ larger of  Cr  or 1  k 

−1.209

Here, k is 0.7 for females and 0.9 for males, and a is −0.329 for females and −0.411 for males. In the first bracketed term, we take the larger of (PCr/k) or 1, whereas in the second bracketed term, we take the smaller of (PCr/k) or 1. Like the MDRD

⋅ (0.993)

Age

⋅ (1.018[if female]) ⋅ (1.159[if African American])

(NE 34-2)  calculation, the CKD-EPI eGFR is normalized to body surface area (i.e., it does not include body weight). The Cockcroft-Gault calculator for eGFR,

kg

eGFR =



mL/min

140 (Age)⋅(Weight)⋅(0.85[if female]) 72 ( PCr )

(NE 34-3) 

mg/dL

takes into account PCr, weight (ideally, lean body mass), sex, and age. For example, for a male aged 22 and weighing 60 kg, the Cockcroft-Gault calculator kg



eGFR = mL/ min

140 (22)⋅(60 kg)⋅(1[for a male]) 72 (1.0)

= 122mL/min = 175L/day

(NE 34-4) 

mg/dL

yields an eGFR of 122 mL/min. The National Kidney Foundation (NKF) recommends that one calculate eGFR with each determination of PCr.

REFERENCES Cockcroft D, Gault MD: Prediction of creatinine clearance from serum creatinine. Nephron 16:31–41, 1976. Levey AS, Stevens LA, Schmid CH, et al; for the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI): A new equation to estimate glomerular filtration rate. Ann Intern Med 150:604–612, 2009. National Kidney Disease Education Program: GFR calculators. Last updated April 25, 2012. http://nkdep.nih.gov/lab -evaluation/gfr-calculators.shtml. National Kidney Disease Education Program: GFR MDRD calculator for adults (conventional units). Last updated

March 1, 2012. http://www.niddk.nih.gov/health-information/ health-communication-programs/nkdep/lab-evaluation/gfr -calculators/Pages/gfr-calculators.aspx. Accessed October 2015. National Kidney Foundation: Calculators for health care professionals. http://www.kidney.org/professionals/KDOQI/ gfr_calculator. Accessed October 2015. QxMD: CKD-EPI eGFR. http://www.qxmd.com/calculate-online/ nephrology/ckd-epi-egfr. Accessed October 2015.

742

SECTION VI  •  The Urinary System

covered with negative charges from anionic proteoglycans. The gene mutations that cause excessive urinary excretion of albumin (nephrotic syndrome; see p. 727) generally affect slit diaphragm proteins, which suggests that the junctions between adjacent podocytes are the predominant barrier to filtration of macromolecules. Table 34-2 summarizes the permselectivity of the glomerular barrier for different solutes, as estimated by the ratio of solute concentration in the ultrafiltrate versus the plasma (UFX/PX). The ratio UFX/PX, also known as the sieving coefficient for the solute X, depends on molecular weight

100 GFR (mL/min) 50 0

Production

1.0 Production and excretion of creatinine 0.5 (mg/min) 0 Plasma creatinine concentration (mg/dL)

Excretion

2 1 0

0

1

2 Days

3

4

Figure 34-3  Effect of suddenly decreasing the GFR on plasma creatinine concentration.

and effective molecular radius. Investigators have used two approaches to estimate UFX/PX. The first, which is valid for all solutes, is the micropuncture technique (see Fig. 33-9A). Sampling fluid from Bowman’s space yields a direct measurement of UFX, from which we can compute UFX/PX. The second approach, which is valid only for solutes that the kidney neither absorbs nor secretes, is to compute the clear­ ance ratio (see p. 733),  N34-3 the ratio of the clearances of X (CX) and inulin (CIn). Inspection of Table 34-2 shows that substances of low molecular weight ( [urea] H2O [NaCl] < [NaCl] H2O Urea H2O

INNER MEDULLA H 2O

600 mOsm NaCl

600 mOsm NaCl 300 mOsm Urea

900

Urea H2O

600 mOsm NaCl 600 mOsm Urea

1200

Figure 38-7  Opposing effects of NaCl and urea gradients on urine concentrating ability during antidiuresis. The numbers in the green boxes indicate the osmolalities (in mOsm) of the interstitial fluid.

causes luminal [urea] to increase in these segments. Because the interstitial [urea] is low in the cortex, a rising luminal [urea] in the ICT and CCT opposes water reabsorption in these segments. Even when the tubule crosses the corticomedullary junction, courses toward the papilla, and is surrounded by interstitial fluid with an ever-increasing [urea], the transepithelial urea gradient still favors water movement into the lumen, which is a handicap for the osmotic concentration of the tubule fluid. The IMCD partially compensates for this problem by acquiring, in response to AVP, a high permeability to urea. The result is a relatively low reflection coefficient for urea (σurea; see p. 468), which converts any transepithelial difference in [urea] into a smaller difference in effective osmotic pressure (see pp. 132–133). Thus, water reabsorption continues from the IMCD even though [urea] in tubule fluid exceeds that in the interstitium. The combination of a high interstitial [NaCl] and high σNaCl (σNaCl = 1.0), along with a low σurea (σurea = 0.74), promotes NaCl-driven water reabsorption. The high AVP-induced urea permeability has the additional effect of raising interstitial [urea], which further reduces the adverse effect of the high luminal [urea] on water reabsorption. If luminal urea opposes the formation of a concentrated urine, why did the mammalian kidney evolve to have high levels of urea in the lumen of the collecting tubules and ducts? At least two reasons are apparent. First, because urea is the body’s major excretable nitrogenous waste, the kidney’s ability to achieve high urinary [urea] reduces the necessity to excrete large volumes of water to excrete nitrogenous waste. Second, as we have already seen, the kidney actually takes advantage of urea—indirectly—to generate maximally concentrated urine. In the presence of AVP, the permeability of the IMCD to urea is high, so that large amounts of urea can enter the medullary interstitium. The high interstitial [urea] energizes the increase in luminal [NaCl] in the tDLH, which, in turn, fuels the single effect in the tALH, thus creating the high inner-medullary [NaCl] that is directly responsible for concentrating the urine. As discussed above in this section, the composition of the inner medullary interstitium determines the composition of the final urine. However, to some extent, the composition of the final urine, as well as the rate of urine flow, also influences the composition of the interstitium. Figure 38-2 shows that the medullary interstitial osmolality is much lower, and the stratification of osmolality from cortex to papillary tip is much less, during water diuresis than during antidiuresis. Two factors contribute to the lesser degree of osmotic stratification under conditions of water diuresis, when levels of AVP are low. First, less urea moves from the IMCD lumen to the interstitium, both because of the low urea permeability of the IMCD and because of the low water permeability of the upstream segments that would otherwise concentrate urea. Second, the MCDs reabsorb some water despite the low AVP levels, and this water dilutes the medullary interstitium. The reasons for this apparent paradox are as follows: (1) even when AVP is low, the water permeability is not zero; (2) the ICT and CCT present a much larger fluid volume to the MCD, because they reabsorb less water when AVP levels are low; and (3) the tubule fluid is more hypo-osmotic, which results in a larger osmotic gradient for transepithelial water

Chapter 38  •  Urine Concentration and Dilution

TABLE 38-2  Factors that Modulate Urinary Concentration and Dilution

movement. With low AVP levels, this larger osmotic gradient overrides the effect of the lower water permeability. Table 38-2 summarizes factors that modulate urinary concentration ability.

1. Osmotic gradient of medullary interstitium from corticomedullary junction to papilla: a. Length of loops of Henle: Species with long loops (e.g., desert rodents) concentrate more than those with short loops (e.g., beaver). b. Rate of active NaCl reabsorption in the TAL: Increased luminal Na+ delivery to the TAL (high GFR or filtration fraction, and low proximal-tubule Na+ reabsorption) enhances NaCl reabsorption, whereas low Na+ delivery (low GFR, increased proximal Na+ and fluid reabsorption) reduces concentrating ability. High Na-K pump turnover enhances NaCl reabsorption, whereas inhibiting transport (e.g., loop diuretics) reduces concentrating ability. c. Protein content of diet: High-protein diet, up to a point, promotes urea production and thus accumulation in the inner medullary interstitium, and increased concentrating ability. 2. Medullary blood flow: Low blood flow promotes high interstitial osmolality. High blood flow washes out medullary solutes. 3. Osmotic permeability of the collecting tubules and ducts to water: AVP enhances water permeability and thus water reabsorption. 4. Luminal flow in the loop of Henle and the collecting duct: High flow (osmotic diuresis) diminishes the efficiency of the countercurrent multiplier, and thus reduces the osmolality of the medullary interstitium. In the MCD, high flow reduces the time available for equilibration of water and urea. 5. Pathophysiology: Central diabetes insipidus (DI) reduces plasma AVP levels, whereas nephrogenic DI reduces renal responsiveness to AVP (see Box 38-1).

REGULATION BY ARGININE VASOPRESSIN Large-bodied neurons in the paraventricular and supraoptic nuclei of the hypothalamus synthesize AVP, a nonapeptide also known as ADH. These neurons package the AVP and transport it along their axons to the posterior pituitary, where they release AVP through a breech in the blood-brain barrier into the systemic circulation (see pp. 844–845). In Chapter 40, we discuss how increased plasma osmolality and decreased effective circulating volume increase AVP release. AVP has synergistic effects on two target organs. First, at rather high circulating levels, such as those seen in hypo­ volemic shock, AVP acts on vascular smooth muscle to cause vasoconstriction (see p. 553) and thus to increase blood pressure. Second, and more importantly, AVP acts on the kidney, where it is the major regulator of water excretion. AVP increases water reabsorption by increasing (1) the water permeabilities of the collecting tubules and ducts, (2) NaCl reabsorption in the TAL, and (3) urea reabsorption by the IMCD.

AVP increases water permeability in all nephron segments beyond the DCT Of the water remaining in the DCT, the kidney reabsorbs a variable fraction in the segments from the ICT to the end of the nephron. Absorption of this final fraction of water is under the control of circulating AVP. Figure 38-8 summarizes the water permeability of various nephron segments. The water permeability is highest in the

GFR, glomerular filtration rate.

Proximal convoluted tubule (PCT) Proximal straight tubule (PST) Thin descending limb (tDLH) Thin ascending limb (tALH) Nephron segments

Medullary thick ascending limb (mTAL) No AVP

Cortical thick ascending limb (cTAL)

AVP

Distal convoluted tubule (DCT) Connecting tubule (CNT) Initial and cortical collecting tubules (ICT & CCT) Outer medullary collecting duct (OMCD) Inner medullary collecting duct (IMCD) 10

100

817

1000

10,000

Osmotic water permeability (µm/s) Figure 38-8  Water permeability in different nephron segments. Note that the x-axis scale is logarithmic. (Modified from Knepper MA, Rector FC: Urine concentration and dilution. In Brenner BM [ed]: The Kidney. Philadelphia, WB Saunders, 1996, pp 532–570.)

818

SECTION VI  •  The Urinary System

proximal tubule and tDLH. The constitutively high water permeability in these segments reflects the abundant presence of AQP1 water channels (see p. 110) in the apical and basolateral cell membranes. In marked contrast to the proximal tubule and tDLH, the following few segments—from the tALH to the connecting tubule—constitutively have very low water permeabilities. In the absence of AVP, the next tubule segments, the ICT and CCT, have rather low water permeabilities, whereas the MCDs are virtually impermeable to water. However, AVP dramatically increases the water permeabilities of the collecting tubules (ICT and CCT) and ducts (OMCD and IMCD) by causing AQP2 water channels to insert into the apical membrane (see below). A third type of water channel, AQP3, is present in the basolateral cell membranes of MCDs. Like AQP1, AQP3 is insensitive to AVP. Given the favorable osmotic gradients discussed in the preceding subchapter, high levels of AVP cause substantial water reabsorption to occur in AVP-sensitive nephron segments. In contrast, when circulating levels of AVP are low, for instance after ingestion of large amounts of water, the water permeability of these nephron segments remains low. Therefore, the fluid leaving the DCT remains hypo-osmotic as it flows down more distal nephron segments. In fact, in the absence of AVP, continued NaCl absorption makes the tubule fluid even more hypo-osmotic, which results in a large volume of dilute urine (see Fig. 38-1).

AVP, via cAMP, causes vesicles containing AQP2 to fuse with apical membranes of principal cells of collecting tubules and ducts AVP binds to V2 receptors in the basolateral membrane of the principal cells from the ICT to the end of the nephron (Fig. 38-9). Receptor binding activates the Gs heterotrimeric G protein, stimulating adenylyl cyclase to generate cAMP (see pp. 56–57). The latter activates protein kinase A, which phosphorylates AQP2 and additional proteins that play a role in the trafficking of intracellular vesicles containing AQP2 and the fusion of these vesicles with the apical membrane. These water channels are AVP sensitive, not in the sense that AVP increases their single channel water conductance, but rather that it increases their density in the apical membrane.  N38-6  In conditions of low AVP, AQP2 water channels are mainly in the membrane of intracellular vesicles just beneath the apical membrane. In the membrane of these vesicles, the AQP2 water channels are present as aggregophores—aggregates of AQP2 proteins. Under the influence of AVP, the vesicles containing AQP2 move to the apical membrane of principal cells of the collecting tubules and ducts. By exocytosis (see pp. 34–35), these vesicles fuse with the apical membrane, thus increasing the density of AQP2. When AVP levels in the blood decline, endocytosis retrieves the water channel–containing aggregates from the apical membrane and shuttles them back to the cytoplasmic vesicle pool. The apical water permeability of principal cells depends not only on AVP levels but also on certain other factors. For example, high [Ca2+]i and high [Li+] both inhibit adenylyl cyclase, thus decreasing [cAMP]i, reducing water permeability, and producing a diuresis. A similar inhibition of AQP2

Clusters of AQP2

V2 receptor Prostaglandins Calcium Protein kinase C Other agents

P Exocytosis

β

γ

AVP

α α

P

AC

Vesicle

Endocytosis

Other proteins

cAMP

Protein kinase A

Protein phosphorylation

Phosphodiesterase

5´ AMP

AQP3 and AQP4

AQP2 synthesis Nucleus P

CREB (CRE-binding protein) DNA

Tubule lumen

CRE site AP1 site

Interstitial space

Figure 38-9  Cellular mechanism of AVP action in the collecting tubules and ducts. AC, adenylyl cyclase; AP1, activator protein 1; CRE, cAMP response element.

insertion, and hence a decrease in water permeability, occurs when agents such as colchicine disrupt the integrity of the cytoskeleton. Conversely, inhibitors of phosphodiesterase (e.g., theophylline), which increase [cAMP]i, tend to increase the osmotic water permeability. In addition to regulating AQP2 trafficking in and out of the apical membrane in the short term, AVP regulates AQP2 protein abundance over the longer term.

AVP increases NaCl reabsorption in the outer medulla and urea reabsorption in the IMCD, enhancing urinary concentrating ability AVP promotes water reabsorption not only by increasing the water permeability of the collecting tubules and ducts, but also by enhancing the osmotic gradients across the walls of the IMCD and perhaps the OMCD. In the outer medulla, AVP acts through the cAMP pathway to increase NaCl reabsorption by the TAL. AVP acts by stimulating apical Na/K/Cl cotransport and K+ recycling across the apical membrane (see p. 768). The net effect is to increase the osmolality of the outer medullary interstitium and thus enhance the osmotic gradient favoring water reabsorption by the OMCD. In addition, AVP stimulates the growth of TAL cells in animals that are genetically devoid of AVP. This hormone also stimulates Na+ reabsorption in the CCT, largely by activating apical Na+ channels (ENaCs). These

Chapter 38  •  Urine Concentration and Dilution

N38-6  Multiple Effects of Arginine Vasopressin on AQP2 Activity Contributed by Erich Windhager and Gerhard Giebisch On page 818, we mentioned that AVP acts through cAMP and protein kinase A (PKA) to phosphorylate AQP2 and other proteins. One result of these phosphorylation events is to increase the trafficking of AQP2 from vesicular pools to the apical membrane of the collecting-duct cells. Thus, AVP increases AQP2 density in the apical membrane; that is, the number of water channels per unit area of apical membrane. In addition, PKA also phosphorylates AQP2 itself as well as cAMP response element–binding protein (CREB; see p. 89). The phosphorylation of CREB, in the longer term, stimulates AQP2 synthesis, as indicated in Figure 38-9.

818.e1

Chapter 38  •  Urine Concentration and Dilution

819

BOX 38-1  Diabetes Insipidus

D

iabetes insipidus (DI) is a fairly rare disorder that occurs in two varieties. The first, neurogenic or central DI, is caused by failure of AVP secretion. The lesion can be at the level of either the hypothalamus (where neurons synthesize AVP) or the pituitary gland (where neurons release AVP). Central DI can be idiopathic, familial, or caused by any disorder of the hypothalamus or pituitary, such as injury, a tumor, infection, or autoimmune processes. In the second variety, nephrogenic DI (NDI), the kidneys respond inadequately to normal or even elevated levels of circulating AVP due to familial or acquired defects. Ninety percent of the familial cases are due to mutations in the X-linked AVPR2 gene that encodes the V2 receptor, and 10%, to mutations in the AQP2 gene. Acquired NDI may be associated with electrolyte abnormalities (e.g., states of K+ depletion or high plasma [Ca2+]  N36-14), the renal disease associated with sickle cell anemia, and various drugs (notably Li+ salts and colchicine). In both central and nephrogenic DI, patients present with polyuria and polydipsia. If patients cannot gain access to water on their own (e.g., infants, bedridden elderly), the disorder can result in marked hypernatremia, hypotension, and shock. Often the physician first suspects the diagnosis when the patient is deprived of access to water or other fluids. The patient may then quickly become dehydrated, and a random determination of plasma [Na+] may yield a very high value. The physician can confirm the diagnosis of DI most easily by a fluid-deprivation test. The patient will continue to produce a large output of dilute urine, despite the need to conserve fluids.

If the patient has central DI, administering a subcutaneous dose of AVP will rapidly increase urine osmolality by >50%. In patients with nephrogenic DI, on the other hand, the increase in urine osmolality will be less. The treatment for central DI is desmopressin acetate (DDAVP) (see Fig. 56-10), a synthetic AVP analog that patients can take intranasally. Nephrogenic DI, in which the kidneys are resistant to the effects of the hormone, does not respond to DDAVP therapy. In these patients, it is best to treat the underlying disease. It can also be helpful to administer a diuretic (to produce natriuresis) and restrict dietary Na+ to induce a state of volume depletion, which in turn enhances proximal NaCl and water reabsorption and thereby moderates the polyuria. The high urine flow in DI is associated with low rates of solute excretion. Therefore, the physician must distinguish DI from states of polyuria accompanied by high rates of solute excretion in the urine (osmotic diuresis). The most frequent cause of the latter is untreated diabetes mellitus. In that case, the polyuria occurs because the high plasma [glucose] leads to the filtration of an amount of glucose that exceeds the capacity of the proximal tubule to retrieve it from the lumen (see pp. 772–773). Another cause of osmotic diuresis is the administration of poorly reabsorbable solutes, such as mannitol. In an entirely distinct class of polyurias is primary polydipsia, a psychoneurotic disorder in which patients drink large amounts of fluid. Whereas simple water deprivation benefits a patient with primary polydipsia, it aggravates the condition of a patient with DI.

BOX 38-2  Role of Aquaporins in Renal Water Transport

W

hereas AQP1 is the water channel responsible for a large amount of transcellular fluid movement in the proximal tubule and the tDLH, three related isoforms of the water channel protein—AQP2, AQP3, and AQP4—are present in the principal cells of the collecting ducts. These channels regulate water transport in collecting tubules and ducts. Apical AQP2 is the basis for AVP-regulated water permeability. AQP3 and AQP4 are present in the basolateral membrane of principal cells, where they provide an exit pathway for water movement into the peritubular fluid. Short-term and long-term regulation of water permeability depends on an intact AQP2 system. In short-term regulation, AVP—via cAMP—causes water channel–containing vesicles from a subapical pool to fuse with the apical membrane (see Fig. 38-9). As a result, the number of channels and the water permeability sharply increase. In long-term regulation, AVP—by enhancing transcription of the AQP2 gene—increases the abundance of AQP2 protein in principal cells.

observations on the TAL and CCT were all made on rodents. In humans these TAL and CCT mechanisms may have only minor significance. In the inner medulla, AVP enhances the urea permea­ bility of the terminal two thirds of the IMCD (see pp. 811– 813). The AVP-dependent increase in [cAMP]i that triggers the apical insertion of AQP2-containing vesicles also leads

Mutations of several AQP genes lead to loss of function and marked abnormalities of water balance. Examples include sharply decreased fluid absorption along the proximal tubule in AQP1 knockout animals and nephrogenic diabetes insipidus (see Box 38-1) in patients with mutations of the gene for AQP2. An interesting situation may develop during the third trimester of pregnancy, when elevated plasma levels of vasopressinase— a placental aminopeptidase that degrades AVP—may lead to a clinical picture of central diabetes insipidus. An acquired increase of AQP2 expression often accompanies states of abnormal fluid retention, such as congestive heart failure, hepatic cirrhosis, nephrotic syndrome, and pregnancy. In addition, some conditions—including acute and chronic renal failure, primary polydipsia, consumption of a low-protein diet, and syndrome of inappropriate antidiuretic hormone secretion (see Box 38-3)—are associated with increased AQP2 levels in the apical membrane.

to a phosphorylation of apical UT-A1 and basolateral UT-A3 urea transporters (see p. 770), increasing their activity. The result is a substantial increase in urea reabsorption and thus the high interstitial [urea] that is indirectly responsible (see p. 816) for generating the osmotic gradient that drives water reabsorption in the inner medulla (Boxes 38-1, 38-2, and 38-3).

820

SECTION VI  •  The Urinary System

BOX 38-3  Syndrome of Inappropriate Antidiuretic Hormone Secretion

T

he syndrome of inappropriate ADH secretion (SIADH) is the opposite of diabetes insipidus. Patients with SIADH secrete ADH (i.e., AVP) or AVP-like substances at levels that are inappropriately high, given the low plasma osmolality and lack of hypovolemia. Thus, the urine osmolality is inappro­ priately high and patients are unable to excrete ingested water loads normally. As a result, total-body water increases, the blood becomes hypo-osmolar, plasma [Na+] drops (hyponatremia), and cells swell. If plasma [Na+] falls substantially, cell swelling can cause headaches, nausea, vomiting, and behavioral changes. Eventually, stupor, coma, and seizures may ensue. Before making the diagnosis of SIADH, the physician must rule out other causes of hyponatremia in which AVP levels may be appropriate. In Chapter 40, we discuss how plasma osmolality (see p. 844) and effective circulating volume (see p. 843) appropriately regulate AVP secretion. SIADH has four major causes: 1. Certain malignant tumors (e.g., bronchogenic carcinoma, sarcomas, lymphomas, and leukemias) release AVP or AVP-like substances.

REFERENCES The reference list is available at www.StudentConsult.com.

2. Cranial disorders (e.g., head trauma, meningitis, and brain abscesses) can increase AVP release. 3. Nonmalignant pulmonary disorders (e.g., tuberculosis, pneumonia, and abscesses) and positive-pressure ventilation also can cause SIADH.  N38-7 4. Several drugs can either stimulate AVP release (e.g., clofibrate, phenothiazines), increase the sensitivity of renal tubules to AVP (e.g., chlorpropamide), or both (e.g., carbamazepine). Treatment is best directed at the underlying disorder, combined, if necessary and clinically appropriate, with fluid restriction. Patients with severe hyponatremia and marked symptoms must receive urgent attention. Infusion of hyperosmotic Na+ is usually effective, but the correction must be gradual or severe neurological damage can result owing to rapid changes in the volume of neurons, especially in the pontine area of the brainstem.

Chapter 38  •  Urine Concentration and Dilution

N38-7  Pulmonary Disorders Causing Syndrome of Inappropriate Antidiuretic Hormone Secretion Contributed by Emile Boulpaep and Walter Boron Several chronic, nonmalignant pulmonary conditions, including positive-pressure ventilation, impede venous return. The result is reduced stretch of the atrial receptors (see Fig. 23-7). As discussed on page 547, the afferent fibers from these stretch receptors project not only to the medulla (where they produce cardiovascular effects) but also to the hypothalamic neurons that synthesize and release AVP. Decreased atrial stretch increases AVP release. Thus, the aforementioned pulmonary conditions result in a syndrome of inappropriate AVP (ADH) release—SIADH.

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Chapter 38  •  Urine Concentration and Dilution

REFERENCES Books and Reviews Agre P, Preston GM, Smyth BL, et al: Aquaporin CHIP: The archetypal molecular water channel. Am J Physiol 265:F463–F476, 1993. Greger R: Transport mechanisms in thick ascending limb of Henle’s loop of mammalian nephron. Physiol Rev 65:760–797, 1985. Knepper MA, Saidel GM, Hascall VC, Dwyer T: Concentration of solutes in the renal inner medulla: Interstitial hyaluronan as a mechano-osmotic transducer. Am J Physiol Renal Physiol 284:F433–F446, 2003. Moeller HB, Fenton RA: Cell biology of vasopressin-regulated aquaporin-2 trafficking. Pflugers Arch 464:133–144, 2012. Sands JM, Layton HE: Urine concentrating mechanism and its regulation. In Seldin DW, Giebisch G (eds): The Kidney: Physiology and Pathophysiology, 3rd ed. Philadelphia, Lippincott Williams & Wilkins, 2000. Sands JM, Layton HE: The physiology of urinary concentration: An update. Semin Nephrol 29:178–195, 2009. Sasaki W, Ishibashi K, Marumo F: Aquaporin-2 and -3: Representatives of two subgroups of the aquaporin family colocalized in the kidney collecting duct. Annu Rev Physiol 60:199– 220, 1998. Seldin DW, Giebisch G (eds): The Kidney: Physiology and Pathophysiology, 3rd ed. Philadelphia, Lippincott Williams & Wilkins, 2000. Shayakul C, Hediger MA: The SLC14 gene family of urea transporters. Pflugers Arch 447:603–609, 2004.

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Tsukaguchi H, Shayakul C, Berger UV, Hediger MA: Urea transporters in kidney: Molecular analysis and contribution to the urinary concentrating process. Am J Physiol 275:F319–F324, 1998. Journal Articles Deen PMT, Verdijk MAJ, Knoers NVAM, et al: Requirement of human renal water channel AQP-2 for vasopressin-dependent concentration of urine. Science 264:92–95, 1994. Gottschalk CW, Mylle M: Micropuncture study of composition of loop of Henle fluid in desert rodents. Am J Physiol 204:532–535, 1959. Grantham JJ, Burg MB: Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules. J Clin Invest 49:1815– 1826, 1970. Lassiter WE, Gottschalk CW, Mylle M: Micropuncture study of net transtubular movement of water and urea in nondiuretic kidney. Am J Physiol 200:1139–1146, 1964. Potter EA, Stewart G, Smith CP: Urea flux across MDCK-mUT-A2 monolayers is acutely sensitive to AVP, cAMP, and [Ca2+]i. Am J Physiol Renal Physiol 291:F122–128, 2006. Sanjana VM, Robertson CR, Jamison RL: Water extraction from the inner medullary collecting tubule system: A role for urea. Kidney Int 10:139–146, 1976. Stewart GS, King SL, Potter EA, Smith CP: Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line. Am J Physiol Renal Physiol 292:F1157–1163, 2007. Pallone TL, Edwards A, Ma, T, et al: The intrarenal distribution of blood flow. Adv Organ Biol 9:75–92, 2000.

C H A P T E R 39  TRANSPORT OF ACIDS AND BASES Gerhard Giebisch, Erich E. Windhager, and Peter S. Aronson

The lungs and the kidneys are largely responsible for regulating the acid-base balance of the blood (see Chapter 28). They do so by independently controlling the two major components of the body’s major buffering system: CO2 and HCO3− (Fig. 39-1). Chapter 31 focuses on how the lungs control plasma [CO2]. In this chapter we see how the kidneys control plasma [HCO3− ].

ACID-BASE BALANCE AND THE OVERALL RENAL HANDLING OF ACID Whereas the lungs excrete the large amount of CO2 formed by metabolism, the kidneys are crucial for excreting nonvolatile acids The kidneys play a critical role in helping the body rid itself of excess acid that accompanies the intake of food or that forms in certain metabolic reactions. By far, the largest potential source of acid is CO2 production (Table 39-1, section A), which occurs during oxidation of carbohydrates, fats, and most amino acids (see pp. 1185–1187). An adult ingesting a typical Western diet produces ~15,000 mmol/day of CO2. This CO2 would act as an acid if it went on to form H+ and HCO3− (see p. 630). Fortunately, the lungs excrete this prodigious amount of CO2 by diffusion across the alveolar-capillary barrier (see p. 673), preventing the CO2 from forming H+. However, metabolism also generates nonvolatile acids— such as sulfuric acid, phosphoric acid, and various organic acids—that the lungs cannot handle (see Table 39-1, section B). In addition, metabolism generates nonvolatile bases, which end up as HCO3− (see Table 39-1, section C). Sub­ tracting the metabolically generated base from the meta­ bolically generated acid leaves a net endogenous H+ production of ~40 mmol/day for a person weighing 70 kg. The strong acids contained in a typical Western acid-ash diet (20 mmol/day of H+ gained) and the obligatory loss of bases in stool (10 mmol/day of OH− lost) represent an additional acid load to the body of 30 mmol/day. Thus, the body is faced with a total load of nonvolatile acids (i.e., not CO2) of ~70 mmol/day—or ~1 mmol/kg body weight—derived from metabolism, diet, and intestinal losses. The kidneys handle this acid load by “dividing” 70 mmol/day of carbonic acid (H2CO3): excreting ~70 mmol/day of H+ into the urine and simultaneously transporting 70 mmol/day of new

HCO3− into the blood. Once in the blood, this new HCO3− neutralizes the daily load of 70 mmol of nonvolatile acid. Were it not for the tightly controlled excretion of H+ by the kidney, the daily load of ~70 mmol of nonvolatile acids would progressively lower plasma pH and, in the process, exhaust the body’s stores of bases, especially HCO3− . The result would be death by relentless acidification. Indeed, one of the characteristic symptoms of renal failure is severe acidosis caused by acid retention.  N39-1  The kidneys continuously monitor the acid-base parameters of the extracellular fluid (ECF) and adjust their rate of acid secretion to maintain the pH of ECF within narrow limits. In summary, although the lungs excrete an extremely large amount of a potential acid in the form of CO2, the kidneys play an equally essential role in the defense of the normal acid-base equilibrium, because they are the sole effective route for neutralizing nonvolatile acids.

To maintain acid-base balance, the kidney must not only reabsorb virtually all filtered HCO3- but also secrete generated nonvolatile acids In terms of acid-base balance, the major task of the kidney is to secrete acid into the urine and thus to neutralize the nonvolatile acids that metabolism produces. However, before the kidney can begin to achieve this goal, it must deal with a related and even more serious problem: retrieving from the tubule fluid virtually all HCO3− filtered by the glomeruli. Each day, the glomeruli filter 180 L of blood plasma, each liter containing 24 mmol of HCO3− , so that the daily filtered load of HCO3− is 180 L × 24 mM = 4320 mmol. If this filtered HCO3− were all left behind in the urine, the result would be equivalent to an acid load in the blood of 4320 mmol, or a catastrophic metabolic acidosis (see p. 635). The kidneys avoid this problem by reclaiming virtually all the filtered HCO3− through secretion of H+ into the tubule lumen and titration of the 4320 mmol/day of filtered HCO3− to CO2 and H2O. After the kidney reclaims virtually all the filtered HCO3− (i.e., 4320 mmol/day), how does it deal with the acid load of 70 mmol/day produced by metabolism, diet, and intestinal losses? If we simply poured 70 mmol of nonvolatile acid into the ~1.5 L of “unbuffered” urine produced each day, urinary [H+] would be 0.070 mol/1.5 L = 0.047 M, which would correspond to a pH of ~1.3. The lowest urine pH that the kidney 821

Chapter 39  •  Transport of Acids and Bases

N39-1  Acidoses of Renal Origin Contributed by Erich Windhager and Gerhard Giebisch Any overall decrease in the ability of the kidneys to excrete the daily load of ~70 mmol of nonvolatile acids will lead to metabolic acidosis. In the strict sense of the term, renal tubular acidosis (RTA) is an acidosis that develops secondary to the dysfunction of renal tubules. In addition, an overall decrease in useful renal mass and GFR—as occurs in endstage renal disease—also leads to an acidosis of renal origin. One system of organizing these maladies recognizes five types of RTAs: • Uremic acidosis or RTA of glomerular insufficiency. The fundamental problem is a decrease in the total amount of NH3 that the proximal tubule can synthesize from glutamine (see pp. 829–831). • Proximal (type 2) RTA. A specific dysfunction of the proximal tubule reduces the total amount of HCO3− that these nephron segments reabsorb. • Classical distal (type 1) RTA. A specific dysfunction of the distal tubule reduces the total amount of HCO3− that these nephron segments reabsorb. The mechanisms can include mutations of key proteins involved in distal H+ secretion, such as H pumps and Cl-HCO3 exchangers. • Generalized (type 4) RTA. A global dysfunction of the distal tubule—secondary to aldosterone deficiency or aldosterone resistance (see p. 835)—leads to a reduced net excretion of acid. • Type 3 RTA. Rare defects in CAII lead to defects in both proximal and distal H+ secretion.

821.e1

822

SECTION VI  •  The Urinary System

Net uptake of acid = 30 mmol/day

Diet + 20 mmol H /day

Absorbed + 20 mmol H /day

15,000 mmol CO2/day ECF pH 7.4

Lung

Metabolism

Gut + H as nonvolatile acids 40 mmol/day

Secreted_ 10 mmol OH /day Feces 10 mmol OH–/day

Reabsorbed 4320 mmol HCO3–/day “New” HCO–3 70 mmol/day

Filtered 4320 mmol HCO3–/day

Expired air 15,000 mmol CO2/day

Kidneys

+

40 mmol NH4/day

30 mmol titratable acid/day Urine + 70 mmol H /day

Figure 39-1  Acid-base balance. All values are for a 70-kg human consuming a typical Western acid-ash diet. The values in the boxes are approximations.

TABLE 39-1  Metabolic Sources of Nonvolatile Acids and Bases A.  Reactions Producing CO2 (merely a potential acid)

1. Complete oxidation of neutral carbohydrate and fat → CO2 + H2O 2. Oxidation of most neutral amino acids → Urea + CO2 + H2O

B.  Reactions Producing Nonvolatile Acids

1. Oxidation of sulfur-containing amino acids → Urea + CO2 + H2O + H2SO4 → 2 H+ + SO2− 4 (Examples: methionine, cysteine) 2. Metabolism of phosphorus-containing compounds → H3PO4 → H+ + H2PO−4 3. Oxidation of cationic amino acids → Urea + CO2 + H2O + H+ (e.g., lysine+, arginine+) 4. Production of nonmetabolizable organic acids → HA → H+ + A− (e.g., uric acid, oxalic acid) 5. Incomplete oxidation of carbohydrate and fat → HA → H+ + A− (e.g., lactic acid, keto acids)

phosphate, creatinine, and urate. Because of its favorable pK of 6.8 and its relatively high rate of excretion, phosphate is the most important nonvolatile filtered buffer. The other major urinary buffer is NH3 /NH +4 , which the kidney synthesizes. After diffusing into the tubule lumen, the NH3 reacts with secreted H+ to form NH +4 . Through adaptive increases in the synthesis of NH3 and excretion of NH +4 , the kidneys can respond to the body’s need to excrete increased loads of H+. The kidney does not simply eliminate the 70 mmol/day of nonvolatile acids by filtering and then excreting them in the urine. Rather, the body deals with the 70-mmol/day acid challenge in three steps: Step 1: Extracellular HCO3− neutralizes most of the H+ load:

can achieve is ~4.4, which corresponds to an [H+] that is three orders of magnitude lower than required to excrete the 70 mmol/day of nonvolatile acids. The kidneys solve this problem by binding the H+ to buffers that the kidney can excrete within the physiological range of urinary pH values. Some of these buffers the kidney filters—for example,

(39-1) 

Acid load

Thus, HCO3− decreases by an amount that is equal to the H+ it consumes, and an equal amount of CO2 is produced in the process. Non-HCO3− buffers (see p. 635) in the blood neutralize most of the remaining H+ load:

C.  Reactions Producing Nonvolatile Bases

1. Oxidation of anionic amino acids → Urea + CO2 + H2O + HCO3− (e.g., glutamate−, aspartate−) 2. Oxidation of organic anions → CO2 + H2O + HCO3− (e.g., lactate−, acetate−)

+ HCO3− + H  → CO2 + H2 O



+ B− + H  → BH

(39-2) 

Acid load

Thus, B−, too, decreases by an amount that is equal to the H+ it consumes. A very tiny fraction of the H+ load (99.9%). As discussed beginning on page 825, the kidney reabsorbs HCO3− at specialized sites along the nephron. However, regardless of the site, the basic mechanism of HCO3− reabsorption is the same (Fig. 39-2A): H+ transported into the lumen by the tubule cell titrates filtered HCO3− to CO2 plus H2O. One way that this titration can occur is by H+ interacting with HCO3− to form H2CO3, which in turn dissociates to yield H2O and CO2. However, the reaction H2CO3 → H2O + CO2 is far too slow to convert the entire filtered load of HCO3− to CO2 plus H2O. The enzyme carbonic anhydrase (CA)  N18-3—which is present in many tubule segments—bypasses this slow reaction by splitting HCO3− into CO2 and OH− (see Table 39-1). The secreted H+ neutralizes this OH− so that the net effect is to accelerate the production of H2O and CO2. The apical membranes of these H+-secreting tubules are highly permeable to CO2, so that the CO2 produced in the lumen, as well as the H2O, diffuses into the tubule cell. Inside the tubule cell, the CO2 and H2O regenerate intra­ cellular H+ and HCO3− with the aid of CA. Finally, the cell exports these two products, thereby moving the H+ out across the apical membrane into the tubule lumen and the HCO3− out across the basolateral membrane into the blood. Thus, for each H+ secreted into the lumen, one HCO3− disappears from the lumen, and one HCO3− appears in the blood. However, the HCO3− that disappears from the lumen and the HCO3− that appears in the blood are not the same molecule! To secrete H+ and yet keep intracellular pH within narrow physiological limits (see pp. 644–645), the cell closely coordinates the apical secretion of H+ and the basolateral exit of HCO3− . Two points are worth re-emphasizing. First, HCO3− reabsorption does not represent net H+ excretion into the urine. It merely prevents the loss of the filtered alkali. Second, even though HCO3− reabsorption is simply a reclamation effort, this process consumes by far the largest fraction of the H+ secreted into the tubule lumen. For example, reclaiming the 4320 mmol of HCO3− filtered each day requires 4320 mmol

Chapter 39  •  Transport of Acids and Bases

N39-2  Urinary Excretion of Carboxylates Contributed by Peter Aronson and Gerhard Giebisch In addition to the loss of filtered HCO3− in the urine, the excretion of organic anions that can undergo conversion to HCO3− (e.g., lactate, citrate) would represent a loss of alkali into the urine, which in principle would need to be taken into account in computing net renal acid excretion. Because the proximal tubule normally reabsorbs nearly all of these carboxylates (see p. 779), this component of alkali loss is minor under most circumstances.

823.e1

824

A

SECTION VI  •  The Urinary System

Figure 39-2  Titration of luminal buffers by secreted H+. A and B, Generic

HCO3– REABSORPTION Tubule lumen

Interstitial space

HCO3–

models of H+ secretion at various sites along the nephron. The red arrows represent diverse transport mechanisms. C, Ammonium handling by the proximal tubule.

Carbonic anhydrases

CO2 –

OH

OH

+

+

H

H

of H+ secretion, far more than the additional 70 mmol/day of H+ secretion necessary for neutralizing nonvolatile acids.

CA IV and XIV CO2 CA II HCO–3

–

Retrieved HCO3–

Titration of Filtered Non-HCO3- Buffers (Titratable-Acid For­ mation)  The H+ secreted into the tubules can interact with

buffers other than HCO3− and NH3. The titration of the nonNH3, non-HCO3− buffers (B−)—mainly HPO2− 4 , creatinine, and urate—to their conjugate weak acids (HB) constitutes the titratable acid discussed on page 823.

H2O



H+ + B− →

BH



(39-4) 

Titratable acid

B

FORMATION OF TITRATABLE ACID Tubule lumen

Interstitial space

CO2 CA II HCO3–

–

+

H

OH

+

H

New HCO3–

HPO42–

H2PO4–

C

AMMONIUM EXCRETION Tubule lumen

Interstitial space

CO2 CA II HCO3– +

H

NH3

+

H

NH3

+

NH4

OH–

Metabolism + NH4

New HCO3–

The major proton acceptor in this category of buffers excreted in the urine is HPO2− 4 , although creatinine also makes an important contribution; urate and other buffers contribute to a lesser extent. Figure 39-2B shows the fate of H+ as it protonates phosphate from its divalent form (HPO2− 4 ) to its monovalent form (H2 PO−4 ). Because low luminal pH inhibits the apical Na/phosphate cotransporter (NaPi) in the proximal tubule, and NaPi carries H2 PO−4 less effectively than + HPO2− 4 (see pp. 785–786), the kidneys tend to excrete H + bound phosphate in the urine. For each H it transfers to the lumen to titrate HPO2− 4 , the tubule cell generates one new HCO3− and transfers it to the blood (see Fig. 39-2B). How much does the “titratable acid” contribute to net acid excretion? The following three factors determine the rate at which these buffers act as vehicles for excreting acid: 1. The amount of the buffer in the glomerular filtrate and final urine. The filtered load (see p. 732) of HPO2− 4 , for example, is the product of plasma [HPO2− 4 ] and glomerular filtration rate (GFR). Plasma phosphate levels may range from 0.8 to 1.5 mM (see p. 1054). Therefore, increasing plasma [HPO2− 4 ] allows the kidneys to excrete more H+ in the urine as H2 PO−4 . Conversely, decreasing the GFR (as in chronic renal failure) reduces the amount of HPO2− 4 available for buffering, lowers the excretion of titratable acid, and thus contributes to metabolic acidosis. Ultimately, the key parameter is the amount of buffer excreted in the urine. In the case of phosphate, the fraction of the filtered load that the kidney excretes increases markedly as plasma [phosphate] exceeds the maximum saturation (Tm; see p. 786). For a plasma [phosphate] of 1.3 mM, the kidneys reabsorb ~90%, and ~30 mmol/day appear in the urine. 2. The pK of the buffer. To be most effective at accepting H+, the buffer (e.g., phosphate, creatinine, urate) should have a pK value that is between the pH of the glomerular filtrate and the pH of the final urine. For example, if blood plasma has a pH of 7.4, then only ~20% of its phosphate (pK = 6.8) will be in the form of H2 PO−4 (Table 39-3). Even if the final urine were only mildly acidic, with a pH of 6.2, ~80% of the phosphate in the urine would be in the form of H2 PO−4 . In other words, the kidney would

Chapter 39  •  Transport of Acids and Bases

TABLE 39-3  Titration of Buffers % PROTONATED BUFFER pH

PHOSPHATE (pK = 6.8)

URATE (pK = 5.8)

CREATININE (pK = 5.0)

7.4

20.1

2.5

0.4

6.2

79.9

28.5

5.9

4.4

99.6

96.2

79.9

have titrated ~60% of the filtered phosphate from HPO2− 4 to H2 PO−4 . Because creatinine has a pK of 5.0, lowering the pH of the tubule fluid from 7.4 to 6.2 increases the fractional protonation of creatinine from ~0.4% to only ~6%. However, urate has a pK of 5.8, so lowering pH from 7.4 to 6.2 would increase its fractional protonation from 2.5% to 28.5%. 3. The pH of the urine. Regardless of the pK of the buffer, the lower is the urinary pH, the more protonated is the buffer and the greater is the amount of acid excreted with this buffer. As discussed, lowering the pH of the tubule fluid from 7.4 to 6.2 increases the protonation of creatinine from 0.4% to only ~6%. However, if the pH of the final urine is 4.4, the fractional protonation of creatinine increases to ~80% (see Table 39-3). Thus, creatinine becomes a much more effective buffer during acidosis, when the kidney maximally acidifies the urine. Titration of Filtered and Secreted NH3 (Ammonium Excre­ tion)  The third class of acceptors of luminal H+ is NH3.

However, unlike either HCO3− or the bases that give rise to “titratable acid” (e.g., HPO2− 4 ), glomerular filtration contributes only a negligible quantity of NH3 because plasma [NH3] concentration is exceedingly low. Instead, urinary NH3 derives mainly from diffusion into the lumen from the proximal-tubule cell (see Fig. 39-2C), with some NH +4 entering the lumen directly via the apical Na-H exchanger NHE3. In the case of the proximal tubule, the conversion of glutamine to α-ketoglutarate (α-KG) generates two NH +4 ions, which form two NH3 and two H+ ions. In addition, the metabolism of α-KG generates two OH− ions, which CA converts to HCO3− ions. This new HCO3− then enters the blood.  N39-3 In summary, when renal-tubule cells secrete H+ into the lumen, this H+ simultaneously titrates three kinds of buffers: (1) HCO3− , (2) HPO2− 4 and other buffers that become the “titratable acid,” and (3) NH3. Each of these three buffers competes with the other two for available H+. In our example, the kidneys secrete 4390 mmol/day of H+ into the tubule lumen. The kidneys use most of this secreted acid— 4320 mmol/day or ~98% of the total—to reclaim filtered HCO3−. The balance of the total secreted H+, 70 mmol/day, the kidneys use to generate new HCO3− .

ACID-BASE TRANSPORT BY DIFFERENT SEGMENTS OF THE NEPHRON Most nephron segments secrete H+ to varying degrees.

825

The nephron reclaims virtually all the filtered HCO3in the proximal tubule (~80%), thick ascending limb (~10%), and distal nephron (~10%) The kidney reabsorbs the largest fraction of filtered HCO3− (~80%) along the proximal tubule (Fig. 39-3A). By the end of the proximal tubule, luminal pH falls to ~6.8, which represents only a modest transepithelial H+ gradient compared with the plasma pH of 7.4. Thus, the proximal tubule is a high-capacity, low-gradient system for H+ secretion. The thick ascending limb of the loop of Henle (TAL) reabsorbs an additional 10% of filtered HCO3− , so that by the time the tubule fluid reaches the distal convoluted tubule (DCT), the kidney has reclaimed ~90% of the filtered HCO3− . The rest of the distal nephron—from the DCT to the inner medullary collecting duct (IMCD)—reabsorbs almost all the remaining ~10% of the filtered HCO3− . Although the latter portion of the nephron reabsorbs only a small fraction of the filtered HCO3− , it can lower luminal pH to ~4.4. Thus, the collecting tubules and ducts are a low-capacity, high-gradient system for H+ transport. The amount of HCO3− lost in the urine depends on urine pH. If the [CO2] in the urine were the same as that in the blood, and if urine pH were 5.4, the [HCO3− ] in the urine would be 0.24 mM, which is 1% of the 24 mM in blood (see p. 630). For a urine production of 1.5 L/day, the kidneys would excrete 0.36 mmol/day of HCO3− . For a filtered HCO3− load of 4320 mmol/day, this loss represents a fractional excretion of ~0.01%. In other words, the kidneys reclaim ~99.99% of the filtered HCO3−. Similarly, at a nearly maximally acidic urine pH of 4.4, urine [HCO3− ] would be only 0.024 mM. Therefore, the kidneys would excrete only 36 µmol/day of filtered HCO3− and would reabsorb ~99.999%.

The nephron generates new HCO3- , mostly in the proximal tubule The kidney generates new HCO3− in two ways (see Fig. 39-3B). It titrates filtered buffers such as HPO2− 4 to produce “titratable acid,” and it titrates secreted NH3 to NH +4 . In healthy people, NH +4 excretion is the more important of the two and contributes ~60% of net acid excretion or new HCO3−. Formation of Titratable Acid  The extent to which a particular buffer contributes to titratable acid (see Fig. 39-2B) depends on the amount of buffer in the lumen and luminal pH. The titratable acid due to phosphate is already substantial at the end of the proximal tubule (Table 39-4), even though the proximal tubule reabsorbs ~80% of the filtered phosphate. The reason is that the luminal pH equals the pK of the buffer at the end of the proximal tubule. The titratable acid due to phosphate rises only slightly along the classical distal tubule (i.e., DCT, connecting tubule [CNT], and initial collecting tubule [ICT]), because acid secretion slightly exceeds phosphate reabsorption. The titratable acid due to phosphate rises further as luminal pH falls to 4.4 along the collecting ducts in the absence of significant phosphate reabsorption. Although the late proximal tubule secretes creatinine, the titratable acid due to creatinine (see Table 39-4) is minuscule at the end of the proximal tubule, because luminal pH is so

Chapter 39  •  Transport of Acids and Bases

825.e1

N39-3  Ammonium Secretion by the Medullary Collecting Duct Contributed by Erich Windhager, Gerhard Giebisch, Emile Boulpaep, and Walter Boron Ammonium secretion by the medullary collecting duct is critical for renal NH+4 excretion. As described in Figure 39-5C, the TAL of juxtamedullary nephrons reabsorbs some NH+4 and deposits this NH+4 in the medullary interstitium, where it is partitioned between ammonium and ammonia according to the equilibrium NH+4 ⇌ NH3 + H+. As pointed out in Figure 39-5D, this interstitial NH+4 (and NH3) can have three fates: (1) some recycles back to the late proximal tubule and descending thin limb of Henle, (2) some bypasses the cortex by being secreted into the medullary collecting duct, and (3) some is washed out by the blood for export to the liver. The mechanism of pathway (2) is depicted in Figure 39-5E. NH3 diffuses from the medullary interstitium, through the tubule cell and into the lumen. The NH3 moves via members of the Rh family at both the basolateral and apical membranes. The parallel extrusion of H+ across the apical membrane of the collecting-duct cell provides the luminal H+ that then titrates the luminal NH3 to NH+4 , which is excreted. This luminal H pumping also generates OH− inside the cells. Although not shown in Figure 39-5E, intracellular CA converts this newly created OH− (along with H2O) to HCO3− , and basolateral Cl-HCO3 exchangers then export this Tubule lumen

newly created HCO3− to the interstitium. The HCO3− , of course, ultimately is washed out by the blood. Thus, for each NH+4 formed in the lumen of the collecting duct by this route, the tubule cell transfers one “new” HCO3− to the blood. Figure 39-5E also shows that the Na-K pump can also transport NH+4 directly into the collecting-duct cell. This intracellular NH+4 can then dissociate into NH3 (which can diffuse into the lumen) and H+ (which moves into the lumen via the apical H pump), with the ultimate formation of NH+4 in the lumen. The NH+4 that enters the collecting-duct lumen by this route does not generate a new HCO3− ion. eFigure 39-1 shows the most recent model for how the TAL handles NH3 and CO2.

REFERENCES Geyer RR, Musa-Aziz R, Qin X, Boron WF: Relative CO2/NH3 selectivities of mammalian aquaporins 0-9. Am J Physiol Cell Physiol 304:C985–C994, 2013. Weiner ID, Verlander JW: Ammonia transport in the kidney by Rhesus glycoproteins. Am J Physiol Renal Physiol 306(10): F1107–F1120, 2014.

Thick Ascending Limb Cell

Collecting Duct

Cl–

Tubule lumen pH ~ 5.5

 intercalated cell

Interstitial space pH ~ 7.4

AE1

pH ~ 7.1

K+ ATPase

H+ + HCO3–

2Cl– NKCC2 Na+ NH4+

NH4+

+

NH4

H+ CA II

H+ NH3

HCO3–

H2O

CO2

K+ channel

RhBG

CO2

diffusion

CO2 NH3

RhCG

CO2 NH3

NH3

HCO3–

H+ ATPase

H2O CO2

NH3 RhCG

NH3

diffusion

NH4+ NH4+

eFigure 39-1  Proposed model for CO2 and NH3 transport across the apical and basolateral membranes of TAL and α-intercalated cells in the collecting duct. Dashed arrows represent the possible diffusion of CO2 or NH3 across plasma membranes. NKCC2, Na/K/Cl cotransporter 2. (Republished with permission from Geyer RR, Parker MD, Toye AM, et al: Relative CO2/NH3 permeabilities of human RhAG, RhBG and RhCG. J Membrane Biology 246(12):915-926, F8, 2013.)

826

A

SECTION VI  •  The Urinary System

–

B

RECLAMATION OF FILTERED HCO3 ALONG THE NEPHRON GFR = 180 L/day – Plasma [HCO3 ] = 24 mM pH = 7.4 Filtered load = 4320 mmol/day 100% remaining

1 80% reabsorbed

2–

Plasma [HPO4 ] ≅ 1.04 mM – Plasma [H2PO4 ] ≅ 0.26 mM + Plasma [NH4 ] ≅ 0 mM

4% remaining 3

6% reabsorbed Distal convoluted tubule

Cortical collecting tubule

H+ to form TA 5 mmol/day H+ to form TA 15 mmol/day

–

New HCO3 5 mmol/day

H+ + NH3 40 mmol/day

10% remaining

–

Proximal convoluted tubule Proximal straight tubule

–

GENERATION OF NEW HCO3

New HCO3 55 mmol/day

2

10% reabsorbed 20% remaining

Thick ascending limb of Henle’s loop (TAL)

4 ~4% reabsorbed

+

NH 4

Outer medullary collecting duct

Inner medullary collecting duct

+

NH 4

H+ to form TA 10 mmol/day –

New HCO 3 10 mmol/day

· V = 1.5 L/day UHCO3– = 0.24 mM pH = 5.4 Urinary excretion of HCO3– ≅ 0.36 mmol/day ~0.01% of filtered load remaining

Excretion of 70 mmol/day of H+ corresponds to the generation of 70 mmol/day of new HCO3–.

Urinary excretion of H+ = 70 mmol/day

Figure 39-3  Acid-base handling along the nephron. In A, the numbered yellow boxes indicate the fraction of the filtered load reabsorbed by various nephron segments. The green boxes indicate the fraction of the filtered load that remains in the lumen at various sites. In B, the red boxes indicate the moieties of acid secretion associated with either the formation of titratable acid (TA) or the secretion of NH+4. The yellow boxes indicate the formation of new HCO3− or NH+4 reabsorption by the TAL. The values in the boxes are approximations.

much higher than creatinine’s pK. However, the titratable acidity due to creatinine increases substantially along the collecting ducts as luminal pH plummets. The urine contains the protonated form of other small organic acids (e.g., uric, lactic, pyruvic, and citric acids) that also contribute to titratable acid. NH+4 Excretion  Of the new HCO3− that the nephron gen­

erates, ~60% (~40 mmol/day) is the product of net NH +4

excretion (see Fig. 39-3B), which is the result of five processes: (1) the proximal tubule actually secretes slightly more than ~40 mmol/day of NH +4 , (2) the TAL reabsorbs some NH +4 and deposits it in the interstitium, (3) some of this interstitial NH +4 recycles back to the proximal tubule and thin descending limb (tDLH), (4) some of the interstitial NH +4 enters the lumen of the collecting duct, and finally, (5) some of the interstitial NH +4 enters the vasa recta and leaves the kidney. As we shall see on p. 831, the liver uses some of

Chapter 39  •  Transport of Acids and Bases

827

TABLE 39-4  Titratable Acidity of Creatinine and Phosphate Along the Nephron* PHOSPHATE

Bowman’s space

pH

FILTERED LOAD REMAINING (%)

7.4

100

CREATININE

TITRATABLE ACID DUE TO Pi (mmol/day) 0

FILTERED LOAD REMAINING (%) 100

End of PT

6.8

20

14.0

120

End of ICT

6.0

10

15.5

Final urine

5.4

10

17.8

TITRATABLE ACID DUE TO CREATININE (mmol/day) 0

†

SUM OF TITRATABLE ACID DUE TO PHOSPHATE AND CREATININE (mmol/day) 0

0.2

14.2

120

1.7

17.2

120

5.5

23.3

*Note that other buffers in the urine contribute to the total titratable acid, which increases with the excreted amount of each buffer and with decreases in urine pH. In this example, we assume a plasma [phosphate] of 1.3 mM, a plasma [creatinine] of 0.09 mM, and a GFR of 180 L/day. † We assume that the proximal tubule secretes an amount of creatinine that is equivalent to 20% of the filtered load. Pi, inorganic phosphate; PT, proximal tubule.

this NH +4 to generate urea, a process that consumes HCO3− . Thus, the net amount of new HCO3− attributable to NH +4 excretion is (1) − (2) + (3) + (4) − (5).

ACID-BASE TRANSPORT AT THE CELLULAR AND MOLECULAR LEVELS The secretion of acid from the blood to the lumen—whether for reabsorption of filtered HCO3− , formation of titratable acid, or NH +4 excretion—shares three steps: (1) transport of H+ (derived from H2O) from tubule cell to lumen, which leaves behind intracellular OH−; (2) conversion of intracellular OH− to HCO3− , catalyzed by CA; and (3) transport of newly formed HCO3− from tubule cell to blood. In addition, because the buffering power of filtered non-HCO3− buffers is not high enough for these buffers to accept sufficient luminal H+, the adequate formation of new HCO3− requires that the kidney generate buffer de novo. This buffer is NH3.

H+ moves across the apical membrane from tubule cell to lumen by Na-H exchange, electrogenic H pumping, and K-H pumping Although the kidney could, in principle, acidify the tubule fluid either by secreting H+ or by reabsorbing OH− or HCO3− , the secretion of H+ appears to be solely responsible for acidifying tubule fluid. At least three mechanisms can extrude H+ across the apical membrane; not all of these are present in any one cell. Na-H Exchanger  Of the known NHE isoforms (see p. 124), NHE3 is particularly relevant for the kidney because it moves more H+ from tubule cell to lumen than any other transporter.  N39-4  NHE3 is present not only throughout the proximal tubule (Fig. 39-4A, B) but also in the TAL (see Fig. 39-4C) and DCT. The apical NHE3 secretes H+ in exchange for luminal + Na . Because a steep lumen-to-cell Na+ gradient drives this exchange process (see p. 115), apical H+ secretion ultimately depends on the activity of the basolateral Na-K pump. The carboxyl termini of the NHEs have phosphorylation sites for various protein kinases. For example, protein kinase

A (PKA) phosphorylates apical NHE in the proximal tubule, inhibiting it. Both parathyroid hormone and dopamine inhibit NHE3 via PKA. Electrogenic H Pump  A second mechanism for apical H+ secretion by tubule cells is the electrogenic H pump, a vacuolar-type ATPase (see pp. 118–119). The ATP-driven H pump can establish steep transepithelial H+ concentration gradients, thus lowering the urine pH to ~4.0 to 5.0. In contrast, NHE3, which depends on the 10-fold Na+ gradient across the apical membrane, cannot generate an H+ gradient in excess of ~1 pH unit. The apical electrogenic H pumps are located mainly in a subpopulation of intercalated cells (α cells) of the CNT, ICT, and cortical collecting tubule (CCT) and in cells of the IMCD and outer medullary collecting duct (OMCD; Fig. 39-4D). However, H pumps are also present in the apical membrane of the proximal tubule (see Fig. 39-4A, B), the TAL (see Fig. 39-4C), and the DCT. In addition, an electrogenic H pump is also present in the basolateral membrane of β-intercalated cells.  N39-5  Mutations in genes encoding subunits of this H pump cause a metabolic acidosis (see p. 635) in the blood—a distal renal tubular acidosis (dRTA). The regulation of the apical H pump involves several mechanisms. First, the transepithelial electrical potential may modulate the H pump rate. For instance, aldosterone induces increased apical Na+ uptake by the principal cells in the CCT (see pp. 765–766), thus causing an increase in the lumen-negative potential, which in turn stimulates the H pump. Second, aldosterone stimulates the H pump independently of changes in voltage. Third, acidosis increases the recruitment and targeting of pump molecules to the apical membranes of α-intercalated cells in the CNT, ICT and CCT, whereas alkalosis has the opposite effect. H-K Exchange Pump  A third type of H+-secretory mecha-

nism is present in the ICT, the CCT, and the OMCD (see Fig. 39-4D): an electroneutral H-K pump (see pp. 117–118) that is related to the Na-K pump. Several isoforms of the H-K pump are present in the kidney and exhibit differential sensitivities to inhibition by drugs such as omeprazole, SCH-28080, and ouabain. The H-K pump probably does not contribute significantly to acid secretion under normal conditions.

Chapter 39  •  Transport of Acids and Bases

N39-4  Renal NHEs

827.e1

N39-5  The β-Intercalated Cell

Contributed by Peter Aronson, Emile Boulpaep, and Walter Boron

Contributed by Walter Boron, Peter Aronson, and Emile Boulpaep

As described on page 124 of the text, several related genes encode NHEs.  N5-20 In the renal proximal tubule, Na-H exchange is blocked by the removal of Na+ from the lumen. Although all NHEs are far less sensitive to amiloride than the ENaC epithelial Na+ channels (see pp. 758–759 and Fig. 35-4D), the apical NHE3 isoform in the proximal tubule is even less amiloride sensitive than the ubiquitous or “housekeeping” NHE1. The NHE1 isoform is present in the basolateral membranes of several nephron segments. The role of basolateral NHEs in acidsecreting nephron segments, such as the proximal tubule, is unclear; they may help regulate pHi independently of transepithelial H+ secretion. Given a 10 : 1 concentration gradient for Na+ from the proximal tubule lumen to the cell interior, a maximal pH gradient of 1 pH unit can be achieved by this gradient. Indeed, the late proximal tubule may have a luminal pH as low as ~6.4. The NHE2 isoform is present at the apical membrane of the DCT, where it may participate in the apical step of H+ secretion.

Electrogenic H pumps are present in β-intercalated cells (see Fig. 39-9B), which, to a first approximation, are backward α-intercalated cells (see Fig. 39-4D). We discuss β-intercalated cells (β-ICs) in the text on page 834. In β-ICs, the electrogenic H pump is present in the basolateral membrane, and the Cl-HCO3 exchanger is in the apical membrane. Thus, unlike the α-ICs, which engage in net HCO3− reabsorption, the β-ICs engage in net HCO3− secretion. An interesting difference between the α-ICs and the β-ICs is that in the α cells, the Cl-HCO3 exchanger is a variant of AE1 (the Cl-HCO3 exchanger in red blood cells, and a member of the SLC4 family), whereas in the β cells the Cl-HCO3 exchanger is molecularly quite different, being a member of the SLC26 family. In addition to the switch from α-IC to β-IC, HCO3− secretion can also be stimulated by increased luminal delivery of Cl−, which promotes the exchange of luminal Cl− for intracellular HCO3− via the apical Cl-HCO3 exchanger. A molecule by the name of hensin controls the conversion from β- to α-intercalated cells. Genetic deletion of hensin in the tubule causes a distal renal tubular acidosis (dRTA) because the mice secrete HCO3− inappropriately and therefore become HCO3− deficient in the blood.

REFERENCE Al-Awqati Q: 2007 Homer W. Smith Award: Control of terminal differentiation in epithelia. J Am Soc Nephrol 19:443– 449, 2008.

828

SECTION VI  •  The Urinary System

A EARLY PROXIMAL CONVOLUTED TUBULE (S1) Tubule lumen Interstitial – space HCO3 OH– CO 2 + Na H

CA IV and XIV CO CA II 3 +

H+

H

Interstitial space

–

HCO3

–

HCO3

–

H+

OH

OH– H+

CO2

H2O

D Interstitial space

Tubule lumen –

HCO3

+

H+

CA II

Cl–

Na+ 3

–

HCO3

CA IV

THICK ASCENDING LIMB (TAL)

Na

–

HCO3

CA II

+

Na

H2O

C

LATE PROXIMAL STRAIGHT TUBULE (S3)

Tubule lumen

Na+

2

+

B

AE2

α-INTERCALATED AND MEDULLARY COLLECTING-DUCT CELLS

Tubule lumen –

HCO3

H

Cl–

+

CA II

Interstitial space AE1 Cl–

+

K H+

H+

Figure 39-4  Cell models of H+ secretion.

However, K+ depletion (see p. 803) induces expression of the H-K pump, which retrieves luminal K+ and, as a side effect, enhances H+ secretion. This H+ secretion contributes to the metabolic alkalosis often observed in patients with hypokalemia—hypokalemic metabolic alkalosis.

CAs in the lumen and cytosol stimulate H+ secretion by accelerating the interconversion of CO2 and HCO3The CAs  N18-3  play an important role in renal acidification by catalyzing the interconversion of CO2 to HCO3−. Inhibition of CAs by sulfonamides, such as acetazolamide, profoundly slows acid secretion. CAs may act at three distinct sites of acid-secreting tubule cells (see Fig. 39-4): the extracellular face of the apical membrane, the cytoplasm, and the extracellular face of the basolateral membrane. Two CAs are especially important for tubule cells. The soluble CA II is present in the cytoplasm, whereas CA IV is coupled via a GPI linkage (see p. 13) to the outside of the apical and basolateral membranes, predominantly in proximal-tubule cells. Apical CA (CA IV)  In the absence of apical CA, the H+

secreted accumulates in the lumen, and Na-H exchange and H+ secretion are inhibited. By promoting the conversion of luminal HCO3− to CO2 plus OH−, apical CA prevents the lumen from becoming overly acidic and thus substantially

relieves this inhibition. Thus, CA promotes high rates of HCO3− reabsorption along the early proximal tubule (see Fig. 39-4A). In the distal nephron (see Fig. 39-4D), H+ secretion is less dependent on luminal CA than it is in the early proximal tubule for two reasons. First, the H+ secretion rate is lower than that in the proximal tubule. Thus, the uncatalyzed conversion of luminal H+ and HCO3− to CO2 and H2O can more easily keep up with the lower H+ secretion rate. Second, in the collecting tubules and ducts the electrogenic H pump can extrude H+ against a very high gradient. Therefore, even in the absence of CA, the collecting ducts can raise luminal [H+] substantially, thereby accelerating the uncatalyzed reaction by mass action. Cytoplasmic CA (CA II)  Cytoplasmic CA accelerates the conversion of intracellular CO2 and OH− to HCO3− (see Fig. 39-4). As a result, CA II increases the supply of H+ for apical H+ extrusion and the supply of HCO3− for the basolateral HCO3− exit step. In the CNT, ICT, and CCT, the intercalated cells (which engage in acid-base transport) contain CA II, whereas the principal cells do not. Basolateral CA (CA IV and CA XII)  The role played by basolateral CA IV and CA XII (an integral membrane protein with an extracellular catalytic domain) is not yet understood.  N39-6

Chapter 39  •  Transport of Acids and Bases

828.e1

N39-6  Carbonic Anhydrase at the Basolateral Membrane Contributed by Walter Boron Although it has been known for years that carbonic anhydrases (CAs) are present at the basolateral membrane of the proximal tubule (CA IV, CA XII), only recently has research begun to shed light on the significance of this observation. Two distinct classes of CAs are present near or at the basolateral membrane: (1) the cytosolic or soluble CA II, and (2) one or more membrane-bound CAs (e.g., CA IV, CA XII) with the catalytic domain facing the interstitial fluid. Renal CA XIV is abundant in rodents but is virtually undetectable in human and rabbit kidneys. The role of CA XIV in rodents may be an adaptation to the relatively low activity of rodent CA IV, owing to a G63Q substitution.

CA II According to several reports, the soluble CA II binds reversibly to a site on the cytosolic carboxyl termini of certain HCO3− transporters in the SLC4 family. Among these is the electrogenic Na/ HCO3 cotransporter NBCe1, which is responsible for the vast majority of HCO3− efflux across the basolateral membrane of the proximal tubule (see Fig. 39-4A). According to one viewpoint, the function of the bound CA II is to provide HCO3− as a substrate for the NBCe1 to export to the basolateral side of the tubule cell according to the reaction

CA II CO2 + OH −   → HCO3−

(NE 39-1) 

Published data are consistent with the hypothesis that bound—but not free—CA II increases HCO3− transport. According to an alternate view that is emerging from the laboratory of Walter Boron, the role of CA II is very different. Preliminary data suggest that NBCe1 transports CO2− 3 . Thus, when operating with an apparent Na+:HCO3− stoichiometry of 1 : 3, as it appears to do in the proximal tubule, NBCe1 might actually − transport 1 Na+, 1 CO2− 3 , and 1 HCO3 out of the cell across the basolateral membrane. You might imagine that 1 Na+ and 3 HCO3− ions approach the basolateral membrane from the bulk cytosol. NBCe1 directly extrudes the Na+ and 1 HCO3− . The second HCO3− dissociates to provide the CO2− 3 that NBCe1 will export:

HCO3− → CO23 − + H+

(NE 39-2) 

The third HCO3− , in a reaction catalyzed by CA II, produces an OH−,

CA II HCO3−   → CO2 + OH −

(NE 39-3) 

and this OH− neutralizes the newly formed H+:

H+ + OH− → H2O



(NE 39-4) 

As a result, NBCe1 would export 1 Na+, 1 HCO3− , and 1 CO2− 3 . Of the original 3 HCO3− ions that approached the basolateral membrane, 1 carbon atom, 2 hydrogen atoms, and 3 oxygen atoms would be left behind in the cytosol in the form of CO2 + H2O. According to the alternate view proposed by the Boron laboratory, the CO2 and H2O would exit across the basolateral membrane via another route. Also according to the alternate view, the role of the bound CA II would be to buffer the H+ ions that accumulate on the intracellular side of the membrane as − CO2− 3 forms from HCO3 . Preliminary data from the Boron laboratory indicate that the presence of CA II does not stimulate the electrical current carried by NBCe1, at least as expressed in Xenopus oocytes.

Extracellular CAs According to the classical view, the role of CAs that face the basolateral ECF would be to consume the HCO3− exported by NBCe1 according to the following reaction:

HCO3−

CA

CO2 + OH −

(NE 39-5) 

According to this view, in consuming the newly exported HCO3− , the CA would stimulate the NBCe1. According to the alternate hypothesis put forward by the Boron laboratory, the role of these extracellular CAs is just the opposite of that of the cytoplasmic CA II. Recall that this hypothesis proposes that NBCe1 directly exports 1 Na+, 1 HCO3− , and 1 CO2− 3 , and that 1 CO2 and 1 H2O exit by a parallel route. The extracellular CA would assist in the reassembly of 1 CO2, 1 H2O, − and 1 CO2− 3 to form 2 HCO3 ions, which would then diffuse away from the membrane into the bulk ECF. Indeed, preliminary data show that expressing CA IV on the surface of a Xenopus oocyte greatly reduces the magnitude of the alkalinization produced as NBCe1 exports “HCO3− ” from the cell. Moreover, blocking the CA IV with acetazolamide increases the magnitude of the alkalinization by more than twofold. Finally, preliminary data show that blockade of the CA IV has virtually no effect on the current carried by NBCe1. Thus, it may be that the role of the extracellular CA is not to stimulate NBCe1, but to minimize the size of pH changes on the extracellular surface of the cell.

Chapter 39  •  Transport of Acids and Bases

Inhibition of CA  The administration of drugs that block CAs, such as acetazolamide, strongly inhibits HCO3− reabsorption along the nephron, leading to the excretion of an alkaline urine. Because acetazolamide reduces the reabsorption of Na+, HCO3− , and water, this drug is also a diuretic (i.e., it promotes urine output).  N39-7  How­ ever, a small amount of H+ secretion and HCO3− reabsorption remains despite the complete inhibition of CA. This remaining transport is related in part to the slow uncatalyzed hydration-dehydration reactions and in part to a buildup of luminal H2CO3, which may diffuse into the cell across the apical membrane (mimicking the uptake of CO2 and H2O).

HCO3- efflux across the basolateral membrane takes place by electrogenic Na/HCO3 cotransport and Cl-HCO3 exchange The regulation of the intracellular pH of acid-secreting tubule cells requires that H+ secretion across the apical membrane be tightly linked to, and matched by, the extrusion of HCO3− across the basolateral membrane. Two mechanisms are responsible for HCO3− transport from the cell into the peritubular fluid: electrogenic Na/HCO3 cotransport and Cl-HCO3 exchange. Electrogenic Na/HCO3 Cotransport  In proximal-tubule cells, the electrogenic Na/HCO3 cotransporter NBCe1 (see p. 122) is responsible for much of the HCO3− transport across the basolateral membrane. NBCe1 (SLC4A4) is expressed at highest levels in the S1 portion of the proximal tubule (see Fig. 39-4A) and gradually becomes less abundant in the more distal proximal-tubule segments (see Fig. 39-4B). NBCe1 is a 1035–amino-acid protein with a molecular weight of ~130 kDa. 4,4′-Diisothiocyanostilbene-2,2′disulfonate (DIDS), an inhibitor of most HCO3− transporters, also inhibits NBCe1. Because, in proximal-tubule cells, this transporter usually transports three HCO3− ions for each Na+ ion, the electrochemical driving forces cause it to carry these ions from cell to blood. Renal NBCe1 carries two net negative charges and is thus electrogenic. Human mutations that reduce either NBCe1 activity or NBCe1 targeting to the basolateral membrane cause a severe metabolic acidosis—proximal renal tubular acidosis N39-8 (pRTA).  Chronic metabolic and respiratory acidosis, hypokalemia, and hyperfiltration all increase NBCe1 activity. As would be expected, several factors cause parallel changes in the activities of the apical NHE3 and basolateral Na/HCO3 cotransporter, minimizing changes in cell pH and [Na+]. Thus, angiotensin II (ANG II) and protein kinase C (PKC) stimulate both transporters, whereas parathyroid hormone and PKA markedly inhibit both. Cl-HCO3 Exchange  In the S3 segment of the proximal tubule, as well as in the TAL and collecting tubules and ducts, Cl-HCO3 exchangers participate in transepithelial acid-base transport. The AE1 anion exchanger (see pp. 124–125) is found in the basolateral membranes of α-intercalated cells of the CNT, the ICT, and the CCT (see Fig. 39-4D). Basolateral AE2 is present in the TAL (see Fig. 39-4C) and the DCT.

829

NH+4 is synthesized by proximal tubules, partly reabsorbed in the loop of Henle, and secreted passively into papillary collecting ducts As we saw in our discussion of the segmental handling of NH +4 (see pp. 826–827 and Fig. 39-3B), the proximal tubule is the main site of renal NH +4 synthesis, although almost all other tubule segments have the capacity to form NH +4 . The proximal tubule forms NH +4 largely from glutamine (Fig. 39-5A), which enters tubule cells both from luminal and peritubular fluid via Na+-coupled cotransporters. Inside the mitochondria, glutaminase splits glutamine into NH +4 and glutamate, and then glutamate dehydrogenase splits the glutamate into α-KG and a second NH +4 . Ammonium is a weak acid that can dissociate to form H+ and NH3. Because the pK of the NH3 /NH +4 equilibrium is ~9.2, the NH3 /NH +4 ratio is 1 : 100 at a pH of 7.2. Whereas the cationic NH +4 does not rapidly cross most cell membranes, the neutral NH3 readily diffuses through most, but not all, cell membranes via gas channels.  N39-3  When NH3 diffuses from a relatively alkaline proximal-tubule or collecting-duct cell into the more acidic lumen, the NH3 becomes “trapped” in the lumen after buffering the newly secreted H+ to form the relatively impermeant NH +4 (see Fig. 39-5A). Not only does NH3 diffuse across the apical membrane, but the apical NHE3 directly secretes some NH +4 into the proximal tubule lumen (with NH +4 taking the place of H+). A second consequence of NH +4 synthesis is that the byproduct α-KG participates in gluconeogenesis, which indirectly generates HCO3− ions. As shown in Figure 39-5A, the metabolism of two glutamines generates four NH3 and two α-KG. Gluconeogenesis of these two α-KG, along with four H+, forms one glucose and four HCO3− ions. Accordingly, for each NH +4 secreted into the tubule lumen, the cell secretes one new HCO3− into the peritubular fluid. In juxtamedullary nephrons, which have long loops of Henle, the tDLH may both reabsorb and secrete NH3, with the secretion dominating. Tubule fluid may become alkaline along the tDLH, titrating NH +4 to NH3 and promoting NH3 reabsorption. On the other hand, reabsorption of NH +4 by the TAL (see following paragraph) creates a gradient favoring NH3 diffusion from the interstitium into the lumen of the tDLH. Modeling of these processes predicts net secretion of NH3 into the tDLH in the outer medulla (see Fig. 39-5D). In the thin ascending limb, NH +4 reabsorption may occur by diffusion of NH +4 into the interstitium. In contrast to the earlier segments, the TAL reabsorbs NH +4 (see Fig. 39-5C). Thus, much of the NH +4 secreted by the proximal tubule and tDLH does not reach the DCT. Because the apical membrane of the TAL is unusual in having a very low NH3 permeability, the TAL takes up NH +4 across the apical membrane by using two transport mechanisms, the Na/K/Cl cotransporter and the K+ channels. Indeed, inhibiting the Na/K/Cl cotransporter blocks a significant fraction of NH +4 reabsorption, which suggests that NH +4 can replace K+ on the cotransporter. Ammonium leaves the cell across the basolateral membrane—probably as NH3, via a gas channel, and as NH +4 carried by the NHE—which leads to accumulation of NH +4 in the renal medulla. The NH +4 that has accumulated in the interstitium of the medulla has three possible fates (see Fig. 39-5D). First,

Chapter 39  •  Transport of Acids and Bases

829.e1

N39-7  Diuretic Action of the CA Inhibitor Acetazolamide Contributed by Gerhard Giebisch and Erich Windhager As described in Box 40-3 and in Table 40-3, the drug acetazolamide (a potent inhibitor of CAs) produces diuresis by inhibiting the component of proximal-tubule Na+ reabsorption that is coupled to HCO3− reabsorption. For further discussion of CAs, consult  N18-3.

N39-8  The Electrogenic Na/HCO3 Cotransporter NBCe1 Contributed by Walter Boron NBCe1 is a member of the SLC4 family of solute transporters. It is believed that all of the family members have the same topology: (1) a large cytoplasmic N terminus (Nt) that comprises about 40% of the protein, (2) a large transmembrane domain (TMD) that includes 10 to 14 transmembrane segments (TMs) and comprises ~50% of the protein, and (3) a short cytoplasmic C terminus (Ct) that comprises ~10% of the protein. The gene SLC4A4 encodes three known variants of NBCe1, which differ from one another at their extreme Nt and Ct. The proximal tubule expresses the variant NBCe1-A, which has a very high functional activity. The other variants—the more ubiquitous NBCe1-B and the “brain” form NBCe1-C—have a different Nt. This difference endows these transporters with a low functional activity—due to either reduced trafficking to the membrane or reduced intrinsic activity. However, a soluble protein called IRBIT appear to reverse this inhibition. The NBCe1-A variant in the proximal tubule, however, is the fast variant. In the proximal tubule, NBCe1-A appears to operate with a stoichiometry of 1 Na+ for 3 HCO3− ions. Thus, each transport event moves two negative charges out of the cell and thereby makes the basolateral membrane potential (Vbl) more positive. The reversal potential for NBCe1-A is very close to Vbl. Accord­ ingly, cell depolarization inhibits Na/HCO3 efflux or can even reverse the direction of transport and cause basolateral Na/HCO3 uptake. At least 10 naturally occurring human mutations of NBCe1 are known. From a molecular perspective, these mutations cause

poor function or poor targeting to the appropriate plasma membrane (i.e., the basolateral membrane in the case of NBCe1-A in the proximal-tubule cell). From a clinical perspective, these naturally occurring mutations have a devastating effect on the patient, causing a severe pRTA and other problems that may— depending on the mutation—lead to short stature, mental retardation, and ocular deficits.

REFERENCES Boron WF, Boulpaep EL: Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3− transport. J Gen Physiol 81:53–94, 1983. Parker MD, Boron WF: The divergence, actions, roles, and relatives of sodium-coupled bicarbonate transporters. Physiol Rev 93:803–959, 2013. Parker MD, Boron WF: Sodium-coupled bicarbonate transporters. In Alpern RJ, Hebert SC (ed): The Kidney. Burlington, MA, Academic Press, pp 1481–1497, 2007. Romero MF, Fulton CM, Boron WF: The SLC4 family of HCO3− transporters. Pflugers Arch 477:495–509, 2004. Romero MF, Hediger MA, Boulpaep EL, Boron WF: Expression cloning of the renal electrogenic Na/HCO3 cotransporter. Nature 387:409–413, 1997. Toye AM, Parker MD, Daly CM, et al: The Human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia. Am J Physiol Cell Physiol 291:788–801, 2006.

830 A

SECTION VI  •  The Urinary System

B

EARLY PROXIMAL TUBULE

Tubule lumen

Glutamine

Mitochondrion

Na+

NH3

2 Glutamine

NH3

CA II

4 NH3

4 NH3

4

NH4+

2

4 OH–

NH4+ 2 Glutamate

4

HCO3–

HCO3–

NBC

H+

H+

Glutamate dehydrogenase

2 NH4+

AQP1

H

+

4 CO2

–

3

+

Na

NH+4

H2O GLUT

+ 4 H

Glucose

2 α-ketoglutarate2 – +

+

H

H

+

Na

4

Interstitial space

Tubule lumen

SNAT3 Na+ (SLC38A3)

SNAT

Glutamine

DESCENDING LIMB OF HENLE (tDLH)

3 Na+

Oxaloacetate2 –

NH4+

2 K+

PEPCK 2 PEP3

–

Interstitial space

C

D

THICK ASCENDING LIMB (TAL)

NH4+

NH4+

3

FATES OF MEDULLARY NH3/NH4+

NH3

+

Na

2 K+

+

H

2

NH3

Cl– +

H2O CO2

Na

NH4+

NH3 +

H

HCO3–

Na+ HCO3–

E

NH4+

MEDULLARY COLLECTING DUCT Interstitial space

Tubule lumen RhCG

NH3

NH4+

H+

2

1

3 To liver

NH3

NH3

1

2

1

2

RhBG NH3

NH3 H+

1

3

Na+

NH4+

RhCG 2 NH4+

40 mmol/day NH3

NH4+

Chapter 39  •  Transport of Acids and Bases

831

Figure 39-5  Ammonium handling. B, In juxtamedullary nephrons, the secretion of NH+4 into the tubule lumen

of the tDLH occurs mainly in the outer portion of the medulla. In D, the three numbered boxes indicate the three fates of the NH+4 reabsorbed by the TAL. GLUT, glucose transporter; NBC, Na/HCO3 cotransporter; PEP, phosphoenolpyruvate.

Metabolism of amino acids ~940 mmol/day of amino groups Amino acids

Glutamate– 20 mmol/ day

NH4+ α keto acid Aspartate 20 mmol/ day

NH2

450 mmol/ day

O

450 mmol/day NH3 + HCO3–

+

H

900 mmol/day of nitrogen Glutamate– + NH4+

Fumarate

C H2N NH2 Urea

Glutamine + H2O

40 mmol/day of nitrogen LIVER KIDNEY

Glutamine 20 mmol/ day

Urea 450 mmol/ day

Glutamine Renal artery 2 NH4+ Renal vein 2

HCO–3 Ureter

α-Ketoglutarate

Urinary excretion of: + NH4 = 40 mmol/day Urea = 450 mmol/day Figure 39-6  Cooperation between the liver and kidney in excreting nitrogen derived from amino-acid breakdown. In this example, we assume a release of 940 mmol/day of amino groups, resulting in the urinary excretion of 450 mmol/day of urea (900 mmol/day of amino nitrogen) and 40 mmol/day of NH+4 . The values in the boxes are approximations.

some dissociates into H+ and NH3, which then enters the lumen of the late proximal tubule and the early tDLH (see Fig. 39-5D). This NH3 probably diffuses across the aquaporin 1 (AQP1) water channel (see Chapter 5) that is present in both the basolateral and apical membranes of these tubules. Luminal H+ then traps the NH3 as NH +4 (see Fig. 39-5B).

Thus, NH +4 recycles between the proximal tubule/tDLH and the TAL. Second, some of the interstitial NH +4 dissociates into H+ and NH3, which then enters the lumen of the medullary collecting ducts (see Fig. 39-5D). NH3 diffuses into the cell across the basolateral membrane via the gas channels RhBG and RhCG, and then enters the lumen via RhCG, where the NH3 combines with secreted H+ to form NH +4 (see Fig. 39-5E). In addition, the Na-K pump may carry NH +4 (in place of K+) into cells of the medullary collecting ducts. To the extent that NH +4 moves directly from the TAL to the medullary collecting duct, it engages in a bypass of the cortical portions of the distal nephron. This bypass prevents cortical portions of the distal nephron from losing NH3 by diffusion from the lumen into the cortical interstitium, and thus minimizes the entry of the toxic NH3 into the circulation. Third, a small fraction of medullary NH +4 enters the vasa recta. This NH +4 washout returns the nitrogen to the systemic circulation for eventual detoxification by the liver. In the steady state, the buildup of NH +4 in the medulla leads to a sharp increase in [NH +4 ] along the corticomedullary axis. Because the liver synthesizes glutamine (see p. 965), the main starting material for NH +4 production in the kidney, hepatorenal interactions are important in the overall process of NH +4 excretion (Fig. 39-6). The liver disposes of ~1000 mmol/day of amino groups during the catabolism of amino acids. Some of these amino groups become NH +4 via deamination reactions, and some end up as amino groups on either glutamate or aspartate via transamination reactions. Of the ~1000 mmol/day of catabolized amino groups, the liver detoxifies ~95% by producing urea (see p. 965), which the kidneys excrete (see p. 770). One −NH2 in urea comes from an NH +4 that had dissociated to form NH3 and H+, the other −NH2 comes from aspartate, and the C=O comes from HCO3− (see Fig. 39-6). The net result is the generation of urea and—considering that the generated H+ consumes another HCO3− —the consumption of two HCO3− . The liver detoxifies the remaining ~5% of catabolized amino groups by converting NH +4 and glutamate to glutamine (see Fig. 39-6). This reaction does not generate acidbase equivalents. The proximal-tubule cells take up this hepatic glutamine and use it as the source of the NH +4 that they secrete into the tubule lumen as they generate one new HCO3− (see Fig. 39-5A). Thus, the two hepatorenal mechanisms for disposing of catabolized amino groups have opposite effects on HCO3− . For each catabolized amino group excreted as urea, the liver consumes the equivalent of one HCO3−. For each catabolized amino group excreted as NH +4 via the glutamine pathway, the proximal tubule produces one new HCO3− (see Fig. 39-6). To the extent that the kidney excretes NH +4 , the liver consumes less HCO3− as it synthesizes urea (Box 39-1).  N39-9

Chapter 39  •  Transport of Acids and Bases

N39-9  Net Renal Ammonium Excretion Contributed by Peter Aronson and Gerhard Giebisch As noted in the text, one component of the “new HCO3− ” created by the proximal tubule parallels the generation of NH+4 in the proximal-tubule lumen. However, this generation of new HCO3− is reversed to the extent that the NH+4 reabsorbed by the TAL into the medullary interstitium is then picked up by the vasa recta and carried back to the liver for urea production (see Fig. 39-6). Thus, the resecretion of NH+4 from the medullary interstitium into the collecting-duct lumen (for excretion into the urine) is crucial to optimize the efficiency of new-HCO3− generation by the kidney and thus to balance net acid production.

831.e1

832

SECTION VI  •  The Urinary System

BOX 39-1  Renal Tubular Acidosis Contributed by Mark D. Parker

R

enal tubular acidosis (RTA) is a broad label applied to a group of disorders that compromise renal acid-base handling. RTA is characterized by a reduced ability to eliminate H+ in the urine or by HCO3− wasting, both of which can result in a lowered plasma pH (i.e., metabolic acidosis) and, in children, severe impairment of physical and intellectual development. RTA can follow a more generalized disruption of renal function (e.g., as a side effect of medication, autoimmune disease, multiple myeloma) or can result from mutations in genes that encode renal acid-base–handling proteins. RTA is classified into four types, each of which has a different set of causes and clinical manifestations. In addition, we can define a fifth type of RTA that is associated with end-stage renal disease.

Type 1 or Distal RTA

Distal RTA (dRTA) results from defective H+ excretion by distal segments of the nephron. Consequently, dRTA patients cannot appropriately acidify their urine and may exhibit a metabolic acidosis. Genetic causes of dRTA include mutations in the Cl-HCO3 exchanger AE1 and in subunits of the H pump, both of which are key components of the H+-secretory machinery in α-intercalated cells (see Fig. 39-4D). In patients with incomplete dRTA, blood pH is unaffected because compensatory mechanisms (e.g., proximal-tubule function) remain intact; in these individuals, metabolic acidosis occurs only following an acid load. Manifestations of dRTA can include hypokalemia, kidney stones, hemolytic anemia (due to loss of AE1 function in red cells), and hearing loss (due to loss of H pump function in the cochlea).

Type 2 or Proximal RTA Proximal RTA (pRTA) results from the inability of proximal-tubule cells to reabsorb filtered HCO3− or to generate new HCO3− . Conse­ quently, pRTA patients exhibit a severe metabolic acidosis and a wasting of HCO3− into the urine. Genetic defects in the Na/HCO3 cotransporter NBCe1 cause pRTA because of the key role of

REGULATION OF RENAL ACID SECRETION A variety of physiological and pathophysiological stimuli can modulate renal H+ secretion as well as NH3 synthesis. Most of these factors produce coordinated changes in apical and basolateral acid-base transport, as well as in NH3 production.

Respiratory acidosis stimulates renal H+ secretion The four fundamental pH disturbances are respiratory acidosis and alkalosis, and metabolic acidosis and alkalosis (see Fig. 28-11A). In each case, the initial and almost instantaneous line of defense is the action of buffers—both in the extracellular and intracellular compartments—to minimize the magnitude of the pH changes (see pp. 628–629). However, restoring the pH to a value as close to “normal” as possible requires slower compensatory responses from the lungs or kidneys. In respiratory acidosis, in which the primary disturbance is an increase in arterial PCO2 , the compensatory response

that protein in mediating HCO3− movement into the bloodstream (see Fig. 39-4A). Other causes include Fanconi syndrome (e.g., due to multiple myeloma, lead poisoning) and acetazolamide toxicity. Manifestations of pRTA can include hypokalemia and—in children—developmental defects, including ocular problems and poor dentition (considered in part to be due to loss of NBCe1 function in the eye and enamel organ).

Type 3 RTA Type 3 RTA is a rare combination of type 1 and type 2 RTAs and is associated with defects in CA II, a shared component of the acid-base–handling mechanisms in the distal and proximal tubules. Clinical manifestations include osteopetrosis due to loss of CA II function in osteoclasts (see p. 1056).

Type 4 or Hyperkalemic RTA (Hypoaldosteronism) Hyperkalemic RTA is a mild form of acidosis caused by aldosterone insufficiency or renal insensitivity to aldosterone. Insufficient stimulation of mineralocorticoid receptors in α-intercalated cells reduces H+ directly (see p. 835); insufficient stimulation of these receptors in principal cells reduces K+ secretion, leading to hyperkalemia, which causes metabolic acidosis by several mechanisms (see p. 835).

Uremic Acidosis In end-stage renal disease, a loss of functional renal mass compromises total ammoniagenesis.  N39-1

Treatment Treatments for RTA vary depending on the clinical signs in each case but generally focus on correcting the metabolic acidosis by administration of HCO3− or citrate salts (oral base therapy). Additional therapies include administration of diuretics (e.g., hydrochlorothiazide) to stimulate renal H+ secretion.

is an increase in renal H+ secretion, which translates to increased production of new HCO3− via NH +4 excretion. The opposite occurs in respiratory alkalosis. These changes in H+ secretion tend to correct the distorted [HCO3− ]/ [CO2] ratios that occur in primary respiratory acid-base derangements. Respiratory acidosis stimulates H+ secretion in at least three ways. First, an acute elevated PCO2 directly stimulates proximal-tubule cells to secrete H+, as shown by applying solutions in which it is possible to change PCO2 without altering basolateral pH or [HCO3−].  N39-10  Thus, proximaltubule cells directly sense basolateral CO2. In part, the mechanism is the exocytotic insertion of H pumps into the apical membranes of proximal-tubule cells. Second, acute respiratory acidosis also causes exocytotic insertion of H pumps into the apical membranes of intercalated cells in distal nephron segments. Third, chronic respiratory acidosis leads to adaptive responses that upregulate acid-base transporters. For example, respiratory acidosis increases the activities of apical NHE3 and basolateral NBCe1 in proximal tubule. These adaptive changes allow the kidney to

Chapter 39  •  Transport of Acids and Bases

832.e1

N39-10  Use of Out-of-Equilibrium Solutions to Probe the Chemosensitivity of the Proximal Tubule Contributed by Walter Boron As described in  N28-4, the laboratory of Walter Boron developed a rapid-mixing technique that makes it possible to generate out-of-equilibrium (OOE) CO2/HCO3− solutions with virtually any combination of [CO2], [HCO3− ], and pH—as long as the desired pH is not more than a few pH units from neutrality. Recently, the laboratory has applied the OOE CO2/HCO3− solutions to learn more about how the proximal tubule senses acute acid-base disturbances and translates that information to alter the rate at which the tubule reabsorbs HCO3− (i.e., moves HCO3− from the lumen to the basolateral side of the tubule). The approach was to isolate a single proximal tubule and perfuse its lumen with a solution of 5% CO2/22 mM HCO3− /pH 7.4 as well as 3 H-methoxyinulin as a volume marker. By collecting the fluid after it had flowed down the lumen and then analyzing this fluid for [HCO3− ] and [3H-methoxyinulin], the investigators were able to compute the rate of volume reabsorption (JV—that is, the rate at which the tubule moves water from the lumen to the basolateral surface of the tubule, measured in nanoliters per minute per millimeter of tubule length) and the rate of HCO3− reabsorption (JHCO3 —measured in picomoles per minute per millimeter of tubule length). The investigators superfused the basolateral (bl) surface of the tubule with a rapidly flowing solution that was either the “standard” equilibrated 5% CO2/22 mM HCO3− /pH 7.4 solution or an OOE solution in which they varied—one at a time—[CO2]bl, [HCO3− ]bl, or pHbl. Thus, they were able to observe the effects of altering basolateral acid-base composition on JV and JHCO3 . What they found was rather striking. When the investigators raised [CO2]bl from 0 to 4.8 mM—at a fixed [HCO3− ]bl of 22 mM and a fixed pHbl of 7.40—they found that JHCO3 increased in a graded fashion. This result is what one might expect from what we learned about a metabolic compensation to a respiratory acidosis (see p. 641). That is, the kidney ought to respond to a rise in [CO2]bl—the “respiratory” part of a respiratory acidosis— by reabsorbing more HCO3− and thereby tending to restore blood pH to a more alkaline value. However, the investigators were quite surprised to find that increases in JHCO3 were not accompanied by the expected increases in JV (i.e., the extra NaHCO3 reabsorbed by the proximal tubule should have been accompanied by osmotically obligated water, which should have raised JV appreciably). When the investigators raised [HCO3− ]bl from 0 to 44 mM— at a fixed [CO2]bl of 1.2 mM and a fixed pHbl of 7.40—they found

that JHCO3 decreased in a graded fashion. This result is what one might expect for the kidney’s response to a metabolic alkalosis caused by an abnormality outside of the kidney. That is, the kidney ought to respond to a rise in [HCO3− ]bl —the “metabolic” part of a metabolic alkalosis—by reabsorbing less HCO3− and thereby tending to restore blood pH to a more acidic value. However, the investigators were quite surprised to find that decreases in JHCO3 were not accompanied by the expected decreases in JV (i.e., because the tubule reabsorbed less NaHCO3, it should also have reabsorbed less osmotically obligated water, so that JV should have fallen appreciably). Finally, when the investigators raised pHbl from 6.8 to 8.0 mM—at a fixed [CO2]bl of 1.2 mM and a fixed [HCO3− ]bl of 22 mM—they found that JHCO3 did not change! One might have expected that a basolateral alkalosis (the “alkalosis” part of a respiratory or metabolic alkalosis) would have caused the tubule to reabsorb less HCO3− and thereby tend to restore blood pH to a more acidic value. In these experiments, the intracellular pH of the tubule cells changed appreciably, but neither change in pH, intracellular or basolateral, triggered a change in JHCO3 or JV. These experiments led the investigators to conclude that the proximal tubule cannot sense pH per se. Instead, they propose that the proximal-tubule cell has sensors for both basolateral CO2 and basolateral HCO3− . In other words, the proximal tubule seems to regulate blood pH by sensing the body’s two most important buffers. When activated, the CO2 sensors would trigger an increase in NaHCO3 reabsorption but a compensating decrease in the reabsorption of other solutes. When activated, the HCO3− sensors would trigger a decrease in NaHCO3 reabsorption but a compensating increase in the reabsorption of other solutes. The compensating effects on the other solutes would serve to stabilize blood pressure.

REFERENCES Zhao J, Zhou Y, Boron WF: Effect of isolated removal of either basolateral HCO3− or basolateral CO2 on HCO3− reabsorption by rabbit S2 proximal tubule. Am J Physiol Renal Physiol 285: F359–F369, 2003. Zhou Y, Zhao J, Bouyer P, Boron WF: Evidence from renal proximal tubules that HCO3− and solute reabsorption are acutely regulated not by pH but by basolateral HCO3− and CO2. Proc Natl Acad Sci U S A 102(10):3875–3880, 2005. Epub February 22, 2005.

Chapter 39  •  Transport of Acids and Bases

↓ pHo

833

0.10

↓ pHi

Protein kinase C

↑ NHE3

Tyrosine kinase pathways

↑ NBCe1

↑ Na/Citrate cotransporter

Immediate early genes

↑ Ammoniagenic enzymes

Chronic metabolic acidosis

Urinary excretion + of NH4 plus NH3 0.05 (mmol/min) Normal

Figure 39-7  Effects of chronic acidosis on proximal-tubule function. Enhanced Na citrate reabsorption is a defense against acidosis by conversion of citrate to HCO3− . The price paid is enhanced stone formation because luminal citrate reduces stone formation by complexing with Ca2+. Indeed, acidotic patients tend to get calcium-containing kidney stones.

0 5.0

6.0

7.0

8.0

Urinary pH Figure 39-8  Effect of chronic metabolic acidosis on total NH+4 excretion

pro­duce a metabolic compensation to the respiratory acidosis (see p. 641).

Metabolic acidosis stimulates both proximal H+ secretion and NH3 production The first compensatory response to metabolic acidosis is increased alveolar ventilation, which blows off CO2 (see p. 710) and thus corrects the distorted [HCO3− ]/[CO2] ratio in a primary metabolic acidosis. The kidneys can also participate in the compensatory response—assuming, of course, that the acidosis is not the consequence of renal disease. Proximal-tubule cells can directly sense an acute fall in basolateral [HCO3− ], which results in a stimulation of proximal H+ secretion.  N39-10  In intercalated cells in the distal nephron, metabolic acidosis stimulates apical membrane H pump insertion and activity. The mechanism may be protonsensitive G protein–coupled receptors on the basolateral membrane of intercalated cells, and an HCO3−-sensitive soluble adenylyl cyclase (sAC) in the cytosol. In chronic metabolic acidosis, the adaptive responses of the proximal tubule are probably similar to those outlined above for chronic respiratory acidosis. These include upregulation of apical NHE3 and electrogenic H pumps, as well as basolateral NBCe1 (Fig. 39-7), perhaps reflecting increases in the number of transporters on the surface membranes. The parallel activation of apical and basolateral transporters may minimize changes in pHi, while increasing transepithelial HCO3− reabsorption. This upregulation appears to involve intracellular protein kinases, including the Src family of receptor-associated tyrosine kinases (see p. 70). Endothelin appears to be essential for the upregulation of NHE3 in chronic metabolic acidosis. In addition to increased H+ secretion, the other ingredi­ ent needed to produce new HCO3− is enhanced NH3 production. Together, the two increase NH +4 excretion. Indeed, the excretion of NH +4 into the urine increases markedly as a result of the adaptive response to chronic metabolic acidosis

into final urine. (Data from Pitts RF: Renal excretion of acid. Fed Proc 7:418–426, 1948.)

(Fig. 39-8). Thus, the ability to increase NH3 synthesis is an important element in the kidney’s defense against acidotic challenges. Indeed, as chronic metabolic acidosis develops, the kidneys progressively excrete a larger fraction of urinary H+ as NH +4 . As a consequence, the excretion of titratable acid becomes a progressively smaller fraction of total acid excretion. The adaptive stimulation of NH3 synthesis, which occurs in response to a fall in pHi, involves a stimulation of both glutaminase and phosphoenolpyruvate carboxykinase (PEPCK). The stimulation of mitochondrial glutaminase increases the conversion of glutamine to NH +4 and glutamate (see Fig. 39-5A). The stimulation of PEPCK enhances gluconeogenesis and thus the conversion of α-KG (the product of glutamate deamination) to glucose.

Metabolic alkalosis reduces proximal H+ secretion and, in the CCT, may even provoke HCO3- secretion Figure 39-9A illustrates the response of the proximal tubule to metabolic alkalosis. As shown in the upper curve, when the peritubular capillaries have a physiological [HCO3−], increasing the luminal [HCO3− ] causes H+ secretion to increase steeply up to a luminal [HCO3− ] of ~45 mM. The reason is that the incremental luminal HCO3− is an additional buffer that minimizes the luminal acidification in the vicinity of the apical H+ transporters. As shown in the lower curve in Figure 39-9A, when [HCO3− ] in the peritubular blood is higher than normal— that is, during metabolic alkalosis—H+ secretion is lower for any luminal [HCO3− ]. The likely explanation is that the proximal-tubule cell directly senses the increase in plasma [HCO3− ], depressing the rates at which NHE3 moves H+ from cell to lumen and NBCe1 moves HCO3− from cell to blood.

834

A

SECTION VI  •  The Urinary System

EFFECT OF BASOLATERAL ALKALOSIS ON H+ SECRETION BY PROXIMAL TUBULES

200

A rise in GFR increases HCO3- delivery to the tubules, enhancing HCO3- reabsorption (glomerulotubular balance for HCO3- )

Normal

+

H secretion pmole mm tubule × min

100 Basolateral metabolic alkalosis 0

B

0

10

20 30 40 50 60 Mean luminal [HCO–3] (millimoles)

70

80

CORTICAL COLLECTING TUBULE (CCT): β INTERCALATED CELL

CCT

Lumen

– NDCBE (SLC4A8)

CO2

H2O

CA

Cl–

H+

OH–

+

Na

2 HCO3– Cl–

HCO3–

Cl– Pendrin (SLC26A4)

Figure 39-9  Effect of chronic metabolic alkalosis on renal acid-base transport. (Data from Alpern RJ, Cogan MG, Rector FC: Effects of extracellular fluid volume and plasma bicarbonate concentration on proximal acidification in the rat. J Clin Invest 71:736–746, 1983.)

So far, we have discussed the effect of metabolic alkalosis on H+ secretion by the proximal tubule. In the ICT and CCT, metabolic alkalosis can cause the tubule to switch from secreting H+ to secreting HCO3− into the lumen. The α-intercalated cells in the ICT and CCT secrete H+ by using an apical H pump and a basolateral Cl-HCO3 exchanger, which is AE1 (SLC4A1; see Fig. 39-4D). Metabolic alkalosis, over a period of days, shifts the intercalated-cell population, increasing the proportion of β-intercalated cells (see Fig. 39-9B)  N39-5  at the expense of α cells. Because β cells have the opposite apical-versus-basolateral distribution of H pumps and Cl-HCO3 exchangers, they secrete HCO3− into the lumen and tend to correct the metabolic alkalosis. The apical Cl-HCO3 exchanger in β cells is pendrin (SLC26A4; see Table 5-4). In contrast to chronic alkalosis, chronic acidosis alters the distribution of intercalated cell types in the distal nephron in favor of acid-secreting α cells (see Fig. 39-4D) over the base-secreting β-intercalated cells.

Increasing either luminal flow or luminal [HCO3− ] significantly enhances HCO3− reabsorption,  N39-11  probably by raising effective [HCO3−] (and thus pH) in the micro­ environment of H+ transporters in the brush-border microvilli. Because a high luminal pH stimulates NHE3 and the H pumps located in the microvilli of the proximal tubule, increased flow translates to enhanced H+ secretion. This flow dependence, an example of glomerulotubular (GT) balance (see p. 763), is important because it minimizes HCO3− loss, and thus the development of a metabolic acidosis, when GFR increases. Conversely, this GT balance of HCO3− reabsorption also prevents metabolic alkalosis when GFR decreases. The flow dependence of HCO3− reabsorption also accounts for the stimulation of H+ transport that occurs after uninephrectomy (i.e., surgical removal of one kidney), when GFR in the remnant kidney rises in response to the loss of renal tissue.

Extracellular volume contraction—via ANG II, aldosterone, and sympathetic activity—stimulates renal H+ secretion A decrease in effective circulating volume stimulates Na+ reabsorption by four parallel pathways (see pp. 838–840), including activation of the renin-angiotensin-aldosterone axis (and thus an increase in ANG II levels) and stimulation of renal sympathetic nerves (and thus the release of norepinephrine). Both ANG II and norepinephrine stimulate Na-H exchange in the proximal tubule. Because the proximal tubule couples Na+ and H+ transport, volume contraction increases not only Na+ reabsorption but also H+ secretion. Similarly, ANG II stimulates acid secretion by α-intercalated cells in the distal nephron. Volume expansion has the opposite effect. On a longer time scale, volume depletion also increases aldosterone levels, thereby enhancing H+ secretion in cortical and medullary collecting ducts (see below). Thus, the regulation of effective circulating volume takes precedence over the regulation of plasma pH.  N39-12

Hypokalemia increases renal H+ secretion As discussed on page 803, acid-base disturbances can cause changes in K+ homeostasis. The opposite is also true. Because a side effect of K+ depletion is increased renal H+ secretion, K+ depletion is frequently associated with metabolic alkalosis. Several lines of evidence indicate that, in the proximal tubule, hypokalemia leads to a marked increase in apical Na-H exchange and basolateral Na/HCO3 cotransport. As in other cells, in tubule cells the pH falls during K+ depletion (see p. 645). The resulting chronic cell acidification may lead to adaptive responses that activate Na-H exchange and electrogenic Na/HCO3 cotransport, presumably by the same mechanisms that stimulate H+ secretion in chronic acidosis (see Fig. 39-7). In the proximal tubule, K+ depletion also markedly increases NH3 synthesis and NH +4 excretion, thus increasing urinary H+ excretion as NH +4 . Finally, K+ depletion stimulates apical K-H exchange in α-intercalated cells

Chapter 39  •  Transport of Acids and Bases

N39-11  Flow Dependence of HCO3- Reabsorption

834.e1

N39-12  Effect of Dietary Na+ Intake on Proximal-Tubule NHE3 Activity

Contributed by Gerhard Giebisch and Erich Windhager

Contributed by Gerhard Giebisch and Erich Windhager

In the text, we point out that raising either luminal [HCO3− ] or luminal flow increases HCO3− reabsorption. One likely mechanism is mentioned in the text: The higher the flow or the higher the bulk luminal [HCO3− ], the higher the pH and [HCO3− ] in the unstirred layer that surrounds the microvilli on the apical membrane. In addition, increasing the flow also increases the shear force that acts on the central cilium present on every proximal-tubule cell. It is believed that the more the cilium bends with flow, the greater the signal to increase the reabsorption of solutes (including NaHCO3) and water. This hypothesis would account for at least a portion of the glomerulotubular balance for both HCO3− reabsorption (see p. 834) and fractional Na+ reabsorption (see p. 763).

Decreased dietary Na+ intake causes a decrease in effective circulating volume (i.e., volume contraction), resulting in increased activity of the apical NHE3. This effect is evident even if one assesses the activity in brush-border membrane vesicles removed from the animal. Consumption of a high-Na+ diet has the opposite effect.

Chapter 39  •  Transport of Acids and Bases

of the ICT and CCT (see p. 799) and enhances H+ secretion as a side effect of K+ retention. Just as hypokalemia can maintain metabolic alkalosis, hyperkalemia is often associated with metabolic acidosis. A contributory factor may be reduced NH +4 excretion, perhaps because of lower synthesis in proximal-tubule cells, possibly due to a higher intracellular pH. In addition, with high luminal [K+] in the TAL, K+ competes with NH +4 for uptake by apical Na/K/Cl cotransporters and K+ channels, thereby reducing NH +4 reabsorption. As a result, the reduced NH +4 levels in the medullary interstitium provide less NH3 for diffusion into the medullary collecting duct. Finally, with high [K+] in the medullary interstitium, K+ competes with NH +4 for uptake by basolateral Na-K pumps in the medullary collecting duct. The net effects are reduced NH +4 excretion and acidosis.

Both glucocorticoids and mineralocorticoids stimulate acid secretion Chronic adrenal insufficiency (see p. 1019) leads to acid retention and, potentially, to life-threatening metabolic acidosis. Both glucocorticoids and mineralocorticoids stimulate H+ secretion, but at different sites along the nephron. Glucocorticoids (e.g., cortisol) enhance the activity of Na-H exchange in the proximal tubule and thus stimulate H+ secretion. In addition, they inhibit phosphate reabsorption, raising the luminal availability of buffer anions for titration by secreted H+. Mineralocorticoids (e.g., aldosterone) stimulate H+ secretion by three coordinated mechanisms—one direct and two indirect. First, mineralocorticoids directly stimulate H+ secretion in the collecting tubules and ducts by increas­ ing the activity of the apical electrogenic H pump and basolateral Cl-HCO3 exchanger (see Fig. 39-4D). Second, mineralocorticoids indirectly stimulate H+ secretion by enhancing Na+ reabsorption in the collecting ducts (see p. 766), which increases the lumen-negative voltage. This increased negativity may stimulate the apical electrogenic H pump in α-intercalated cells to secrete acid. Third, mineralocorticoids—particularly when administered for longer periods of time and accompanied by high Na+ intake—cause K+ depletion and indirectly increase H+ secretion (see pp. 834–835).

835

Diuretics can change H+ secretion, depending on how they affect transepithelial voltage, ECF volume, and plasma [K+] The effects of diuretics on renal H+ secretion  N39-13  vary substantially from one diuretic to another, depending on both the site and the mechanism of action. From the point of view of acid-base balance, diuretics fall broadly into two groups: those that promote the excretion of a relatively alkaline urine and those that have the opposite effect. To the first group belong CA inhibitors and K+-sparing diuretics. The CA inhibitors lead to excretion of an alkaline urine by inhibiting H+ secretion. Their greatest effect is in the proximal tubule, but they also inhibit H+ secretion by the TAL and intercalated cells in the distal nephron. K+-sparing diuretics—including amiloride, triamterene, and the spironolactones—also reduce acid excretion. Both amiloride and triamterene inhibit the apical epithelial Na+ channels (ENaCs; see pp. 758–759) in the collecting tubules and ducts, rendering the lumen more positive so that it is more difficult for the electrogenic H pump to secrete H+ ions into the lumen. Spironolactones decrease H+ secretion by interfering with the action of aldosterone. The second group of diuretics—those that tend to increase urinary acid excretion and often induce alkalosis—includes loop diuretics such as furosemide (which inhibits the apical Na/K/Cl cotransporter in the TAL) and thiazide diuretics such as chlorothiazide (which inhibits the apical Na/Cl cotransporter in the DCT). These diuretics act by three mechanisms. First, all cause some degree of volume contraction, and thus lead to increased levels of ANG II and aldosterone (see pp. 841–842), both of which enhance H+ secretion. Second, these diuretics enhance Na+ delivery to the collecting tubules and ducts, thereby increasing the electrogenic uptake of Na+, raising lumen-negative voltage, and enhancing H+ secretion. Third, this group of diuretics causes K+ wasting; as discussed on pages 834–835, K+ depletion enhances H+ secretion.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 39  •  Transport of Acids and Bases

N39-13  Effect of Diuretics on Renal H+ Excretion Contributed by Erich Windhager and Gerhard Giebisch Box 40-3, as well as Table 40-3, summarizes some of the effects of various classes of diuretics and lists the protein targets of these diuretics in the kidney.

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REFERENCES Books and Reviews Alper SL: Genetic diseases of acid-base transporters. Annu Rev Physiol 64:899–923, 2002. Bobulescu IA, Moe OW: Na+/H+ exchangers in renal regulation of acid-base balance. Semin Nephrol 26:334–344, 2006. Brown D, Wagner CA: Molecular mechanisms of acid-base sensing by the kidney. J Am Soc Nephrol 23:774–780, 2012. Fry AC, Karet FE: Inherited renal acidoses. Physiology (Bethesda) 22:202–211, 2007. Good DW: Ammonium transport by the thick ascending limb of Henle’s loop. Annu Rev Physiol 56:623–647, 1994. Igarashi T, Inatomi J, Sekine T, et al: Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities. Nat Genet 23:264–266, 1999. Karet FE: Mechanisms in hyperkalemic renal tubular acidosis. J Am Soc Nephrol 20:251–254, 2009. Laing CM, Toye AM, Capasso G, Unwin RJ: Renal tubular acidosis: Developments in our understanding of the molecular basis. Int J Biochem Cell Biol 37:1151–1161, 2005. Moe OW: Acute regulation of proximal tubule apical membrane Na/H exchanger NHE-3: Role of phosphorylation, protein, trafficking, and regulatory factors. J Am Soc Nephrol 10:2412–2425, 1999. Purkerson JM, Schwartz GJ: The role of carbonic anhydrases in renal physiology. Kidney Int 71:103–115, 2007. Rose BD, Post TW: Clinical Physiology of Acid-Base and Electrolyte Disorders, 5th ed. New York, McGraw-Hill, 2001. Seldin DW, Giebisch G (eds): The Kidney: Physiology and Pathophysiology, 3rd ed. Philadelphia, Lippincott Williams & Wilkins, 2000. Stone DK, Xie XS: Proton translocating ATPases: Issues in structure and function. Kidney Int 33:767–774, 1988. Wakabayashi S, Shigekawa M, Pouysségur J: Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77:51–74, 1997. Wall SM: Recent advances in our understanding of intercalated cells. Curr Opin Nephrol Hypertens 14:480–484, 2005. Journal Articles Aronson PS, Nee J, Suhm MA: Modifier role of internal H in activating the Na-H exchanger in renal microvillus membrane vesicles. Nature 299:161–163, 1982. Boron WF, Boulpaep EL: Intracellular pH regulation in the renal proximal tubule of the salamander: Basolateral HCO3− transport. J Gen Physiol 81:53–94, 1983. Bruce LJ, Cope DL, Jones GK, et al: Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene. J Clin Invest 100:1693–1707, 1997. Fry AC, Karet FE: Inherited renal acidoses. Physiology (Bethesda) 22:202–211, 2007. Geibel J, Giebisch G, Boron WF: Angiotensin II stimulates both Na-H exchange and Na/HCO3 cotransport in the rabbit proximal tubule. Proc Natl Acad Sci U S A 87:7917–7920, 1990. Igarashi T, Inatomi J, Sekine T, et al: Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities. Nat Genet 23:264–266, 1999.

Karet FE: Mechanisms in hyperkalemic renal tubular acidosis. J Am Soc Nephrol 20:251–254, 2009. Karet FE, Finberg KE, Nelson RD, et al: Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet 21:84–90, 1999. Laing CM, Toye AM, Capasso G, Unwin RJ: Renal tubular acidosis: Developments in our understanding of the molecular basis. Int J Biochem Cell Biol 37:1151–1161, 2005. McKinney TD, Burg MB: Bicarbonate transport by rabbit cortical collecting tubules: Effect of acid and alkali loads in vivo on transport in vitro. J Clin Invest 60:766–768, 1977. Petrovic S, Wang Z, Ma L, Soleimani M: Regulation of the apical Cl − /HCO3− exchanger pendrin in rat cortical collecting duct in metabolic acidosis. Am J Physiol Renal Physiol 284:F103–F112, 2003. Piermarini PM, Verlander JW, Royaux IE, Evans DH: Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am J Physiol Regul Integr Comp Physiol 283:R983– R992, 2002. Quentin F, Chambrey R, Trinh-Trang-Tan MM, et al: The Cl − /HCO3− exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance. Am J Physiol Renal Physiol 287:F1179–1188, 2004. Romero MF, Hediger MA, Boulpaep EL, Boron WF: Expression cloning of the renal electrogenic Na/HCO3 cotransporter. Nature 387:409–413, 1997. Royaux IE, Wall SM, Karniski LP, et al: Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci U S A 98:4221–4226, 2001. Schwartz GJ, Al-Awqati Q: Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules. J Clin Invest 75:1638–1644, 1985. Sly WS, Hewett-Emmett D, Whyte MP, et al: Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci U S A 80:2752– 2756, 1983. Smith AN, Skaug J, Choate KA, et al: Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nat Genet 26:71–75, 2000. Sun X, Yang LV, Tiegs BC, et al: Deletion of the pH sensor GPR4 decreases renal acid excretion. J Am Soc Nephrol 21:1745–1755, 2010. Verlander JW, Hassell KA, Royaux IE, et al: Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney. Role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003. Wall SM, Hassell KA, Royaux IE, et al: Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284:F229–F241, 2003. Wang T, Malnic G, Giebisch G, Chan YL: Renal bicarbonate reabsorption in the rat. IV. Bicarbonate transport mechanisms in the early and late distal tubule. J Clin Invest 91:2776–2784, 1993. Zhou Y, Zhao J, Bouyer P, Boron WF: Evidence from renal proximal tubules that HCO3− and solute reabsorption are acutely regulated not by pH but by basolateral HCO3− and CO2. Proc Natl Acad Sci U S A 102:3875–3880, 2005.

C H A P T E R 40  INTEGRATION OF SALT AND WATER BALANCE Gerhard Giebisch, Erich E. Windhager, and Peter S. Aronson

Two separate but closely interrelated control systems regulate the volume and osmolality of the extracellular fluid (ECF). It is important to regulate the ECF volume to maintain blood pressure, which is essential for adequate tissue perfusion and function. The body regulates ECF volume by adjusting the total-body content of NaCl. It is important to regulate the extracellular osmolality because hypotonic (see pp. 131–132) or hypertonic (see p. 131) osmolalities cause changes in cell volume that seriously compromise cell function, especially in the central nervous system (CNS). The body regulates extracellular osmolality by adjusting totalbody water content. These two homeostatic mechanisms— for ECF volume and osmolality—use different sensors, different hormonal transducers, and different effectors (Table 40-1). However, they have one thing in common: some of their effectors, although different, are located in the kidney. In the case of the ECF volume, the control system modulates the urinary excretion of Na+. In the case of osmolality, the control system modulates the urinary excretion of solutefree water or simply free water (see pp. 806–807). Sodium Balance  The maintenance of the ECF volume, or Na+ balance, depends on signals that reflect the adequacy of the circulation—the so-called effective circulating volume, discussed below. Low- and high-pressure baroreceptors send afferent signals to the brain (see pp. 536–537), which translates this “volume signal” into several responses that can affect ECF volume or blood pressure over either the short or the long term. The short-term effects (over a period of seconds to minutes) occur as the autonomic nervous system and humoral mechanisms modulate the activity of the heart and blood vessels to control blood pressure. The long-term effects (over a period of hours to days) consist of nervous, humoral, and hemodynamic mechanisms that modulate renal Na+ excretion (see pp. 763–769). In the first part of this chapter, we discuss the entire feedback loop, of which Na+ excretion is the effector. Why is the Na+ content of the body the main determinant of the ECF volume? Na+, with its associated anions, Cl− and HCO3− , is the main osmotic constituent of the ECF volume; when Na salts move, water must follow. Because the body generally maintains ECF osmolality within narrow limits (e.g., ~290 milliosmoles/kg, or 290 mOsm), it follows that whole-body Na+ content—which the kidneys control—must be the major determinant of the ECF volume. A simple example illustrates the point. If the kidney were to enhance

836

the excretion of Na+ and its accompanying anions by 145 milliequivalents (meq) each—the amount of solute normally present in 1 L of ECF—the kidneys would have to excrete an additional liter of urine to prevent a serious fall in osmolality. Alternatively, the addition of 145 mmol of “dry” NaCl to the ECF obligates the addition of 1 L of water to the ECF; this addition can be accomplished by ingestion of water or reduction of renal excretion of free water. Relatively small changes in Na+ excretion lead to marked alterations in the ECF volume. Thus, precise and sensitive control mechanisms are needed to safeguard and regulate the body’s content of Na+. Water Balance  The maintenance of osmolality, or water balance, depends on receptors in the hypothalamus that detect changes in the plasma osmolality. These receptors send signals to areas of the brain that (1) control thirst and thus regulate free-water intake and (2) control the production of arginine vasopressin (AVP)—also known as antidiuretic hormone (ADH)—and thus regulate free-water excretion by the kidneys. We discuss renal water excretion beginning on page 806. In the second part of this chapter, we discuss the entire feedback loop, of which water excretion is merely the end point. Why is the water content of the body the main determinant of osmolality? Total-body osmolality is defined as the ratio of total-body osmoles to total-body water (see p. 102). Although the ECF volume control system can regulate the amount of extracellular osmoles, it has little effect on totalbody osmoles. Total-body osmoles are largely a function of the intracellular milieu because the intracellular compartment is larger than the ECF and its solute composition is highly regulated. Total-body osmoles do not change substantially except during growth or during certain disease states, such as diabetes mellitus (in which excess glucose increases total-body osmolality). Only by controlling water independent of Na+ control can the body control osmolality.

CONTROL OF EXTRACELLULAR FLUID VOLUME In the steady state, Na+ intake via the gastrointestinal tract equals Na+ output from renal and extrarenal pathways The two principal solutes in the ECF are Na+ and Cl−. Sodium is one of the most abundant ions in the body, totaling

Chapter 40  •  Integration of Salt and Water Balance

837

TABLE 40-1  Comparison of the Systems Controlling ECF Volume and Osmolality REGULATION OF ECF VOLUME AND BLOOD PRESSURE

REGULATION OF OSMOLALITY

What is sensed?

Effective circulating volume

Plasma osmolality

Sensors

Carotid sinus, aortic arch, renal afferent arteriole, atria

Hypothalamic osmoreceptors

Efferent pathways

Renin-angiotensin-aldosterone axis, sympathetic nervous system, AVP, ANP

AVP

Thirst

Effector

Short term: Heart, blood vessels Long term: Kidney

Kidney

Brain: drinking behavior

What is affected?

Short term: Blood pressure Long term: Na+ excretion

Renal water excretion

Water intake

~58 meq/kg body weight. Approximately 65% of the total Na+ is located in the ECF, and an additional 5% to 10%, in the intracellular fluid (ICF). Extracellular and intracellular Na+, comprising 70% to 75% of the total-body pool, is readily exchangeable, as determined by its ability to equilibrate rapidly with injected radioactive Na+. The remaining 25% to 30% of the body’s Na+ pool is bound as Na+-apatites in bone. The concentration of Na+ in the plasma and interstitial fluid typically ranges between 135 and 145 mM. Chloride totals ~33 meq/kg body weight. Approximately 85% is extracellular, and the remaining 15% is intracellular. Thus, all Cl− is readily exchangeable. The [Cl−] of plasma and interstitial fluid normally varies between 100 and 108 mM. Changes in total-body Cl− are usually influenced by the same factors, and in the same direction, as changes in total-body Na+. Exceptions arise during acid-base disturbances, when Cl− metabolism may change independently of Na+. By definition, in the steady state, the total-body content of water and electrolytes is constant. For Na+, this concept can be expressed as

Oral Na + intake = Renal Na + output + Extrarenal Na + output

(40-1) 

Under normal circumstances, extrarenal Na+ output is negligible. However, large fluid losses from the gastrointestinal tract (e.g., vomiting, diarrhea) or skin (e.g., excessive sweating, extensive burns) can represent substantial extrarenal Na+ losses. The kidney responds to such deficits by reducing renal Na+ excretion. Conversely, in conditions of excessive Na+ intake, the kidneys excrete the surfeit of Na+.

The kidneys increase Na+ excretion in response to an increase in ECF volume, not to an increase in extracellular Na+ concentration In contrast to many other renal mechanisms of electrolyte excretion, the renal excretion of Na+ depends on the amount of Na+ in the body and not on the Na+ concentration in ECF. Because the amount of Na+ is the product of ECF volume and the extracellular Na+ concentration, and because the osmoregulatory system keeps plasma osmolality constant within very narrow limits, it is actually the volume of ECF that acts as the signal for Na+ homeostasis. Figure 40-1A demonstrates the renal response to an abrupt step increase and step decrease in Na+ intake. A subject weighing 70 kg starts with an unusually low Na+

intake of 10 mmol/day, matched by an equally low urinary output. When the individual abruptly increases dietary Na+ intake from 10 to 150 mmol/day—and maintains it at this level for several days—urinary Na+ output also increases, but at first it lags behind intake. This initial period during which Na+ intake exceeds Na+ output is a state of positive Na+ balance. After ~5 days, urinary Na+ output rises to match dietary intake, after which total-body Na+ does not increase further. In this example, we assume that the cumulative retention of Na+ amounts to 140 mmol. The abrupt increase in dietary Na+ initially elevates plasma osmolality, thus stimulating thirst and release of AVP. Because the subject has free access to water, and because the kidneys salvage water in response to AVP (see pp. 817–819), the volume of free water rises. This increase in free water not only prevents a rise in [Na+] and osmolality, but also produces a weight gain that, in this example, is 1 kg (see Fig. 40-1A). This weight gain corresponds, in our example, to the accumulation of 140 mmol of Na+ and the accompanying free water, which makes 1 L of isotonic saline. In the new steady state, only the extracellular compartment has increased in volume. Intracellular volume does not change because, in the end, no driving force exists for water to cross cell membranes (i.e., extracellular osmolality is normal). Instead, the slight expansion of ECF volume signals the kidney to increase its rate of Na+ excretion. The extracellular Na+ concentration is unchanged during this period and thus cannot be the signal to increase Na+ excretion. When the subject in our example later reduces Na+ intake to the initial level of 10 mmol/day (see Fig. 40-1A), Na+ excretion diminishes until the initial balanced state (input = output) is established once again. Immediately after the reduction in Na+ intake, Na+ is temporarily out of balance. This time, we have a period of negative Na+ balance, in which output exceeds input. During this period, the ECF volume falls by 1 L, and body weight returns to normal. Again, the extracellular Na+ concentration is unchanged during this transient period. Ingestion of increasingly larger amounts of Na+ results in retention of progressively larger amounts in the steady state and thus accumulation of progressively more ECF volume. Urinary Na+ excretion increases linearly with this rise in retained Na+, as shown in Figure 40-1B. The control system that so tightly links urinary Na+ excretion to ECF volume is extremely sensitive. In our hypothetical example (see Fig. 40-1A)—a 70-kg individual with an initial ECF volume of 17 L—expanding ECF volume by 1 L, or ~6%, triggers a

838

A

SECTION VI  •  The Urinary System

+ EFFECT OF ABRUPT CHANGES IN Na INTAKE

Weight (kg)

B

EFFECT OF POSITIVE Na+ BALANCE ON Na+ EXCRETION 1400

71 1200

70

1000

150 +

Na (mmol/ day)

100 50 10

Output Intake

Negative balance

Positive balance

–3 –2 –1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Days

Incremental urinary Na+ excretion (mmol/day)

800 600 400 200 0

0

4 Gain in extracellular water (L)

8

0

800 1000 1200 200 400 600 + Amount of Na retained by body (mmol cumulative Na+ balance)

Figure 40-1  Na+ balance. In A, the red curve shows the time course of dietary Na+ intake, and the green

curve shows Na+ excretion. The gold area between the two curves at the beginning of the experiment corresponds to the accumulated total-body Na+ of 140 mmol. This additional Na+, dissolved in ~1 L of ECF, accounts for the 1-kg gain in body weight (blue curve). (B, Data from Walser M: Phenomenological analysis of renal regulation of sodium and potassium balance. Kidney Int 27:837–841, 1985.)

15-fold increase in steady-state urinary Na+ excretion (i.e., from 10 mmol/day to 150 mmol/day in Fig. 40-1A). Physiologically normal individuals can be in Na+ balance on a nearly Na+-free diet (1 to 2 mmol/day) without overt signs of ECF volume depletion. Conversely, even with consumption of a high-Na+ diet (200 mmol/day versus the “normal” ~100 mmol/day for a Western diet), clinical signs of ECF volume excess, such as edema, are absent.

in Na+ retention. For example, in congestive heart failure, particularly when edema is extensive, the total ECF volume is greatly increased. However, the low cardiac output fails to expand the key blood-filled compartments. As a result, Na+ reabsorption by the renal tubules remains high (i.e., urinary Na+ excretion is inappropriately low compared with Na+ intake), which exacerbates the systemic congestion (Box 40-1).  N40-1 

It is not the ECF volume as a whole, but the effective circulating volume, that regulates Na+ excretion

Decreases in effective circulating volume trigger four parallel effector pathways to decrease renal Na+ excretion

Although we have referred to the overall expansion of the ECF volume as the signal for increased urinary Na+ excretion, this is an oversimplification. Only certain regions of the ECF compartment are important for this signaling. For an expansion in ECF volume to stimulate Na+ excretion—either acutely or chronically—the expansion must make itself evident in parts of the ECF compartment where the ECF volume sensors are located, namely, in blood-filled compartments. ECF volume per se is not the critical factor in regulating renal Na+ excretion. The critical parameter that the body recognizes is the effective circulating volume (see pp. 554–555)—not something that we can identify anatomically. Rather, effective circulating volume is a functional blood volume that reflects the extent of tissue perfusion in specific regions, as evidenced by the fullness or pressure within their blood vessels. Normally, changes in effective circulating volume parallel those in total ECF volume. However, this relationship may be distorted in certain diseases, such as congestive heart failure, nephrotic syndrome, or liver cirrhosis. In all three cases, total ECF volume is grossly expanded (e.g., edema or ascites). In contrast, the effective circulating volume is low, resulting

Figure 40-2 shows the elements of the feedback loop that controls the effective circulating volume. As summarized in Table 40-2, sensors that monitor changes in effective circulating volume are baroreceptors located in both highpressure (see pp. 534–536) and low-pressure (see pp. 546– 547) areas of the circulation. Although most are located within the vascular tree of the thorax, additional baroreceptors are present in the kidney—particularly in the afferent arterioles (see p. 730)—as well as in the CNS and liver. Of the pressures at these sites, it is renal perfusion pressure that is most important for long-term regulation of Na+ excretion, and thus blood pressure, because increased resistance to renal blood flow (e.g., renal artery stenosis) causes sustained hypertension (Box 40-2). The sensors shown in Figure 40-2 generate four distinct hormonal or neural signals (pathways 1 to 4 in the figure). In the first pathway, the kidney itself senses a reduced effective circulating volume and directly stimulates a hormonal effector pathway, the renin-angiotensinaldosterone system, discussed in the section beginning on page 841. In addition, increased renal perfusion pressure

Chapter 40  •  Integration of Salt and Water Balance

N40-1  Effect of Posture and Water Immersion on Na+ Excretion Contributed by Gerhard Giebisch and Erich Windhager On page 838, we introduce congestive heart failure as an example of the nonparallel behavior of ECF volume on the one hand and effective circulating volume on the other. Two additional examples that depend upon gravity are posture and water immersion. Urinary Na+ excretion is lowest when one is standing (i.e., when thoracic perfusion is lowest), higher when one is lying down (recumbency), and highest when one is immersed up to the chin for several hours in warm water. During immersion, the hydrostatic pressure of the water compresses the tissues—and thus the vessels, particularly the veins—in the extremities and abdomen and consequently enhances venous return to the thorax. Recumbency—and, to a greater extent, water immersion—shifts blood into the thoracic vessels, increasing the so-called central blood volume (see p. 449). In contrast, the upright position depletes the intrathoracic blood volume. The thoracic vessels are immune to this compression because their extravascular pressure (i.e., intrapleural pressure; see p. 606) is unaffected by the water. Thus, it is the enhanced venous return alone that stimulates vascular sensors to increase Na+ excretion. This example clearly demonstrates that only special portions within the ECF compartment play critical roles in the sensing of ECF volume.

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Chapter 40  •  Integration of Salt and Water Balance

BOX 40-1  Volume Expansion and Contraction

W

hen Na+ intake persists in the face of impaired renal Na+ excretion (e.g., during renal failure), the body retains isosmotic fluid. The result is an expansion of plasma volume and of the interstitial fluid compartment. In the extreme, the interstitial volume increase can become so severe that the subepidermal tissues swell (e.g., around the ankles). When the physician presses with a finger against the skin and then removes the finger, the finger imprint remains in the tissue—pitting edema. Not all cases of lower-extremity edema reflect total-body Na+ and fluid retention. For example, venous obstruction to return of blood from the lower extremities can cause local edema in the lower legs. Patients with this condition should elevate their feet whenever possible and should wear compression stockings. Fluid can also accumulate in certain transcellular spaces (see p. 102), such as the pleural cavity (pleural effusion) or the peritoneal cavity (ascites); such conditions reflect derangements of local Starling forces or an increase in protein permeability due to inflammation, which alters the fluid distribution between the plasma and the ECF (see Box 20-1). In cases of abnormal Na+ retention, putting the subject on a low-Na+ diet can partially correct the edema. Administration of diuretics  N40-2  can also reduce volume overload, as long as the kidney retains sufficient function to respond to them. An excessive loss of Na+ into the urine can be caused by disturbances of Na+ reabsorption along the nephron and leads to a dramatic shrinkage of the ECF volume. Because the plasma volume is part of the ECF volume, significant reductions can severely affect the circulation, culminating in hypovolemic shock (see p. 583). Renal causes of reduced ECF volume include the prolonged use of powerful loop diuretics (see p. 757), osmotic diuresis (see Box 35-1) during poorly controlled diabetes mellitus, adrenal insufficiency with low aldosterone levels, and the recovery phase following acute renal failure or relief of urinary obstruction.

TABLE 40-2  ECF Volume Receptors “Central” vascular sensors High pressure JGA (renal afferent arteriole) Carotid sinus Aortic arch Low pressure Cardiac atria Pulmonary vasculature Sensors in the CNS (less important) Sensors in the liver (less important)

itself can increase Na+ excretion independent of the reninangiotensin-aldosterone system, as we shall see beginning on page 843. The second and third effector pathways are neural. Baroreceptors detect decreases in effective circulating volume and communicate these via afferent neurons to the medulla of the brainstem. Emerging from the medulla are two types of efferent signals that ultimately act on the kidney. In one, increased activity of the sympathetic division of the autonomic nervous system reduces renal blood flow and directly

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BOX 40-2  Renal Hypertension

I

n the 1930s, Goldblatt produced hypertension experimentally in unilaterally nephrectomized animals by placing a surgical clip around the renal artery of the remaining kidney (one-kidney Goldblatt hypertension). The constriction can be adjusted so that it results not in renal ischemia, but only in a reduction of the perfusion pressure distal to the clip. This maneuver stimulates the renal baroreceptors, leading to a rapid increase in synthesis and secretion of renin from the clipped kidney. The renin release reaches a peak after 1 hour. As renin cleaves ANG I from angiotensinogen, systemic ANG I levels rise quickly. ACE, present mainly in the lungs but also in the kidneys, then rapidly converts ANG I into ANG II. Thus, within minutes of clamping the renal artery, one observes a sustained rise in systemic arterial pressure. The newly established stable elevation in systemic pressure then normalizes the pressure in the renal artery downstream from the constriction. From this time onward, circulating renin and ANG II levels decline toward normal over 5 to 7 days, while the systemic arterial pressure remains abnormally high. The early rise in blood pressure is the result of the renin-angiotensin vasoconstrictor mechanism, which is activated by the experimentally induced reduction in pressure and flow in the renal artery distal to the constriction. The later phase of systemic hypertension is the result of aldosterone release and of the retention of salt and water. Unilateral partial clamping of a renal artery in an otherwise healthy animal also produces hypertension (two-kidney Goldblatt hypertension). As in the one-kidney model, the clipped kidney increases its synthesis and secretion of renin. Renin then causes ANG II levels to increase systemically and will, in addition to the effect on the clamped kidney, cause the nonclamped contralateral kidney to retain salt and water. As in the one-kidney model, the resulting hypertension has an early vasoconstrictive phase and a delayed volume-dependent phase. These models of hypertension show that the kidney can be critical as a long-term baroreceptor. Thus, when increased resistance in a renal artery leads to reduced intrarenal perfusion pressure, the rest of the body, including central baroreceptors, experiences—and cannot counteract—the sustained hypertension. In both types of Goldblatt hypertension, administration of ACE inhibitors can lower arterial blood pressure. In fact, inhibiting ACE is therapeutically effective even after circulating renin and ANG II levels have normalized. The reason is that maintained hypertension involves an increased intrarenal conversion of ANG I to ANG II (via renal ACE), with the ANG II enhancing proximal Na+ reabsorption. Indeed, direct measurements show that, even after circulating renin and ANG II levels have returned to normal, the intrarenal levels of ACE and ANG II are elevated. ACE inhibitors lower systemic and intrarenal ANG II levels. These experimental models correspond to some forms of human hypertension, including hypertension produced by renin-secreting tumors of the JGA and by all types of pathological impairment of renal arterial blood supply. Thus, coarctation of the aorta, in which the aorta is constricted above the renal arteries but below the arteries to the head and upper extremities, invariably leads to hypertension. Renal hypertension also results from stenosis of a renal artery, caused, for example, by arteriosclerotic thickening of the vessel wall.

Chapter 40  •  Integration of Salt and Water Balance

N40-2  Sensitivity of the Natriuretic Response to Increased Extracellular Fluid Volume Contributed by Erich Windhager and Gerhard Giebisch Figure 40-1B shows a hypothetical example of how urinary Na+ excretion (y-axis) changes in response to increases in isotonic extracellular water volume (upper x-axis) or amount of Na+ retained by the body (lower x-axis). In the example in the figure, the urinary Na+ excretion increases by 120 mmol/ day for every 100 mmol of cumulative Na+ retention. This proportionality is indicated by the slope of the line. However, this slope need not be the same for every person. In a patient with abnormal Na+ retention, the natriuretic response must be less sensitive than normal (i.e., the slope of the line in Fig. 40-1B must be less steep). In other words, in response to an increase in Na+ intake, the patient would have to accumulate more Na+ and water (i.e., he or she would have to become more volume expanded than would a normal person) in order to sufficiently stimulate the kidneys to elicit the natriuretic response necessary for coming into Na+ balance (i.e., achieving a steady state in which urinary excretion balances dietary intake).

839.e1

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SECTION VI  •  The Urinary System

Increased renal Na+ retention counteracts decreased effective circulating volume.

Effective circulating volume

Renal baroreceptor

Liver

Aortic arch

Carotid sinus

Cardiac atria

Central nervous system

Atrial myocytes

GFR

Juxtaglomerular apparatus (JGA)

Atrial lowpressure receptor

Pulmonary lowpressure receptor

Brain 1 Renin

Angiotensin II (ANG II)

2 Sympathetic division of ANS

3 Posterior pituitary

Aldosterone

4

Atrial natriuretic peptide (ANP)

Arginine vasopressin (AVP or ADH)

Changes in hemodynamics and tubule transport

Na+ excretion Figure 40-2  Feedback control of effective circulating volume. A low effective circulating volume triggers four parallel effector pathways (numbered 1 to 4) that act on the kidney, either by changing the hemodynamics or by changing Na+ transport by the renal-tubule cells. ANS, autonomic nervous system.

stimulates Na+ reabsorption, thereby reducing Na+ excretion (discussed on pp. 842–843). In the other effector pathway, the posterior pituitary increases its secretion of AVP, which leads to conservation of water (discussed on p. 843). This AVP mechanism becomes active only after large declines in effective circulating volume. The final pathway is hormonal. Reduced effective circulating volume decreases the release of atrial natriuretic peptide (ANP), thus reducing Na+ excretion (discussed on p. 843). All four parallel effector pathways correct the primary change in effective circulating blood volume. An increase in effective circulating volume promotes Na+ excretion (thus

reducing ECF volume), whereas a decrease in effective circulating volume inhibits Na+ excretion (thus raising ECF volume). An important feature of renal Na+ excretion is the two-way redundancy of control mechanisms. First, efferent pathways may act in concert on a single effector within the kidney. For instance, both sympathetic input and hemodynamic/ physical factors often act on proximal tubules. Second, one efferent pathway may act at different effector sites. For example, angiotensin II (ANG II) enhances Na+ retention directly by stimulating apical Na-H exchange in tubule cells (see Fig. 35-4) and indirectly by lowering renal plasma flow (see p. 746).

Chapter 40  •  Integration of Salt and Water Balance

841

Effective circulating volume Lungs

ACE

Angiotensin II

Adrenal Increased renal Na+ retention counteracts decreased effective circulating volume.

Hypothalamus

Aldosterone Angiotensin I

Kidneys

Thirst and AVP Renin

JGA

Angiotensinogen

Liver

+

Na excretion H2O excretion

Figure 40-3  Renin-angiotensin-aldosterone axis.

Increased activity of the renin-angiotensin-aldosterone axis is the first of four parallel pathways that correct a low effective circulating volume The renin-angiotensin-aldosterone axis (Fig. 40-3) promotes Na+ retention via the actions of both ANG II and aldosterone. For a consideration of this axis in the context of the physiology of the adrenal cortex, see page 1029. Angiotensinogen,  N23-12  also known as renin substrate, is an α2-globulin that is synthesized by the liver and released into the systemic circulation. The liver contains only small stores of angiotensinogen. Another protein, renin,  N40-4  is produced and stored in distinctive granules by the granular cells of the renal juxtaglomerular apparatus (JGA; see p. 727). As discussed below (see p. 841), decreases in effective circulating volume stimulate these cells to release renin, which is a protease that cleaves a peptide bond near the C terminus of angiotensinogen, releasing the decapeptide angiotensin I (ANG I). Angiotensin-converting enzyme (ACE) rapidly removes the two C-terminal amino acids from the physiologically inactive ANG I to form the physiologically active octapeptide ANG II. ACE is present on the luminal surface of vascular endothelia throughout the body and is abundantly present in the endothelium-rich lungs. ACE in the kidney—particularly in the endothelial cells of the afferent and efferent arterioles, and also in the proximal tubule—can produce enough ANG II to exert local vascular effects. Thus, the kidney receives ANG II from three sources: (1) Systemic ANG II comes from the general circulation, originating largely from the pulmonary circulation. (2) Renal vessels generate ANG II from ANG I. (3) Proximaltubule cells, which contain renin and ACE, secrete ANG II into its lumen. Both in the circulation and in the tubule

lumen, aminopeptidases further cleave ANG II to the heptapeptides ANG III [ANG-(2-8)] and ANG-(1-7), which are biologically active. The principal factor controlling plasma ANG II levels is renin release from JGA granular cells. A decrease in effective circulating volume manifests itself to the JGA—and thus stimulates renin release—in three ways (see Fig. 40-2): 1. Decreased systemic blood pressure (sympathetic effect on JGA). A low effective circulating volume, sensed by baroreceptors located in the central arterial circulation (see p. 534), signals medullary control centers to increase sympathetic outflow to the JGA, which in turn increases renin release. Renal denervation or β-adrenergic blocking drugs (e.g., propranolol) inhibit renin release. 2. Decreased NaCl concentration at the macula densa (NaCl sensor). Decreased effective circulating volume tends to increase filtration fraction (the inverse of the sequence shown in Fig. 34-10), thereby increasing Na+ and fluid reabsorption by the proximal tubule (see p. 842) and reducing the flow of tubule fluid through the loop of Henle. Na+ reabsorption in the thick ascending limb (TAL) then decreases luminal [Na+] more than if tubular flow were higher. The resulting decrease in luminal [NaCl] at the macula densa stimulates renin release. 3. Decreased renal perfusion pressure (renal baroreceptor). Stretch receptors in the granular cells (see p. 727) of the afferent arterioles sense the decreased distention associated with low effective circulating volume. This decreased stretch lowers [Ca2+]i, which increases renin release and initiates a cascade that tends to promote Na+ reabsorption and thus increase blood pressure. Conversely, increased distention (high extracellular volume) inhibits renin release.

Chapter 40  •  Integration of Salt and Water Balance

N40-4  Renin Release from Granular Cells Contributed by Erich Windhager and Gerhard Giebisch As pointed out in the text, the granular cells are one of two cell types in which the exocytosis of a hormone decreases in response to a rise in [Ca2+]i. For example, if one raises [K+]o, the granular cell depolarizes. This depolarization probably opens voltage-gated Ca2+ channels (see p. 190) or decreases Ca2+ extrusion via an Na-Ca exchanger (see pp. 123–124). In either case, [Ca2+]i rises and blocks renin release. Similarly, applying Ca2+ ionophores—compounds that increase the permeability of the cell membrane to Ca2+—also raises [Ca2+]i and reduces renin release. Increases in intracellular levels of cAMP have the opposite effect of raising [Ca2+]i—increases in [cAMP]i stimulate renin release from granular cells. Conversely, agents that inhibit adenylyl cyclase activity (e.g., β-adrenergic antagonists, α-adrenergic agonists, and A1 adenosine receptor agonists) decrease [cAMP]i and thereby inhibit renin release.

REFERENCE Kurtz A: Cellular control of renin secretion. Rev Physiol Biochem Pharmacol 113:1–38, 1989.

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SECTION VI  •  The Urinary System

Renal blood flow Afferent Efferent arteriolar resistance

Filtration fraction

Peritubular capillary colloid osmotic pressure

Proximal Na+ reabsorption

Peritubular capillary hydrostatic pressure

Na+ excretion H2O excretion

ANG II Vasa recta blood flow

Washout of urea from medullary interstitium

[Urea] [Na+] in medullary interstitium

Gradient for passive NaCl reabsorption by the thin ascending limb of Henle

+

Loop Na reabsorption

Figure 40-4  Hemodynamic actions of ANG II on Na+ reabsorption.

The above stimulation of renin release by a decrease in [Ca2+]i  N40-4  stands in contrast to most Ca2+-activated secretory processes, in which an increase in [Ca2+]i stimulates secretion (see p. 221). Another exception is the chief cell of the parathyroid gland, in which an increase in [Ca2+]i inhibits secretion of parathyroid hormone (see pp. 1060–1061). Intracellular cAMP also appears to be a second messen­ ger for renin release. Agents that activate adenylyl cyclase  N40-5  enhance renin secretion, presumably via protein kinase A. The question whether the effects of [cAMP]i and [Ca2+]i are independent or sequential remains open.  N40-3

Additional factors also modulate renin release. Prosta­ glandins E2 and I2 and endothelin all activate renin release. Agents that blunt renin release include ANG II (which represents a short feedback loop), AVP, thromboxane A2, high plasma levels of K+, and nitric oxide. ANG II has several important actions as follows: 1. Stimulation of aldosterone release from glomerulosa cells in the adrenal cortex (see p. 1028). In turn, aldosterone promotes Na+ reabsorption in the distal tubule and collecting tubules and ducts (see p. 766). 2. Vasoconstriction of renal and other systemic vessels. ANG II increases Na+ reabsorption by altering renal hemodynamics, probably in two ways (Fig. 40-4). First, at high concentrations, ANG II constricts the efferent more than the afferent arterioles, thus increasing filtration fraction and reducing the hydrostatic pressure in the downstream peritubular capillaries. The increased filtration fraction also increases the protein concentration in the downstream blood and hence raises the colloid osmotic pressure of the peritubular capillaries. The changes in each of these two Starling forces favor the uptake of reabsorbate from peritubular interstitium into peri­tubular capillaries (see pp. 763–765) and hence enhance the reabsorption of Na+ and fluid by the proximal tubule. Second, ANG II decreases medullary blood flow through the vasa recta. Low blood flow decreases the medullary washout of NaCl and urea (see pp. 813–815), thus raising [urea] in the medullary interstitium and enhancing Na+ reabsorption along the thin ascending limb of Henle’s loop (see p. 811). 3. Enhanced tubuloglomerular feedback. ANG II raises the sensitivity and lowers the set-point of the tubuloglo-

merular feedback mechanism (see pp. 750–751), so that an increase in Na+ and fluid delivery to the macula densa elicits a more pronounced fall in the glomerular filtration rate (GFR). 4. Enhanced Na-H exchange. ANG II promotes Na+ reabsorption in the proximal tubule, TAL, and initial collecting tubule (see pp. 765–766). 5. Renal hypertrophy. Over a prolonged time, ANG II induces hypertrophy of renal-tubule cells. 6. Stimulated thirst and AVP release. ANG II acts on the hypothalamus, where it increases the sensation of thirst and stimulates secretion of AVP from the posterior pituitary, both of which increase total-body free water. This ANG II effect represents an intersection between the systems for regulating effective circulating volume and osmolality.

Increased sympathetic nerve activity, increased AVP, and decreased ANP are the other three parallel pathways that correct a low effective circulating volume Renal Sympathetic Nerve Activity  The second of the four parallel effector pathways for the control of effective circulating volume is the sympathetic nervous system. Enhanced activity of the renal sympathetic nerves has two direct effects on Na+ reabsorption (see pp. 766–768): (1) increased renal vascular resistance, and (2) increased Na+ reabsorption by tubule cells. In addition, increased sympathetic tone has an indirect effect—enhancing renin release from granular cells (see previous section). These multiple actions of sympathetic traffic to the kidney reduce GFR and enhance Na+ reabsorption, thereby increasing Na+ retention and increasing effective circulating volume. In everyday life (i.e., the unstressed state), the role of sympathetic nerve activity in kidney function appears to be modest at best. However, sympathetic innervation may play a role during challenges to volume homeostasis. For example, low Na+ intake triggers reduced renal Na+ excretion; renal denervation blunts this response. Another example is hemorrhage, in which renal sympathetic nerves emerge as important participants in preserving ECF volume.

Chapter 40  •  Integration of Salt and Water Balance

842.e1

N40-3  Systemic versus Local Roles of the Juxtaglomerular Apparatus Contributed by Emile Boulpaep and Walter Boron The JGA performs two apparently opposite functions: main­ taining a constant GFR (tubuloglomerular feedback, or TGF) and maintaining a constant whole-body blood pressure by modulating renin release. TGF (see pp. 750–751) is a local phenomenon, whereas the release of renin has systemic consequences (see pp. 841–842). In the case of tubuloglomerular feedback (i.e., the local response), decreased renal perfusion pressure, reduced filtered load, or enhanced proximal fluid reabsorption all lead to a decrease in the flow of tubule fluid past the macula densa, as well as to a decrease in Na+ delivery and Na+ concentration. Within seconds after such a transient disturbance, and by an unknown mechanism, TGF dilates the afferent arteriole of the same nephron in an attempt to increase single-nephron glomerular filtration rate (SNGFR) and restore fluid and Na+ to that particular macula densa. In the case of renin release (i.e., the systemic response), by contrast, a sustained fall in arterial pressure or a contraction of the extracellular volume reduces fluid delivery to many maculae

N40-5  Other Factors that Activate Adenylyl Cyclase in Granular Cells Contributed by Gerhard Giebisch and Erich Windhager Agents that activate adenylyl cyclase in the granular cells of the JGA—and thus stimulate renin release—include forskolin, β-adrenergic agonists, A2 adenosine receptor agonists, dopamine, and glucagon. In addition, exogenous cAMP and phosphodiesterase inhibitors enhance renin secretion. All of these agents presumably act through protein kinase A.

densae, leading to the release of renin. Renin, in turn, causes an increase in local and systemic concentrations of ANG II. Besides causing general vasoconstriction, ANG II constricts the afferent and efferent glomerular arterioles, thereby decreasing GFR. This effect is opposite to that of TGF: TGF dilates a single afferent arteriole, whereas renin release constricts many afferent and efferent arterioles. TGF may be viewed as a mechanism designed to maintain a constant SNGFR, whereas renin release is aimed at maintaining blood pressure by both systemic and renal vasoconstriction (i.e., hemodynamic effects), as well as by reducing SNGFR and enhancing tubule Na+ reabsorption (Na+-retaining effects). TGF is a minute-to-minute, fine control of SNGFR that can be superseded by the intermediate- to long-term effects of the powerful renin response, which comes into play whenever plasma volume and blood pressure are jeopardized. It must be emphasized that renin release is governed not only by the JGA but also by other mechanisms, in particular by changes in the activity of sympathetic nerves (see pp. 842–843).

Chapter 40  •  Integration of Salt and Water Balance

Conversely, expansion of the intravascular volume increases renal Na+ excretion; renal denervation sharply reduces this response as well. Arginine Vasopressin (Antidiuretic Hormone)  As discussed below (see p. 844), the posterior pituitary releases AVP primarily in response to increases in extracellular osmolality. Indeed, AVP mainly increases distal-nephron water per­ meability, promoting water retention (see pp. 817–818). However, the posterior pituitary also releases AVP in response to large reductions in effective circulating volume (e.g., hemorrhage), and secondary actions of AVP— vasoconstriction (see p. 553) and promotion of renal Na+ retention (see p. 768)—are appropriate for this stimulus. Atrial Natriuretic Peptide  Of the four parallel effectors

that correct a low effective circulating volume (see Fig. 40-2), ANP is the only one that does so by decreasing its activity. As its name implies, ANP promotes natriuresis (i.e., Na+ excretion). Atrial myocytes synthesize and store ANP and release ANP in response to stretch (a low-pressure volume sensor; see p. 547). Thus, reduced effective circulating volume inhibits ANP release and reduces Na+ excretion. ANP plays a role in the diuretic response to the redistribution of ECF and plasma volume into the thorax that occurs during water immersion and space flight (see p. 1233). Acting through a receptor guanylyl cyclase (see pp. 66– 67), ANP has many synergistic effects (see p. 768) on renal hemodynamics and on transport by renal tubules that promote renal Na+ and water excretion.  N40-6  Although ANP directly inhibits Na+ transport in the inner medullary collecting duct, its major actions are hemodynamic— increased GFR and increased cortical and medullary blood flow. ANP also decreases the release of renin, independently inhibits aldosterone secretion by the adrenal gland, and decreases release of AVP. In summary, a decrease in effective circulating volume leads to a fall in ANP release and a net decrease in Na+ and water excretion.

High arterial pressure raises Na+ excretion by hemodynamic mechanisms, independent of changes in effective circulating volume We have seen that expanding the effective circulating volume stimulates sensors that increase Na+ excretion via four parallel effector pathways (see Fig. 40-2). However, the kidney can also modulate Na+ excretion in response to purely hemodynamic changes, as in the following two examples. Large and Acute Decrease in Arterial Blood Pressure  If glomerulotubular (GT) balance (see p. 763) were perfect, decreasing the GFR would cause Na+ excretion to fall linearly (Fig. 40-5, blue line). However, acutely lowering GFR by partial clamping of the aorta causes a steep, nonlinear decrease in urinary Na+ excretion (see Fig. 40-5, red curve). When GFR falls sufficiently, the kidneys excrete only traces of Na+ in a small volume of urine. This response primarily reflects the transport of the classical distal tubule (see p. 765), which continues to reabsorb Na+ at a high rate despite the decreased Na+ delivery.

843

+ As GFR rises, Na excretion rises very rapidly (“pressure diuresis”).

175

150

Normal GFR and + Na excretion

125

Percent 100 of Na+ excretion

Ideal GT balance (i.e., fractional excretion of Na+ is constant)

75

50

As GFR falls, Na+ excretion falls even faster.

25

0

0

25 50 75 100 Percent of control glomerular filtration rate

125

Figure 40-5  Effect of changes in GFR on urinary Na+ excretion. The blue line represents ideal glomerulotubular (GT) balance. The red curve summarizes data from dogs. The investigators reduced GFR by inflating a balloon in the aorta, above the level of the renal arteries. They increased GFR by compressing the carotid arteries and thus increased blood pressure. (Data from Thompson DD, Pitts RF: Effects of alterations of renal arterial pressure on sodium and water excretion. Am J Physiol 168:490– 499, 1952.)

Large Increase in Arterial Pressure  In some cases, an increased effective circulating volume is accompanied by an increase in arterial pressure. Examples include primary hyperaldosteronism and Liddle disease  N23-14,  states of abnormally high distal Na+ reabsorption. The excess Na+ reabsorption leads to high blood pressure and compensatory pressure-induced natriuresis. One reason for this pressure diuresis is that hypertension increases GFR, increasing the filtered load of Na+, which by itself would increase urinary Na+ excretion (see Fig. 40-5, blue line). However, at least four other mechanisms contribute to the natriuresis (see Fig. 40-5, red curve). First, the increased effective circulating volume inhibits the renin-angiotensin-aldosterone axis and thus reduces Na+ reabsorption (see pp. 765–766). Second, the high blood pressure augments blood flow in the vasa recta, thereby washing out medullary solutes and reducing interstitial hypertonicity in the medulla (see pp. 813–815) and ultimately reducing passive Na+ reabsorption in the thin ascending limb (see p. 811). Third, an increase in arterial pressure leads, by an unknown mechanism, to prompt reduction in the number of apical Na-H exchangers in the proximal tubule. Normalizing the blood pressure rapidly reverses this effect. Finally, hypertension leads to increased pressure in the peritubular capillaries, thereby reducing proximal-tubule reabsorption (physical factors; see p. 763).

Chapter 40  •  Integration of Salt and Water Balance

N40-6  Renal Sites of Action of Atrial Natriuretic Peptide Contributed by Erich Windhager and Gerhard Giebisch 1 GFR is increased. 2 + Na reabsorption is directly or indirectly inhibited. 3 ANG II−stimulated Na+ reabsorption is inhibited.

10 Na+ reabsorption is inhibited owing to decrease in plasma aldosterone levels.

Na+ Na Na+

+

Macula densa

5 Passive water efflux is decreased.

Hypertonicity of the medullary interstitium is decreased. 6

Cl– Na+

12 Na+ load to inner medullary collecting duct is increased.

8 Load to macula densa is increased. Outer medulla Inner medulla

H2O

Thin descending limb of loop of Henle

Na+

9 Renin secretion is inhibited.

Glomerulus

Proximal convoluted tubule Thick ascending limb of loop of Henle

4 Na+ load to loop of Henle is increased.

11 Thiazide-sensitive Na/Cl cotransport is inhibited.

Na+

+

Na

13 Amiloridesensitive Na+ reabsorption is inhibited.

7 Passive Na+ efflux is decreased. Urinary Na+ excretion is increased. 15

Na+ –

Cl K+

⋅ UNaV

14 Furosemidesensitive Na/K/Cl cotransport in basolateral membranes is stimulated.

 urinary sodium excretion rate. (Data from Atlas SA, Maack T: eFigure 40-1  Sites of action of ANP. UNaV, Atrial natriuretic factor. In Windhager E (ed): Handbook of Physiology, Section 8: Renal Physiology. New York, Oxford University Press [for American Physiological Society], 1992, pp 1577–1674.)

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SECTION VI  •  The Urinary System

CONTROL OF WATER CONTENT (EXTRACELLULAR OSMOLALITY) Water accounts for half or more of body weight (~60% in men and 50% in women; see p. 102) and is distributed between the ICF and ECF compartments. Changes in totalbody water content in the absence of changes in total-body solute content lead to changes in osmolality, to which the CNS is extremely sensitive. Osmolality deviations of ±15% lead to severe disturbances of CNS function. Thus, osmoregulation is critical. Two elements control water content and thus whole-body osmolality: (1) the kidneys, which control water excretion (see pp. 806–807); and (2) thirst mechanisms, which control the oral intake of water. These two effector mechanisms are part of negative-feedback loops that begin within the hypothalamus. An increase in osmolality stimulates separate osmoreceptors to secrete AVP (which reduces renal excretion of free water) and to trigger thirst (which, if fulfilled, increases intake of free water). As a result, the two complementary feedback loops stabilize osmolality and thus [Na+].

Increased plasma osmolality stimulates hypothalamic osmoreceptors that trigger the release of AVP, inhibiting water excretion An increase in the osmolality of the ECF is the primary signal for the secretion of AVP from the posterior pituitary gland. An elegant series of animal studies by Verney in the 1940s established that infusing a hyperosmotic NaCl solution into the carotid artery abruptly terminates an established water diuresis (Fig. 40-6A). Infusing the same quantity of hyperosmotic NaCl into the peripheral circulation has little effect because the hyperosmolar solution becomes diluted by the time it reaches the cerebral vessels. Therefore, the osmosensitive site is intracranial. Surgically removing the posterior pituitary abolishes the effect of infusing hyperosmotic NaCl into the carotid artery (see Fig. 40-6B). However, injecting posterior-pituitary extracts into the animal inhibits the diuresis, regardless of whether the posterior pituitary is intact. Later work showed that Verney’s posterior-pituitary extract contained an “antidiuretic hormone”—now known to be AVP—that the posterior pituitary secretes in response to increased plasma osmolality. Ingesting large volumes of water causes plasma osmolality to fall, thus leading to reduced AVP secretion. In healthy individuals, plasma osmolality is ~290 mOsm. The threshold for AVP release is somewhat lower, ~280 mOsm (Fig. 40-7, red curve). Increasing the osmolality by only 1% higher than this level is sufficient to produce a detectable increase in plasma [AVP], which rises steeply with further increases in osmolality. Thus, hyperosmolality leads to increased levels of AVP, which completes the feedback loop by causing the kidneys to retain free water (see pp. 817–818). Although changes in plasma [NaCl] are usually responsible for changes in plasma osmolality, other solutes can do the same. For example, hypertonic mannitol resembles NaCl in stimulating AVP release. However, an equivalent increase in extracellular osmolality by urea has little effect on plasma AVP levels. The reason is that urea readily permeates cell membranes and hence exerts a low effective

A

BEFORE REMOVAL OF POSTERIOR PITUITARY H2O (p.o.)

6

Hyperosmotic Posterior pituitary NaCl (i.a.) extract (i.v.)

4 Urine flow (mL/min) 2

0

B

0

60

120 180 Time (min)

240

300

AFTER REMOVAL OF POSTERIOR PITUITARY H2O (p.o.)

6

Hyperosmotic Posterior pituitary NaCl (i.a.) extract (i.v.)

4 Urine flow (mL/min) 2

0

0

60

120 180 Time (min)

240

300

Figure 40-6  Sensing of blood osmolality in the dog brain. i.a., intraarterial (carotid) injection; i.v., intravenous injection; p.o., per os (by mouth). (Data from Verney EG: The antidiuretic hormone and the factors which determine its release. Proc Royal Soc Lond B 135:25–106, 1947.)

12 Volume contraction 8 Plasma AVP (pg/mL)

Volume expansion

4

0 260

Euvolemia (normal)

270

280 290 300 Plasma osmolality (mOsm)

310

Figure 40-7  Dependence of AVP release on plasma osmolality. (Data from Robertson GL, Aycinena P, Zerbe RL: Neurogenic disorders of osmoregulation. Am J Med 72:339–353, 1982.)

osmolality or tonicity (see pp. 132–133) and is thus poorly effective in shrinking cells.

Hypothalamic neurons synthesize AVP and transport it along their axons to the posterior pituitary, where they store it in nerve terminals prior to release Osmoreceptors of the CNS appear to be located in two areas that breech the blood-brain barrier: the organum

Chapter 40  •  Integration of Salt and Water Balance

Osmoreceptors in OVLT and SFO Paraventricular nucleus Magnocellular neurons

Hypothalamus Baroreceptor input from NTS

Supraoptic nucleus

Anterior lobe of pituitary Posterior lobe of pituitary

AVP

Preproneurophysin II Proneurophysin II 1

2

3

4 Arg

Gly Lys Arg

10 11 12 1. Signal peptide 19 aa 2. AVP 9 aa 3. Neurophysin II 95 aa AVP

4. Glycopeptide 39 aa

S

Gly in the 10 position (which is removed) is necessary for amidation of the Gly residue in the 9 position of AVP.

S

N– Cys Tyr Phe Gln Asn Cys Pro Arg Gly

–Amide

1 2 3 4 5 6 7 8 9 Arginine vasopressin Figure 40-8  Control of AVP synthesis and release by osmoreceptors. Osmoreceptors are located in the OVLT and SFO, two areas that breech the blood-brain barrier. Signals from atrial low-pressure baroreceptors travel with the vagus nerve to the nucleus tractus solitarii (NTS); a second neuron carries the signal to the hypothalamus. aa, amino acids.

vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO), two of the circumventricular organs (see pp. 284–285). Specific neurons in these regions (Fig. 40-8) are able to sense changes in plasma osmolality. Elevated osmolality increases the activity of mechanosensitive cation

845

channels located in the neuronal membrane, which results in depolarization and thus an increased frequency of action potentials. Hypo-osmolality causes a striking decrease of frequency. The osmosensitive neurons project to large-diameter neurons in the supraoptic and paraventricular nuclei of the anterior hypothalamus (see Fig. 40-8). These neurons synthesize AVP, package it into granules, and transport the granules along their axons to nerve terminals in the posterior lobe of the pituitary, which is part of the brain (see pp. 979– 981). When stimulated by the osmosensitive neurons, these magnocellular neurons release the stored AVP into the posterior pituitary—an area that also lacks a blood-brain barrier—and AVP enters the general circulation. In humans and most mammals, the antidiuretic hormone is AVP, which is encoded by the messenger RNA for pre­ proneurophysin II. After cleavage of the signal peptide, the resulting prohormone proneurophysin II contains AVP, neurophysin II (NpII), and a glycopeptide (see Fig. 40-8). Cleavage of the prohormone within the secretory granule yields these three components. AVP has nine amino acids, with a disulfide bridge connecting two cysteine residues. Mutations of NpII impair AVP secretion, which suggests that NpII assists in the processing or secretion of AVP. Levels of circulating AVP depend on both the rate of AVP release from the posterior pituitary and the rate of AVP degradation. The major factor controlling AVP release is plasma osmolality. However, as discussed below, other factors also can modulate AVP secretion. Two organs, the liver and the kidney, contribute to the breakdown of AVP and the rapid decline of AVP levels when secretion has ceased. The half-life of AVP in the circulation is 18 minutes. Diseases of the liver and kidney may impair AVP degradation and may thereby contribute to water retention. For example, the congestion of the liver and impairment of renal function that accompany heart failure can compromise AVP breakdown, leading to inappropriately high circulating levels of AVP. Conversely, in pregnancy, placental vasopressinase activity can accelerate degradation of AVP.

Increased osmolality stimulates a second group of osmoreceptors that trigger thirst, which promotes water intake The second efferent pathway of the osmoregulatory system is thirst, which regulates the oral intake of water. Like the osmoreceptors that trigger AVP release, the osmoreceptors that trigger thirst are located in two circumventricular organs, the OVLT and the SFO. Also like the osmoreceptors that trigger AVP release, those that trigger thirst respond to the cell shrinkage that is caused by hyperosmolar solutions. However, these thirst osmoreceptor neurons are distinct from the adjacent AVP osmoreceptor neurons in the OVLT and SFO. Hyperosmolality triggers two parallel feedback-control mechanisms that have a common end point (Fig. 40-9): an increase in whole-body free water. In response to hyperosmolality, the AVP osmoreceptors in the hypothalamus trigger other neurons to release AVP. The result is the insertion of aquaporin 2 (AQP2) water channels in the collecting duct of the kidney, an increase in the reabsorption of water, and, therefore, a reduced excretion of free water. In response

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SECTION VI  •  The Urinary System

Increased oral intake and renal recovery of free water counteract hyperosmolality.

Arterial pressure

Osmolality Effective circulating volume

Brain Carotid sinus

Juxtaglomerular apparatus (JGA)

OVLT & SFO Thirst osmoreceptor

AVP osmoreceptor

Thirst

AVP neurons (PVN & SON)

Atrial low-pressure receptors Na+ appetite

Renin

Angiotensin II

Increased Na+ intake counteracts decreased effective circulating volume.

AVP

Kidneys

Figure 40-9  Feedback systems involved in the control of osmolality. PVN, paraventricular nucleus; SON, supraoptic nucleus of the hypothalamus.

H2O intake

H2O excretion

Free water

to hyperosmolality, the thirst osmoreceptors stimulate an appetite for water that leads to the increased intake of free water. The net effect is an increase in whole-body free water and, therefore, a reduction in osmolality.

Several nonosmotic stimuli also enhance AVP secretion Although an increase in plasma osmolality is the primary trigger for AVP release, several other stimuli increase AVP release, including a decrease in effective circulating volume or arterial pressure and pregnancy. Conversely, volume expansion diminishes AVP release. Reduced Effective Circulating Volume  As noted above on page 846, a mere 1% rise in plasma osmolality stimulates AVP release by a detectable amount. However, fairly large reductions in effective circulating volume (5% to 10%) are required to stimulate AVP release of similar amounts. Nevertheless, once the rather high threshold for nonosmotic release of AVP is exceeded, AVP release rises steeply with further volume depletion. The interaction between osmotic and volume stimuli on AVP release is illustrated in Figure 40-7, which shows that the effective circulating volume modifies the slope of the relationship between plasma AVP levels

and osmolality, as well as the osmotic threshold for AVP release. At a fixed osmolality, volume contraction (see Fig. 40-7, green curve) increases the rate of AVP release. Therefore, during volume depletion, a low plasma osmolality (e.g., 280 mOsm) that would normally suppress AVP release allows AVP secretion to continue (see Fig. 40-7, green dot). This leftward shift of the osmolality threshold for AVP release is accompanied by an increased slope, reflecting an increased sensitivity of the osmoreceptors to changes in osmolality. Figure 40-9 summarizes the three pathways by which decreased effective circulating volume and low arterial pressure enhance AVP release: (1) A reduction in left atrial pressure—produced by volume depletion—via low-pressure receptors in the left atrium decreases the firing rate of vagal afferents (see p. 547). These afferents signal brainstem neurons in the nucleus tractus solitarii, causing magnocellular neu­rons in the hypothalamus to release AVP (see Fig. 40-8). Indeed, at constant osmolality, AVP secretion varies inversely with left atrial pressure. (2) Low effective circulating volume triggers granular cells in the JGA to release renin. This leads to the formation of ANG II, which acts on receptors in the OVLT and the SFO to stimulate AVP release. (3) More importantly, a fall in the arterial pressure similarly causes high-pressure carotid sinus baroreceptors to stimulate AVP release (see pp. 534–536).

Chapter 40  •  Integration of Salt and Water Balance

Two clinical examples in which reduced effective circulating volume leads to increases in AVP are severe hemorrhagic shock and hypovolemic shock (e.g., shock resulting from excessive loss of ECF, as in cholera). In both cases, the water retention caused by AVP release accounts for the accompanying hyponatremia. In the first part of this chapter, we said that the appropriate renal response to decreased effective circulating volume is to retain Na+ (i.e., isotonic saline). Why is it that, in response to shock, the body also retains free water? Compared with isotonic saline, free water is less effective as an expander of the ECF volume (see p. 135). Nevertheless, in times of profound need, the body uses free-water retention to help expand extracellular (and plasma) volume. Clearly, the body is willing to tolerate some hypo-osmolality of the body fluids as the price for maintaining an adequate blood volume. A clinical example in which reduced effective circulating volume can lead to an inappropriate increase in AVP levels is congestive heart failure (see p. 838). In this situation, the water retention may be so severe that the patient develops hyponatremia (i.e., hypo-osmolality).

847

Volume Expansion  In contrast to volume contraction, chronic volume expansion reduces AVP secretion, as a consequence of the rightward shift of the threshold to higher osmolalities and of a decline in the slope (see Fig. 40-7, blue curve). In other words, volume expansion decreases the sensitivity of the central osmoreceptors to changes in plasma osmolality. A clinical example is hyperaldosteronism. With normal thirst and water excretion, the chronic Na+ retention resulting from the hyperaldosteronism would expand the ECF volume isotonically, thus leaving plasma [Na+] unchanged. However, because chronic volume expansion downregulates AVP release, the kidneys do not retain adequate water, which results in slight hypernatremia (i.e., elevated plasma [Na+]) and very modest hyperosmolality (Box 40-3). Pregnancy  Leftward shifts in the threshold for AVP release and thirst often occur during pregnancy. These changes probably reflect the action of chorionic gonadotropin on the sensitivity of the osmoreceptors. Pregnancy is therefore often associated with a decrease of 8 to 10 mOsm

BOX 40-3  Diuretics

D

iuretics reversibly inhibit Na+ reabsorption at specific sites along the nephron, increasing the excretion of Na+ and water, creating a state of negative Na+ balance, and thereby contracting ECF volume. Properly speaking, these agents should be called natriuretic to emphasize this use to promote Na+ excretion. This is in contrast to aquaretic agents (e.g., vasopressin receptor antagonists, or VRAs) that promote water excretion with little or no effect on Na+ excretion. Nevertheless, it has been customary to refer to natriuretics as diuretics. Clinicians use diuretics to treat hypertension as well as edema (see Box 20-1) caused by heart failure, cirrhosis of the liver, or nephrotic syndrome. Common to these latter edematous diseases is an abnormal shift of ECF away from the effective circulating volume, which thereby activates the feedback pathways. The results are Na+ retention and expansion of total extracellular volume. However, this expansion, which results in edema formation, falls short of correcting the underlying decrease in the effective circulating volume. The reason that most of this added extracellular volume remains ineffective—and does not restore the effective circulating volume—is not intuitive but reflects the underlying pathologic condition that initiated the edema in the first place. Thus, treating these edematous diseases requires generating a negative Na+ balance, which can often be achieved by rigid dietary Na+ restriction or the use of diuretics. Diuretics are also useful in treating hypertension. Even though the primary cause of the hypertension may not always be an increase in the effective circulating volume, enhanced Na+ excretion is frequently effective in lowering blood pressure.

Classification The site and mechanism of a diuretic’s action determine the magnitude and nature of the response (Table 40-3). Both chemically and functionally, diuretics are very heterogeneous. For example, acetazolamide produces diuresis by inhibiting carbonic anhydrase and thus the component of proximal-tubule Na+

reabsorption that is coupled to HCO3− reabsorption. The diuretic effect of hydrochlorothiazide is largely the result of its ability to inhibit Na/Cl cotransport in the distal convoluted tubule. Spironolactone (which resembles aldosterone) competitively inhibits mineralocorticoid receptors in principal cells of the initial and cortical collecting tubule. Mannitol (reduced fructose) is a powerful osmotic diuretic (see Box 35-1) that reduces net Na+ transport in the proximal tubule and TAL by causing retention of water in the lumen and reduction in luminal [Na+]. An ideal diuretic should promote the excretion of urine whose composition resembles that of the ECF. Such diuretics do not exist. In reality, diuretics not only inhibit the reabsorption of Na+ and its osmotically obligated water, but also interfere with the renal handling of Cl−, H+, K+, and Ca2+, as well as with urinary concentrating ability.  N40-7  Thus, many diuretics disturb the normal plasma electrolyte pattern. Table 40-4 summarizes the most frequent side effects of diuretic use on the electrolyte composition of the ECF. These electrolyte derangements are the predictable consequences of the mechanism of action of individual diuretics at specific tubule sites.

Delivery of Diuretics to Their Sites of Action Diuretics generally inhibit transporters or channels at the apical membranes of tubule cells. How do the diuretics get there? Plasma proteins bind many diuretics so that the free concentration of the diuretic in plasma water may be fairly low. Thus, glomerular filtration may deliver only a modest amount to the tubule fluid. However, organic anion or organic cation trans­ porters in the S3 segment of the proximal tubule can secrete diuretics and can thereby produce high luminal concentrations. For example, the basolateral organic anion transporter system that carries para-aminohippurate (see pp. 779–781) also secretes thiazide diuretics, furosemide, and ethacrynic acid. Organic cation transporters (see pp. 783–784) secrete amiloride. The subsequent reabsorption of fluid along the nephron further concentrates diuretics in the tubule lumen. Not surprisingly, renal Continued

Chapter 40  •  Integration of Salt and Water Balance

847.e1

N40-7  Secondary Effects of Diuretic Drugs Contributed by Erich Windhager and Gerhard Giebisch As noted in the text, the perfect diuretic—which does not exist— would produce an increase in the urinary excretion of protein-free fluid with a composition otherwise identical to that of the ECL. However, diuretics not only inhibit the reabsorption of Na+ and the osmotically obligated water, but also interfere with the renal handling of Cl−, H+, K+, and Ca2+, as well as with urinary concentrating ability. 1. Urine [Cl−]. With the exception of carbonic anhydrase inhibitors, all diuretics promote the excretion of urine having a high [Cl−]. The ratio [Cl−]/[Na+] is greater in the urine than in the plasma. 2. Urine pH. Because of its inhibition of proximal-tubule HCO3− reabsorption, acetazolamide leads to excretion of a relatively alkaline urine. Thus, acetazolamide produces a mild metabolic acidosis. In contrast, the loop diuretics and thiazides cause the excretion of a Cl−-rich, HCO3− -poor urine, which tends to induce a metabolic alkalosis. 3. Urine [K+]. Some diuretics are called K+-sparing because they tend to conserve body K+. These diuretics—which include amiloride, triamterene, and spironolactone—block

only a small fraction of Na+ reabsorption, but reduce K+ secretion through apical K+ channels by hyperpolarizing the apical cell membrane. By inhibiting passive cation movement, they may induce hyperkalemia. This hyperkalemia may lead to metabolic acidosis (see p. 835). 4. Urine [Ca2+]. With the exception of the chlorothiazides, most diuretics enhance Ca2+ excretion. They interfere with the passive reabsorption of Ca2+ through the paracellular pathway in both the proximal tubule and TAL (see p. 787). In the proximal tubule, the high luminal flow rate produced by the diuresis reduces the reabsorption of Ca2+ via solvent drag. In the TAL, loop diuretics diminish the lumen-positive potential that normally drives the passive reabsorption of Ca2+. 5. Urine osmolality. Loop diuretics diminish the urinary concentrating ability by inhibiting Na+ transport in the TAL (see p. 811). Clinical side effects of diuretic therapy are summarized in Table 40-4.

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SECTION VI  •  The Urinary System

BOX 40-3  Diuretics—cont’d disease may compromise the delivery of diuretics and cause resistance to the actions of diuretics.  N40-8

Response of Nephron Segments Downstream from a Diuretic’s Site of Action

The proximal tubule reabsorbs the largest fraction of filtered Na+; the loop of Henle, the distal convoluted tubule, and the collecting ducts retrieve smaller fractions. Thus, intuition could suggest that the proximal tubule would be the best target for diuretics. However, secondary effects in downstream nephron segments can substantially mitigate the primary effect of a diuretic. Inhibiting Na+ transport by the proximal tubule raises Na+ delivery to downstream segments and almost always stimulates Na+ reabsorption there (see p. 765). As a result of this downstream Na+ reclamation, the overall diuretic action of proximally acting diuretics (e.g., acetazolamide) is relatively weak. A diuretic is most potent if it acts downstream of the proximal tubule, a condition met by loop diuretics, which inhibit Na+ transport along the TAL. Although the TAL normally reabsorbs only 15% to 25% of the filtered load of Na+, the reabsorptive capacity of the more distal nephron segments is limited. Thus, the loop diuretics are currently the most powerful diuretic agents. Because nephron segments distal to the TAL have only modest rates of

Na+ reabsorption, diuretics that target these segments are not as potent as loop diuretics. Nevertheless, distally acting diuretics are important because their effects are long lasting. Moreover, agents acting on the connecting and collecting tubules are K+ sparing (i.e., they tend to conserve body K+). It is sometimes advantageous to use two diuretics that act at different sites along the nephron, generating a synergistic effect. Thus, if a loop diuretic alone is providing inadequate diuresis, one could complement its action by adding a thiazide, which will block the compensating effect of the distal convoluted tubule to reabsorb Na+.

Blunting of Diuretic Action with Long-Term Use The prolonged administration of a diuretic may lead to a sustained loss of body weight but only transient natriuresis.  N40-9  Most of the decline in Na+ excretion occurs because the druginduced fall in effective circulating volume triggers Na+ retention mediated by increased sympathetic outflow to the kidneys (which lowers GFR), increased secretion of ANG II and aldosterone, and decreased secretion of ANP. Hypertrophy or increased activity of tubule segments downstream of the main site of action of the diuretic can also contribute to the diminished efficacy of the drug during long-term administration.

TABLE 40-3  Action of Diuretics PHYSIOLOGICAL REGULATION OF “TARGET”

PAGE REFERENCE FOR TARGET

SITE

DRUG

FINAL MOLECULAR “TARGET”

PCT

Acetazolamide

Carbonic anhydrase

PCT

Dopamine

Na-H exchanger (NHE3)

ANG II, sympathetic nerve activity, α-adrenergic agonists

Dopamine

p. 827

TAL

Loop diuretics:   Furosemide   Bumetanide   Ethacrynic acid

Na/K/Cl cotransporter (NKCC2)

Aldosterone

PGE2

p. 757

DCT

Thiazides Metolazone

Na/Cl cotransporter (NCC)

ANG II Aldosterone

CCT

Amiloride Triamterene

Na+ channel (ENaC)

ANG II

CCT

Spironolactone

Mineralocorticoid receptor

Aldosterone

IMCD

Amiloride

cGMP-gated cation channel

Aldosterone

Water-permeable segments

Osmotic diuretics (mannitol)

STIMULATOR

INHIBITOR

pp. 828–829

p. 758 PGE2

pp. 758–759 p. 766

ANP

p. 768

CCT, cortical collecting tubule; DCT, distal convoluted tubule; IMCD, inner medullary collecting duct; PCT, proximal convoluted tubule; PGE2, prostaglandin E2.

in plasma osmolality. A similar but smaller change may also occur in the late phase of the menstrual cycle. Other Factors  Pain, nausea, and several drugs (e.g., morphine, nicotine, and high doses of barbiturates) stimulate AVP secretion. In contrast, alcohol and drugs that block the effect of morphine (opiate antagonists) inhibit AVP secretion

and thus promote diuresis. Of great clinical importance is the hypersecretion of AVP that may occur postoperatively. In addition, some malignant tumors secrete large amounts of AVP. Such secretion of inappropriate amounts of “antidiuretic hormone” leads to pathological retention of water with dilution of the plasma electrolytes, particularly Na+. If progressive and uncorrected, this condition may lead

Chapter 40  •  Integration of Salt and Water Balance

N40-8  Reduced Delivery of Diuretics in Renal Disease

848.e1

N40-9  Blunting of Diuretic Action Contributed by Erich Windhager and Gerhard Giebisch

Contributed by Erich Windhager and Gerhard Giebisch As noted in the text, diuretics cannot have their intended effects unless they have appropriate access to their protein targets in the tubule cells. The two access routes are filtration and secretion, of which secretion is usually the most important. Not surprisingly, renal disease may compromise the net secretion of diuretics in three ways. First, the capability of the diseased cells to secrete diuretics may be impaired (i.e., decreased transport). Second, renal failure leads to a buildup in the blood of organic anions that would otherwise be secreted. These organic anions may competitively inhibit the transport of diuretics by the proximal tubule (i.e., competition). Third, in renal diseases in which breakdown of the glomerular filtration barrier leads to proteinuria, albumin and other proteins not normally present in the tubule lumen bind the diuretics and greatly reduce the concentration of unbound drug (i.e., binding).

Let us assume that a patient has a fixed daily intake of Na+. As noted in the text, the administration of a diuretic will cause an initial period of increased Na+ excretion (negative Na+ balance), peaking within a few days, that leads to a loss in weight. During prolonged administration of the diuretic, urinary Na+ excretion will fall back toward normal over a period of many days, and the patient will reach a steady state (neutral Na+ balance) in which Na+ intake and excretion are equal, and in which the initial weight loss is maintained. When the drug is discontinued, the patient will experience a transient period of diminished urinary Na+ excretion, reaching a nadir after a few days. During this time he or she is in positive Na+ balance. As a result, the patient will regain the weight that was lost during the initial phase of the diuretic treatment. However, over a period of many days, the Na+ excretion eventually rises back to a normal level as the patient achieves a new steady state (neutral Na+ balance) in which Na+ intake and excretion are again equal, and the patient maintains a prediuretic weight.

Chapter 40  •  Integration of Salt and Water Balance

849

TABLE 40-4  Complications of Diuretic Therapy COMPLICATION

CAUSATIVE DIURETICS

SYMPTOMS

CAUSATIVE FACTORS

ECF volume depletion

Loop diuretics and thiazides

Lassitude, thirst, muscle cramps, hypotension

Rapid reduction of plasma volume

K+ depletion

Acetazolamide, loop diuretics, thiazides

Muscle weakness, paralysis, cardiac arrhythmias

Flow and Na+-related stimulation of distal K+ secretion

K+ retention

Amiloride, triamterene, spironolactone

Cardiac arrhythmias, muscle cramps, paralysis

Block of ENaC in the collecting duct

Hyponatremia

Thiazides, furosemide

CNS symptoms, coma

Block of Na+ transport in waterimpermeable nephron segment

Metabolic alkalosis

Loop diuretics, thiazides

Cardiac arrhythmias, CNS symptoms

Excessive Cl− excretion, secondary volume contraction

Metabolic acidosis

Acetazolamide, amiloride, triamterene

Hyperventilation, muscular and neurological disturbances

Interference with H+ secretion

Hypercalcemia

Thiazides

Abnormal tissue calcification, disturbances of nerve and muscle function

Increased Ca2+ reabsorption in distal convoluted tubule

Hyperuricemia

Thiazides, loop diuretics

Gout

Decreased ECF volume, which activates proximal fluid and uric acid reabsorption

ENaC, epithelial Na+ channel.

to life-threatening deterioration of cerebral function (see Box 38-3).

Defense of the effective circulating volume usually has priority over defense of osmolality

Decreased effective circulating volume and low arterial pressure also trigger thirst

Under physiological conditions, the body regulates plasma volume and plasma osmolality independently. However, as discussed on page 847, this clear separation of defense mechanisms against volume and osmotic challenges breaks down when more dramatic derangements of fluid or salt metabolism occur. In general, the body defends volume at the expense of osmolality. Examples include severe reductions in absolute blood volume (e.g., hemorrhage) and decreases in effective circulating volume even when absolute ECF volume may be expanded (e.g., congestive heart failure, nephrotic syndrome, and liver cirrhosis). All are conditions that strongly stimulate both Na+- and water-retaining mechanisms. However, hyponatremia can be the consequence.  N40-10

Large decreases in effective circulating volume and blood pressure not only stimulate the release of AVP, they also profoundly stimulate the sensation of thirst. In fact, hemorrhage is one of the most powerful stimuli of hypovolemic thirst: “Thirst among the wounded on the battlefield is legendary” (Fitzsimons). Therefore, three distinct stimuli— hyperosmolality, profound volume contraction, and large decreases in blood pressure—lead to the sensation of thirst. Low effective circulating volume and low blood pressure stimulate thirst centers in the hypothalamus via the same pathways by which they stimulate AVP release (see Fig. 40-9). In addition to stimulating thirst, some of these hypothalamic areas are also involved in stimulating the desire to ingest salt (i.e., Na+ appetite). We discuss the role of the hypothalamus in the control of appetite on page 1001.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 40  •  Integration of Salt and Water Balance

849.e1

N40-10  Defense of Osmolality at the Expense of Effective Circulating Volume During Dehydration Contributed by Gerhard Giebisch, Erich Windhager, Emile Boulpaep, and Walter Boron An exception to the rule of defending volume over osmolality occurs during severe water loss (i.e., dehydration; see p. 1215). In this case, the hyperosmolality that accompanies the dehydration maximally stimulates AVP secretion and thirst (see Fig. 40-9). Of course, severe dehydration also reduces total-body volume. However, this loss of free water occurs at the expense of both intracellular water (~60%) and extracellular water (~40%). Thus, dehydration does not put the effective circulating volume at as great a risk as the acute loss of an equivalent volume of blood. Because dehydration reduces effective circulating volume, one might think that the renin-angiotensin-aldosterone axis would lead to Na+ retention during dehydration. However, the opposite effect may occur, possibly because hyperosmolality makes the glomerulosa cells of the adrenal medulla less sensitive to ANG II and thereby reduces the release of aldosterone. Thus, the kidneys fail to retain Na+ appropriately. Accordingly, in severe

dehydration, the net effect is an attempt to correct hyperosmolality by both water intake and retention, as well as by the loss of Na+ (i.e., natriuresis) that occurs because aldosterone levels are inappropriately low for the effective circulating volume. Therefore, in severe dehydration, the body violates the principle of defending volume over osmolality. If the dehydration occurs during exercise, the drive to preserve effective circulating volume will trump temperature regulation (see p. 1215), offsetting the earlier vasodilation of the skin and active muscle. We can infer that the exercise-induced dehydration, by triggering thirst and AVP secretion (see previous paragraph), leads to a correction of the hyperosmolality and an increase in effective circulating volume that, once again, allows the individual to sweat and effectively regulate whole-body temperature.

849.e2

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REFERENCES

Schrier RW: Use of diuretics in heart failure and cirrhosis. Semin Nephrol 31(6):503–512, 2011.

Books and Reviews Bernstein PL, Ellison DH: Diuretics and salt transport along the nephron. Semin Nephrol 31(6):475–482, 2011. Bonny O, Rossier BC: Disturbances of Na/K balance: Pseudohypoaldosteronism revised. J Am Soc Nephrol 13:2399– 2414, 2002. Bourque CW: Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci 9(7):519–531, 2008. Epub May 29, 2008. Bourque CW, Oliet SHR: Osmoreceptors in the central nervous system. Annu Rev Physiol 59:601–619, 1997. Crowley SD, Coffman TM: In hypertension, the kidney rules. Curr Hypertens Rep 9(2):148–153, 2007. DiBona GF: Physiology in perspective: The wisdom of the body. Neural control of the kidney. Am J Physiol Regul Integr Comp Physiol 289(3):R633–R641, 2005. Fitzsimons JT: Angiotensin, thirst and sodium appetite. Physiol Rev 78:583–686, 1998. Gutkowska J, Antunes-Rodrigues J, McCann SM: Atrial natriuretic peptide in brain and pituitary gland. Physiol Rev 77:465–515, 1997. Nader PC, Thompson JR, Alpern RJ: Complications of diuretic use. Semin Nephrol 8:365–387, 1988. Navar LG, Zou L, Von Thun A, et al: Unraveling the mystery of Goldblatt hypertension. News Physiol Sci 13:170–176, 1998. Rennke HG, Denker BD: Renal Pathophysiology: The Essentials, 3rd ed. Baltimore, MD, Lippincott Williams & Wilkins, 2009. Rolls BJ, Rolls ET: Thirst. Cambridge, UK, Cambridge University Press, 1982.

Journal Articles Chou CL, Marsh DJ: Role of proximal convoluted tubule in pressure diuresis in the rat. Am J Physiol 251:F283–F289, 1986. Clark BA, Brown RS, Epstein FH: Effect of atrial natriuretic peptide on potassium-stimulated aldosterone secretion: Potential relevance to hypoaldosteronism in man. J Clin Endocrinol Metab 75:399–403, 1992. Gurley SB, Riquier-Brison AD, Schnermann J, et al: AT1A angiotensin receptors in the renal proximal tubule regulate blood pressure. Cell Metab 13(4):469–475, 2011. Iino Y, Imai M: Effects of prostaglandins on Na transport in isolated collecting tubules. Pflugers Arch 373(2):125–132, 1978. Mason WT: Supraoptic neurones of rat hypothalamus are osmosensitive. Nature 287:154–157, 1980. Oliet SHR, Bourque CW: Mechanosensitive channels transduce osmosensitivity in supraoptic neurons. Nature 364:341–343, 1993. Rabkin R, Share L, Payne PA, et al: The handling of immunoreactive vasopressin by the isolated perfused rat kidney. J Clin Invest 63:6–13, 1979. Verney EG: The antidiuretic hormone and the factors which determine its release. Proc R Soc London B Biol Sci 135:25–106, 1947. Yang LE, Maunsbach AB, Leong PKK, McDonough AA: Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes. Am J Physiol Renal Physiol 287:F896–F906, 2004.

C H A P T E R 41  ORGANIZATION OF THE GASTROINTESTINAL SYSTEM Henry J. Binder

OVERVIEW OF DIGESTIVE PROCESSES The gastrointestinal tract is a tube that is specialized along its length for the sequential processing of food The gastrointestinal (GI) tract consists of both the series of hollow organs stretching from the mouth to the anus and the several accessory glands and organs that add secretions to these hollow organs (Fig. 41-1). Each of these hollow organs, which are separated from each other at key locations by sphincters, has evolved to serve a specialized function. The mouth and oropharynx are responsible for chopping food into small pieces, lubricating it, initiating carbohydrate and fat digestion, and propelling the food into the esophagus. The esophagus acts as a conduit to the stomach. The stomach (see Chapter 42) temporarily stores food and also initiates digestion by churning and by secreting proteases and acid. The small intestine (see Chapters 44 and 45) continues the work of digestion and is the primary site for the absorption of nutrients. The large intestine (see Chapters 44 and 45) reabsorbs fluids and electrolytes and also stores the fecal matter before expulsion from the body. The accessory glands and organs include the salivary glands, pancreas, and liver. The pancreas (see Chapter 43) secretes digestive enzymes into the duodenum, in addition to secreting HCO3− to neutralize gastric acid. The liver secretes bile (see Chapter 46), which the gallbladder stores for future delivery to the duodenum during a meal. Bile contains bile acids, which play a key role in the digestion of fats. Although the anatomy of the wall of the GI tract varies along its length, certain organizational themes are common to all segments. Figure 41-2, a cross section through a generic piece of stomach or intestine, shows the characteristic layered structure of mucosa, submucosa, muscle, and serosa. The mucosa consists of the epithelial layer, as well as an underlying layer of loose connective tissue known as the lamina propria, which contains capillaries, enteric neurons, and immune cells (e.g., mast cells), as well as a thin layer of smooth muscle known as the lamina muscularis mucosae (literally, the muscle layer of the mucosa). The surface area of the epithelial layer is amplified by several mechanisms. Most cells have microvilli on their apical surfaces. In addition, the layer of epithelial cells can be evaginated to form villi or invaginated to form crypts (or glands). Finally, on a larger scale, the mucosa is organized into large folds. The submucosa consists of loose connective tissue and larger blood vessels. The submucosa may also contain glands that secrete material into the GI lumen. 852

The muscle layer, the muscularis externa, includes two layers of smooth muscle. The inner layer is circular, whereas the outer layer is longitudinal. Enteric neurons are present between these two muscle layers. The serosa is an enveloping layer of connective tissue that is covered with squamous epithelial cells.

Assimilation of dietary food substances requires digestion as well as absorption The sedentary human body requires ~30 kcal/kg body weight each day (see p. 1170). This nutrient requirement is normally acquired by the oral intake of multiple food substances that the GI tract then assimilates. Although antigenic amounts of protein enter the body via the skin and across the pulmonary epithelium, caloric uptake by routes other than the GI tract is not thought to occur. Both the small and large intestines absorb water and electrolytes, but only the small intestine absorbs lipids, carbohydrates, and amino acids. However, even without effective GI function, parenteral (i.e., intravenous) alimentation can provide sufficient calories to sustain adults and to support growth in premature infants. Total parenteral nutrition has been used successfully on a long-term basis in many clinical settings in which oral intake is impossible or undesirable. Food substances are not necessarily—and often are— consumed in a chemical form that the small intestine can directly absorb. To facilitate absorption, the GI tract digests the food by both mechanical and chemical processes. Mechanical disruption of ingested food begins in the mouth with chewing (mastication). Individuals without teeth usually require their solid food to be cut into smaller pieces before eating. The mechanical processes that alter food composition to facilitate absorption continue in the stomach (see p. 865), both to initiate protein and lipid enzymatic digestion and to allow passage of gastric contents through the pylorus into the duodenum. This change in the size and consistency of gastric contents is necessary because solids that are >2 mm in diameter do not pass through the pylorus. The chemical form in which different nutrients are ingested and absorbed varies according to the specific nutrient in question. For example, although most lipids are consumed in the form of triacylglycerols, it is fatty acids and monoacylglycerols, not triacylglycerols, that are absorbed by the small intestine. Thus, a complex series of chemical reactions (i.e., lipid digestion) are required to convert dietary

CHAPTER 41  •  Organization of the Gastrointestinal System

Parotid gland

Mouth Salivary glands

Upper esophageal sphincter (UES)

Esophagus

Lower esophageal sphincter (LES)

Liver Gallbladder Pyloric sphincter

Stomach Pancreas

Duodenum

Transverse colon

Ascending colon

Jejunum Descending colon Ileum

Haustra

Anus

Ileocecal sphincter Appendix

Internal and external anal sphincters Figure 41-1  Major components of the human digestive system.

triacylglycerols to these smaller lipid forms (see pp. 927– 928). Similarly, amino acids are present in food as proteins and large peptides, but only amino acids and small peptides— primarily dipeptides and tripeptides—are absorbed by the small intestine. Carbohydrates are present in the diet as starch, disaccharides, and monosaccharides (e.g., glucose). However, because the small intestine absorbs all carbohydrates as monosaccharides, most dietary carbohydrates require chemical digestion before their absorption.

Digestion requires enzymes secreted in the mouth, stomach, pancreas, and small intestine Digestion involves the conversion of dietary food nutrients to a form that the small intestine can absorb. For carbohydrates and lipids, these digestive processes are initiated in the mouth by salivary and lingual enzymes: amylase for carbohydrates and lipase for lipids. Protein digestion is initiated in the stomach by gastric proteases (i.e., pepsins), whereas additional lipid digestion in the stomach occurs primarily as a result of the lingual lipase that is swallowed, although some gastric lipase is also secreted. Carbohydrate digestion does not involve any secreted gastric enzymes. Digestion is completed in the small intestine by the action of both pancreatic enzymes and enzymes at the brush border of the small intestine. Pancreatic enzymes, which include

853

lipase, chymotrypsin, and amylase, are critical for the digestion of lipids, protein, and carbohydrates, respectively. The enzymes on the luminal surface of the small intestine (e.g., brush-border disaccharidases and dipeptidases) complete the digestion of carbohydrates and proteins. Digestion by these brush-border enzymes is referred to as membrane digestion. The material presented to the small intestine includes both dietary intake and secretory products. The food material entering the small intestine differs considerably from the ingested material because of the mechanical and chemical changes just discussed. The load to the small intestine is also significantly greater than that of the ingested material. Dietary fluid intake is 1.5 to 2.5 L/day, whereas the fluid load presented to the small intestine is 8 to 9 L/day. The increased volume results from substantial quantities of salivary, gastric, biliary, pancreatic, and small-intestinal secretions. These secretions contain large amounts of protein, primarily in the form of the digestive enzymes discussed above.

Ingestion of food initiates multiple endocrine, neural, and paracrine responses Digestion of food involves multiple secretory, enzymatic, and motor processes that are closely coordinated with one another. The necessary control is achieved by neural and hormonal processes that are initiated by dietary food substances; the result is a coordinated series of motor and secretory responses. For example, chemoreceptors, osmoreceptors, and mechanoreceptors in the mucosa in large part generate the afferent stimuli that induce gastric and pancreatic secretions. These receptors sense the luminal contents and initiate a neurohumoral response. Endocrine, neural, and paracrine mechanisms all contribute to digestion. All three include sensor and transmitter processes. An endocrine mechanism (see p. 47) involves the release of a transmitter (e.g., peptide) into the blood. For example, protein in the stomach stimulates the release of gastrin from antral G cells. Gastrin then enters the blood and stimulates H+ release from parietal cells in the body of the stomach. A neural mechanism involves the activation of nerves and neurotransmitters that influence either secretory or motor activity. Neural transmission of these responses may involve the enteric nervous system (ENS; see pp. 339– 340) or the central nervous system (CNS). An example of neural control is activation of the vagus nerve in response to the smell of food. The resultant release of the neurotransmitter acetylcholine (ACh) also releases H+ from parietal cells in the stomach. The third mechanism of neurohumoral control is paracrine (see p. 47). In this mechanism, a transmitter is released from a sensor cell, and it affects adjacent cells without either entering the blood or activating neurons. For example, paracrine mechanisms help regulate gastric acid secretion by parietal cells: the histamine released from so-called enterochromaffin-like (ECL) cells in the body of the stomach stimulates H+ release from neighboring parietal cells. In addition to the primary response that leads to the release of one or more digestive enzymes, other signals terminate these secretory responses. Enteric neurons are

854 A

SECTION VII  •  The Gastrointestinal System

MACROSCOPIC VIEW OF THE WALL OF THE DUODENUM Stomach Common bile duct Mesentery

Submucosa Submucosal blood vessels Lamina propria

Gland in submucosa

Serosa Intestinal villi with epithelial lining Muscularis externa

B

Outer longitudinal muscle layer Inner circular muscle layer

Muscularis mucosae

Gland in lamina propria Mucosa

MICROSCOPIC VIEW OF THE WALL OF THE COLON

Large intestine Surface absorptive cell

Crypt of ¨ Lieberkuhn

Goblet cell

Lamina propria Enteric endocrine cell

Muscularis mucosae Submucosa Circular muscle of muscularis externa Longitudinal muscle of muscularis externa

Crypt

Stem/progenitor cell

Undifferentiated crypt cell

Figure 41-2  Wall of the GI tract. A, The wall of a segment of the duodenum consists of the following structures, from inside to outside: an epithelial layer with crypts, lamina propria, muscularis mucosae, submucosa, circular and then longitudinal layer of the muscularis externa, and serosa. B, The colon has the same basic structure as the small intestine. Some of the epithelial cells are on the surface and others are in the crypts that penetrate into the wall of the colon.

CHAPTER 41  •  Organization of the Gastrointestinal System

important throughout the initiation and termination of the responses. Although the endocrine, neural, and paracrine responses are most often studied separately, with considerable effort made to isolate individual events, these responses do not occur as isolated events. Rather, each type is part of an integrated response to a meal that results in the digestion and absorption of food. This entire series of events that results from the ingestion of food can best be described as an integrated response that includes both afferent and efferent limbs.

In addition to its function in nutrition, the GI tract plays important roles in excretion, fluid and electrolyte balance, and immunity Although its primary roles are digesting and absorbing nutrients, the GI tract also excretes waste material. Fecal material includes nondigested/nonabsorbed dietary food products, colonic bacteria and their metabolic products, and several excretory products. These excretory products include (1) heavy metals such as iron and copper, whose major route of excretion is in bile; and (2) several organic anions and cations, including drugs, that are excreted in bile but are reabsorbed either poorly or not at all by either the small or large intestine. As noted above, the small intestine is presented with 8 to 9 L/day of fluid, an amount that includes ~1 L/day that the intestine itself secretes. Almost all this water is reabsorbed in the small and large intestine; therefore, stool has relatively small amounts of water (~0.1 L/day). Diarrhea (an increase in stool liquidity and weight, >200 g/day) results from either increased fluid secretion by the small or large intestine, or decreased fluid reabsorption by the intestines. An important clinical example of diarrhea is cholera, especially in developing countries. Cholera can be fatal because of the water and electrolyte imbalance that it creates. Thus, the GI tract plays a crucial role in maintaining overall fluid and electrolyte balance (see Chapter 44). The GI tract also contributes to immune function. The mucosal immune system, or gut-associated lymphoid tissue (GALT), consists of both organized aggregates of lymphoid tissue (e.g., Peyer’s patches; see Fig. 41-2B) and diffuse populations of immune cells. These immune cells include lymphocytes that reside between the epithelial cells lining the gut, as well as lymphocytes and mast cells in the lamina propria. GALT has two primary functions: (1) to protect against potential microbial pathogens, including bacteria, protozoans, and viruses; and (2) to permit immunological tolerance to both the potentially immunogenic dietary substances and the bacteria that normally reside primarily in the lumen of the large intestine. The mucosal immune system is important because the GI tract has the largest area of the body in potential direct contact with infectious, toxic, and immunogenic material. Approximately 80% of the immunoglobulin-producing cells are found in the small intestine. Although GALT has some interaction with the systemic immune system, GALT is operationally distinct. Finally, evidence indicates communication between the GALT and mucosal immune systems at other mucosal surfaces, such as the pulmonary epithelia.

855

Certain nonimmunological defense processes are also important in protecting against potential luminal pathogens and in limiting the uptake of macromolecules from the GI tract. The nonimmunological mechanisms that are critical in maintaining the ecology of intestinal flora include gastric acid secretion, intestinal mucin, peristalsis, and the epithelial-cell permeability barrier. Thus, whereas relatively low levels of aerobic bacteria are present in the lumen of the small intestine of physiologically normal subjects, individuals with impaired small-intestinal peristalsis often have substantially higher levels of both aerobic and anaerobic bacteria in their small intestine. A consequence may be diarrhea or steatorrhea (i.e., increased fecal fat excretion). The clinical manifestation of impaired intestinal peristalsis is referred to as either blind loop syndrome or stagnant bowel syndrome.

REGULATION OF GASTROINTESTINAL FUNCTION The ENS is a “minibrain” with sensory neurons, interneurons, and motor neurons The ENS (see pp. 339–340) is the primary neural mechanism that controls GI function and is one of the three divisions of the autonomic nervous system (ANS), along with the sympathetic and parasympathetic divisions. One indication of the importance of the ENS is the number of neurons consigned to it. The ENS consists of ~100 million neurons, roughly the number in the spinal cord or in the rest of the entire ANS. The ENS is located solely within GI tissue, but it can be modified by input from the brain. Neurons of the ENS are primarily, but not exclusively, clustered in one of two collections of neurons (Fig. 41-3A): the submucosal plexus and the myenteric plexus. The submucosal (or Meissner’s) plexus is found in the submucosa only in the small and large intestine. The myenteric (or Auerbach’s) plexus is located between the circular and longitudinal muscle layers throughout the GI tract from the proximal end of the esophagus to the rectum. The ENS is a complete reflex circuit and can operate totally within the GI tract, without the participation of either the spinal cord or the cephalic brain. As with other neurons, the activity of the ENS is the result of the generation of action potentials by single neurons and the release of chemical neurotransmitters that affect either other neurons or effector cells (i.e., epithelial or muscle cells). The ENS consists of sensory circuits, interneuronal connections, and secretomotor neurons (see Fig. 41-3B). Sensory (or afferent) neurons monitor changes in luminal activity, including distention (i.e., smooth-muscle tension), chemistry (e.g., pH, osmolality, levels of specific nutrients), and mechanical stimulation. These sensory neurons activate interneurons, which relay signals that activate efferent secretomotor neurons that in turn stimulate or inhibit a wide range of effector cells: smooth-muscle cells, epithelial cells that secrete or absorb fluid and electrolytes, submucosal blood vessels, and enteric endocrine cells. The largely independent function of the ENS has given rise to the concept of a GI “minibrain.” Because the efferent responses to several different stimuli are often quite similar,

856 A

SECTION VII  •  The Gastrointestinal System

B

LOCATION OF THE ENS

Longitudinal muscle of muscularis externa

Paravascular nerve

CONNECTIONS OF ENS NEURONS Longitudinal muscle

Perivascular nerve

Circular muscle

SENSORY

Myenteric (Auerbach’s) plexus

Blood vessels

Sensory

Tertiary plexus

Muscularis mucosae

Endocrine cells

PARASYMPATHETIC Motor

Circular muscle of muscularis externa

Vagus nerve

Mechanoreceptors Motor

Deep muscular plexus

Motor

Submucosal (Meissner’s) plexus Submucosal artery

Chemoreceptors

Pelvic nerve Muscularis mucosae

Mucosal plexus

Secretory cells

SYMPATHETIC

Mucosa

Motor

Motor

Brainstem Sympathetic or spinal cord ganglia

Myenteric plexus

Submucosal plexus

Mucosa

Figure 41-3  Schematic representation of the ENS. A, The submucosal (or Meissner’s) plexus is located between the muscularis mucosae and the circular muscle of the muscularis externa. The myenteric (or Auerbach’s) plexus is located between the circular and longitudinal layers of the muscularis externa. In addition to these two plexuses that have ganglia, three others—the mucosal, deep muscular, and tertiary plexuses—are present. B, The ENS consists of sensory neurons, interneurons, and motor neurons. Some sensory signals travel centrally from the ENS. Both the parasympathetic and the sympathetic divisions of the ANS modulate the ENS. This figure illustrates some of the typical circuitry of ENS neurons.

a generalized concept has developed that the ENS possesses multiple preprogrammed responses. For example, both mechanical distention of the jejunum and the presence of a bacterial enterotoxin in the jejunum can elicit identical responses: stimulation of profuse fluid and electrolyte secretion, together with propagated, propulsive, coordinated smooth-muscle contractions. Such preprogrammed efferent responses are probably initiated by sensory input to the enteric interneuronal connections. However, efferent responses controlled by the ENS may also be modified by input from autonomic ganglia, which are in turn under the influence of the spinal cord and brain (see p. 336).  N41-1  In addition, the ENS receives input directly from the brain via parasympathetic nerves (i.e., the vagus nerve).

ACh, peptides, and bioactive amines are the ENS neurotransmitters that regulate epithelial and motor function ACh is the primary preganglionic and postganglionic neurotransmitter regulating both secretory function and smooth-muscle activity in the GI tract. In addition, many other neurotransmitters are present in enteric neurons. Among the peptides, vasoactive intestinal peptide (VIP)

has an important role in both inhibition of intestinal smooth muscle and stimulation of intestinal fluid and electrolyte secretion. Although VIP was first identified in the GI tract, it is now appreciated that VIP is also an important neurotransmitter in the brain (see Table 13-1). Also playing an important role in GI regulation are other peptides (e.g., enkephalins, somatostatin, and substance P), amines (e.g., serotonin), and nitric oxide (NO). Our understanding of ENS neurotransmitters is evolving, and the list of identified agonists grows ever longer. In addition, substantial species differences exist. Frequently, chemical neurotransmitters are identified in neurons without a clear-cut demonstration of their physiological role in the regulation of organ function. More than one neurotransmitter has been identified within single neurons, a finding suggesting that regulation of some cell functions may require more than one neurotransmitter.

The brain-gut axis is a bidirectional system that controls GI function via the ANS, GI hormones, and the immune system Well recognized, but poorly understood, is the modification of several different aspects of GI function by the brain. In

CHAPTER 41  •  Organization of the Gastrointestinal System

N41-1  Hierarchical Reflex Loops in the ANS Contributed by George Richerson 5 Higher CNS centers (hypothalamus)

Descending

Ascending 4 Brainstem Descending Ascending Afferent ascending

3 Spinal cord

Preganglionic 2 Autonomic ganglion

Postganglionic

End organ (colon)

1 ENS

eFigure 41-1  At the lowest level, the ENS is an independent system consisting of afferent neurons, interneurons, and motor neurons. One level up, the autonomic ganglia control the autonomic end organs, including the ENS. One further level up, the spinal cord controls certain autonomic ganglia and integrates response among different levels of the spinal cord. The brainstem receives inputs from visceral afferents and coordinates the control of all viscera. Finally, forebrain CNS centers receive input from the brainstem and coordinate the activity of the ANS via input to the brainstem.

856.e1

CHAPTER 41  •  Organization of the Gastrointestinal System

other words, neural control of the GI tract is a function of not only intrinsic nerves (i.e., the ENS) but also nerves that are extrinsic to the GI tract. These extrinsic pathways are composed of elements of both the parasympathetic and, to a lesser extent, the sympathetic nervous system and are under the control of autonomic centers in the brainstem (see p. 338). Parasympathetic innervation of the GI tract from the pharynx to the distal colon is through the vagus nerve; the distal third of the colon receives its parasympathetic innervation from the pelvic nerves (see Fig. 14-4). The preganglionic fibers of the parasympathetic nerves use ACh as their neurotransmitter and synapse on some neurons of the ENS (see Fig. 41-3B). These ENS neurons are thus postganglionic parasympathetic fibers, and their cell bodies are, in a sense, the parasympathetic ganglion. These postganglionic parasympathetic fibers use mainly ACh as their neurotransmitter; however, as noted in the previous section, many other neurotransmitters are also present. Parasympathetic stimulation—after one or more synapses in a very complex ENS network—increases secretion and motility. The parasympathetic nerves also contain afferent fibers (see p. 339) that carry information to autonomic centers in the medulla from chemoreceptors, osmoreceptors, and mechanoreceptors in the mucosa. The loop that is initiated by these afferents, integrated by central autonomic centers, and completed

TABLE 41-1  GI Peptide Hormones 

857

by the aforementioned parasympathetic efferents, is known as a vagovagal reflex. The preganglionic sympathetic fibers to the GI tract synapse on postganglionic neurons in the prevertebral ganglia (see Fig. 14-3); the neurotransmitter at this synapse is ACh (see p. 341). The postganglionic sympathetic fibers either synapse in the ENS or directly innervate effector cells (see Fig. 41-3B). In addition to the control that is entirely within the ENS, as well as control via autonomic centers in the medulla, the GI tract is also under the control of higher CNS centers. Examples of cerebral function that affects GI behavior include the fight-or-flight response, which reduces blood flow to the GI tract, and the sight and smell of food, which increase gastric acid secretion. Communication between the GI tract and higher CNS centers is bidirectional. For example, cholecystokinin from the GI tract mediates, in part, the development of food satiety in the brain. In addition, gastrin-releasing peptide, a neurotransmitter made in ENS cells (see p. 868), inhibits gastric acid secretion when experimentally injected into the ventricles of the brain. Table 41-1 summarizes peptide hormones made by the GI tract as well as their major actions. In addition to the “hard-wired” communications involved in sensory input and motor output, communication via the

N41-2

HORMONE

SOURCE

TARGET

ACTION

Cholecystokinin

I cells in duodenum and jejunum and neurons in ileum and colon

Pancreas Gall bladder

↑ Enzyme secretion ↑ Contraction

Gastric inhibitory peptide

K cells in duodenum and jejunum

Pancreas

Exocrine: ↓ fluid absorption Endocrine: ↑ insulin release

Gastrin

G cells, antrum of stomach

Parietal cells in body of stomach

↑ H+ secretion

Gastrin-releasing peptide

Vagal nerve endings

G cells in antrum of stomach

↑ Gastrin release

Guanylin

Ileum and colon

Small and large intestine

↑ Fluid absorption

Motilin

Endocrine cells in upper GI tract

Esophageal sphincter Stomach Duodenum

↑ Smooth-muscle contraction

Neurotensin

Endocrine cells, widespread in GI tract

Intestinal smooth muscle

Vasoactive stimulation of histamine release

Peptide YY

Endocrine cells in ileum and colon

Stomach Pancreas

↓ Vagally mediated acid secretion ↓ Enzyme and fluid secretion

Secretin

S cells in small intestine

Pancreas

↑ HCO3− and fluid secretion by pancreatic ducts ↓ Gastric acid secretion

Somatostatin

D cells of stomach and duodenum, δ cells of pancreatic islets

Stomach Intestine

Stomach

Pancreas Liver

↓ ↑ ↑ ↓ ↓

Gastrin release Fluid absorption/↓ secretion Smooth-muscle contraction Endocrine/exocrine secretions Bile flow

Substance P

Enteric neurons

Enteric neurons

Neurotransmitter

VIP

ENS neurons

Small intestine

↑ Smooth-muscle relaxation ↑ Secretion by small intestine ↑ Secretion by pancreas

Pancreas

CHAPTER 41  •  Organization of the Gastrointestinal System

N41-2  GI Peptide Hormones Contributed by Emile Boulpaep and Walter Boron The amino-acid sequences of several of the peptide hormones listed in Table 41-1 are presented elsewhere in the text or below: • Cholecystokinin (CCK): The amino-acid sequence is presented in Figure 42-7C. • Cholecystokinin-like peptide (CCK-8): The amino-acid sequence is presented in Figure 13-9. This is one of several cleavage products of CCK. • Gastric inhibitory peptide: See Table 41-1. A peptide consisting of 42 amino acids. The single-letter code for these amino acids is YAEGTFISD YSIAMDKIHQ QDFVNWLLAQ KGKKNDWKHN ITQ. • Gastrin (“little” and “big”): The amino-acid sequences are presented in Figure 42-7. • Gastrin-releasing peptide (GRP): The amino-acid sequence is presented in Figure 13-9. • Guanylin (guanylyl cyclase activator 2A): A peptide consisting of 15 amino acids. The single-letter code for these amino acids is PGTCEICAYA ACTGC. • Neurotensin: The amino-acid sequence is presented in Figure 13-9. • Peptide YY: Peptide YY (also known as PYY-I) consists of 36 amino acids. The single-letter code for these amino acids is YP IKPEAPGEDA SPEELNRYYA SLRHYLNLVT RQRY. Notice that the sequence starts and ends with a Y (i.e., tyrosine). PYY-II lacks the first two residues of PYY-I (i.e., YP) and thus is only 34 residues in length (see p. 1005). • Secretin: This peptide (see p. 876) consists of 27 amino acids: HSD GTFTSELSRL REGARLQRLL QGLV. • Somatostatin: The amino-acid sequence is presented in Figure 13-9. • Substance P: The amino-acid sequence is presented in Figure 13-9. • Vasoactive intestinal peptide (VIP): The amino-acid sequence is presented in Figure 13-9.

857.e1

858

SECTION VII  •  The Gastrointestinal System

gut-brain axis also requires significant participation of the immune system. Neuroimmune regulation of both epithelial and motor function in the small and large intestine primarily involves mast cells in the lamina propria of the intestine. Because the mast cells are sensitive to neurotransmitters, they can process information from the brain to the ENS and can also respond to signals from interneurons of the ENS. Mast cells also monitor sensory input from the intestinal lumen by participating in the immune response to foreign antigens. In turn, chemical mediators released by mast cells (e.g., histamine) directly affect both intestinal smooth-muscle cells and epithelial cells. Our understanding of how the immune system modulates the neural control of GI function is rapidly evolving. In conclusion, three parallel components of the gut-brain axis—the ENS, GI hormones, and the immune system— control GI function, an arrangement that provides substantial redundancy. Such redundancy permits fine-tuning of the regulation of digestive processes and provides “backup” or “fail-safe” mechanisms that ensure the integrity of GI function, especially at times of impaired function (i.e., during disease).

GASTROINTESTINAL MOTILITY Tonic and rhythmic contractions of smooth muscle are responsible for churning, peristalsis, and reservoir action The motor activity of the GI tract performs three primary functions. First, it produces segmental contractions that are associated with nonpropulsive movement of the luminal contents. The result is the increased mixing—or churning—that enhances the digestion and absorption of dietary nutrients. Second, GI motor activity produces peristalsis, a progressive wave of relaxation followed by contraction. The result is propulsion, or the propagated movement of food and its digestive products in a caudal direction, ultimately eliminating nondigested, nonabsorbed material. Third, motor activity allows some hollow organs— particularly the stomach and large intestine—to hold the luminal content, exerting a reservoir function. This reservoir function is made possible by sphincters that separate the organs of the GI tract. All these functions are primarily accomplished by the coordinated activity of smooth muscle (see pp. 243–249). The electrical and mechanical properties of intestinal smooth muscle needed for these functions include both tonic (i.e., sustained) contractions and rhythmic contractions (i.e., alternating contraction and relaxation) of individual muscle cells. The intrinsic rhythmic contractility is a function of the membrane voltage (Vm) of the smoothmuscle cell. Vm can either oscillate in a subthreshold range at a low frequency (several cycles per minute), referred to as slow-wave activity, or reach a threshold for initiating a true action potential (see Fig. 9-14). The integrated effect of the slow waves and action potentials determines the smoothmuscle activity of the GI tract. Slow-wave activity apparently occurs as voltage-gated Ca2+ channels depolarize the cell and increase [Ca2+]i, followed by the opening of Ca2+-activated K+ channels, which repolarize the cell (see p. 244).

These activities are regulated, in large part, by both neural and hormonal stimuli. Modulation of intestinal smoothmuscle contraction is largely a function of [Ca2+]i (see pp. 246–247). Several agonists regulate [Ca2+]i by one of two mechanisms: (1) activating G protein–linked receptors, which results in the formation of inositol 1,4,5-trisphosphate (IP3) and the release of Ca2+ from intracellular stores; or (2) opening and closing of plasma-membrane Ca2+ channels. Both excitatory and inhibitory neurotransmitters can modulate smooth-muscle [Ca2+]i and thus contractility. In general, ACh is the predominant neurotransmitter of excitatory motor neurons, whereas VIP and NO are the neurotransmitters of inhibitory motor neurons. Different neural or hormonal inputs probably increase (or decrease) the frequency with which Vm exceeds threshold and produces an action potential and thus increases (or decreases) muscle contractility. An additional, unique factor in the aforementioned regulatory control is that luminal food and digestive products activate mucosal chemoreceptors and mechanoreceptors, as discussed above, thus inducing hormone release or stimulating the ENS and controlling smooth-muscle function. For example, gastric contents with elevated osmolality or a high lipid content entering the duodenum activate mucosal osmoreceptors and chemoreceptors that increase the release of cholecystokinin and thus delay gastric emptying (see p. 878).

Segments of the GI tract have both longitudinal and circular arrays of muscles and are separated by sphincters that consist of specialized circular muscles The muscle layers of the GI tract consist almost entirely of smooth muscle. Exceptions are the striated muscle of (1) the upper esophageal sphincter (UES), which separates the hypopharynx from the esophagus; (2) the upper third of the esophagus; and (3) the external anal sphincter. As shown above in Figure 41-2, the two smooth-muscle layers are arranged as an inner circular layer and an outer longitudinal layer. The myenteric ganglia of the ENS are located between the two muscle layers. The segments of the GI tract through which food products pass are hollow, low-pressure organs that are separated by specialized circular muscles or sphincters. These sphincters function as barriers to flow by maintaining a positive resting pressure that serves to separate the two adjacent organs, in which lower pressures prevail. Sphincters thus regulate both antegrade (forward) and retrograde (reverse) movement. For example, the resting pressure of the pyloric sphincter controls, in part, the emptying of gastric contents into the duodenum. On the opposite end of the stomach, the resting pressure of the lower esophageal sphincter (LES) prevents gastric contents from refluxing back into the esophagus and causing gastroesophageal reflux disease (GERD). As a general rule, stimuli proximal to a sphincter cause sphincteric relaxation, whereas stimuli distal to a sphincter induce sphincteric contraction. Changes in sphincter pressure are coordinated with the smooth-muscle contractions in the organs on either side. This coordination depends on both the intrinsic properties of sphincteric smooth muscle and neurohumoral stimuli. Sphincters effectively serve as one-way valves. Thus, the act of deglutition (or swallowing) induces relaxation of the

CHAPTER 41  •  Organization of the Gastrointestinal System

UES, whereas the LES remains contracted. Only when the UES returns to its initial pressure does the LES begin to relax, ~3 seconds after the start of deglutition. Disturbances in sphincter activity are often associated with alterations in one or more of these regulatory processes.

Dry swallow At rest After swallowing

UES

100 mm Hg 0

1

100 mm Hg 0

Location of a sphincter determines its function Six sphincters are present in the GI tract (see Fig. 41-1), each with a different resting pressure and different response to various stimuli. An additional sphincter, the sphincter of Oddi, regulates movement of the contents of the common bile duct into the duodenum. Upper Esophageal Sphincter  Separating the pharynx and the upper part of the esophagus is the UES, which consists of striated muscle and has the highest resting pressure of all the GI sphincters. The swallowing mechanism, which involves the oropharynx and the UES, is largely under the control of the swallowing center in the medulla via cranial nerves V (trigeminal), IX (glossopharyngeal), X (vagus), and XII (hypoglossal). Respiration and deglutition are closely integrated (see p. 720). The UES is closed during inspiration, thereby diverting atmospheric air to the glottis and away from the esophagus. During swallowing, the situation reverses, with closure of the glottis and inhibition of respiration, but with relaxation of the UES (Fig. 41-4). These changes permit the entry of food contents into the esophagus and not into the airways of the respiratory tract. Lower Esophageal Sphincter  The esophagus is separated from the stomach by the LES, which is composed of specialized smooth muscle that is both anatomically and physiologically distinct from adjacent smooth muscle in the distal end of the esophagus and proximal portion of the stomach. The primary functions of the LES are (1) to permit coordinated movement of ingested food into the stomach from the esophagus after swallowing or deglutition, and (2) to prevent reflux of gastric contents into the esophagus. Either deglutition or distention of the esophagus results in a reduction in LES pressure (see Fig. 41-4), thereby permitting entry of food into the stomach. Relaxation of the LES occurs after the UES has already returned to its resting pressure. The LES maintains a resting tone that is the result of both intrinsic myogenic properties of the sphincteric muscle and cholinergic regulation. Relaxation of the LES is mediated both by the vagus nerve and by intrinsic properties of the smooth muscle, including important inhibitory effects by VIP and by NO. Abnormalities of both resting LES pressure and its relaxation in response to deglutition are often associated with significant symptoms. Thus, a reduced resting LES pressure often results in gastroesophageal reflux, which may cause esophagitis (i.e., inflammation of the esophageal mucosa). A defect in LES relaxation is a major component of a condition called achalasia (Box 41-1), which often results in dilation of the esophagus (megaesophagus) and is associated with difficulty in swallowing (dysphagia). Swallowing and the function of the UES and LES are closely integrated into the function of the esophagus. Under normal circumstances, esophageal muscle contractions are

859

2

100 mm Hg 0 3

100 mm Hg 0

4 Diaphragm LES

6

100 mm Hg 0

5

100 mm Hg 0 0 5s

Figure 41-4  Esophageal pressures during swallowing. The swallowing center in the medulla that initiates deglutition includes the nucleus ambiguus (cranial nerves [CN] IX and X), the dorsal motor nucleus of the vagus (CN X), and others. Shown are recordings of intraluminal pressures at different sites along the esophagus, from the UES (record 1) to the LES (record 6). The left side of the graph shows the pressures at rest. As shown on the right side, after a dry swallow, the pressure wave of a “primary peristalsis” moves sequentially down the esophagus. (Data from Conklin JL, Christensen J: Motor functions of the pharynx and esophagus. In Johnson LR [ed]: Physiology of the Gastrointestinal Tract, 3rd ed. New York, Lippincott-Raven, 1994, pp 903–928.)

BOX 41-1  Achalasia

A

chalasia is a relatively uncommon condition associated with difficulty swallowing (dysphagia) and a dilated esophagus proximal to a narrowed, tapered area at the gastroesophageal junction. The term achalasia is derived from Greek words meaning “absence of relaxation.” The distal narrowed area of the esophagus suggests the presence of a stricture. However, it is easy to introduce an esophagoscope into the stomach through the narrowed area. Subsequent studies of esophageal motility in which investigators measured intraesophageal pressure demonstrated the presence of two defects in patients with achalasia: (1) failure of the LES to relax, and (2) impaired peristalsis in the distal two thirds of the body of the esophagus (i.e., the portion that consists of smooth muscle). Peristalsis is intact in the proximal third of the esophagus, which consists of striated muscle. In essence, the smooth-muscle portions of the esophagus behave as a denervated structure. The fundamental defect in achalasia is likely related to selective loss of intramural inhibitory neurons that regulate the LES, the neurotransmitters for which are VIP and NO. Treatment is either physical distention (or stretching) of the LES with a pneumatic-bag dilator or surgical cutting of the LES (i.e., an esophageal Heller myotomy via a laparoscopic approach).

860

SECTION VII  •  The Gastrointestinal System

almost exclusively peristaltic and are initiated by swallowing. Deglutition initiates relaxation of the UES and propagated contractions, first of the UES and then of the muscles along the esophagus (see Fig. 41-4). In the meantime, the LES has already relaxed. The result of the advancing peristaltic wave is the caudad propulsion of a bolus toward the stomach. Distention of the esophagus (in the absence of swallowing) also initiates propulsive esophageal contractions distal to the site of distention, as well as relaxation of the LES. Reflux of gastric contents into the lower part of the esophagus also produces such a local distention, without a swallow, and elicits the same response: peristaltic contractions that clear the esophagus of refluxed gastric material. Peristalsis that is initiated by swallowing is called primary peristalsis, whereas that elicited by distention of the esophagus is referred to as secondary peristalsis. Esophageal contractions after a swallow are regulated by the medullary swallowing center, intramural esophageal plexuses, the vagus nerve, and intrinsic myogenic processes. Pyloric Sphincter  The pylorus is the sphincter that sepa-

rates the stomach from the duodenum. The pressure of the pyloric sphincter regulates, in part, gastric emptying and prevents duodenal-gastric reflux. However, although a specific pyloric sphincter is present, it is quite short and is a relatively poor barrier (i.e., it can resist only a small pressure gradient). The stomach, duodenum, biliary tract, and pancreas—which are closely related embryologically— function as an integrated unit. Indeed, coordinated contraction and relaxation of the antrum, pylorus, and duodenum (which is sometimes referred to as the antroduodenal cluster unit) are probably more important than simply the pressure produced by the pyloric smooth muscle per se. Regulation of gastric emptying is discussed further on pp. 877–878. Ileocecal Sphincter  The valve-like structure that separates the ileum and cecum is called the ileocecal sphincter. Similar to other GI sphincters, the ileocecal sphincter maintains a positive resting pressure and is under the control of the vagus nerve, sympathetic nerves, and the ENS. Distention of the ileum results in relaxation of the sphincter, whereas distention of the proximal (ascending) colon causes contraction of the ileocecal sphincter. As a consequence, ileal flow into the colon is regulated by luminal contents and pressure, both proximal and distal to the ileocecal sphincter. Internal and External Anal Sphincters  The “anal sphincter” actually consists of both an internal and an external sphincter. The internal sphincter has both circular and longitudinal smooth muscle and is under involuntary control. The external sphincter, which encircles the rectum, contains only striated muscle but is controlled by both voluntary and involuntary mechanisms. The high resting pressure of the overall anal sphincter predominantly reflects the resting tone of the internal anal sphincter. Distention of the rectum (Fig. 41-5A), either by colonic contents (i.e., stool) or experimentally by balloon inflation, initiates the rectosphincteric reflex by relaxing the internal sphincter (see Fig. 41-5B). If defecation is not desired, continence is maintained by an involuntary reflex—orchestrated by the sacral spinal cord— that contracts the external anal sphincter (see Fig. 41-5C). If

A

If passive distention of the rectum is sufficiently large…

RECTUM

Rectal distention Active

40 30

Passive

Change in 20 pressure (mm Hg) 10

…it triggers an active contraction of the rectal smooth muscles.

0 Time B

INTERNAL ANAL SPHINCTER Passive rectal distention also triggers relaxation of the smooth muscle of the internal anal sphincter (rectosphincteric reflex).

Change in 0 pressure (mm Hg) –10 –20 Time C

EXTERNAL ANAL SPHINCTER

30

If defecation is not desired, the skeletal muscle of the external anal sphincter contracts by an involuntary reflex.

15 Change in pressure 10 (mm Hg) 5 0 Time Figure 41-5  Pressure changes initiated by rectal distention. (Data from Schuster MM: Simultaneous manometric recording of internal and external anal sphincteric reflexes. Johns Hopkins Med J 116:70–88, 1965.)

defecation is desired, a series of both voluntary and involuntary events occurs that includes relaxation of the external anal sphincter, contraction of abdominal wall muscles, and relaxation of pelvic wall muscles. Flexure of the hips and descent of the pelvic floor then facilitate defecation by minimizing the angle between the rectum and anus. In contrast, if a delay in defecation is needed or desired, voluntary contraction of the external anal sphincter is usually sufficient to override the series of reflexes initiated by rectal distention.

Motility of the small intestine achieves both churning and propulsive movement, and its temporal pattern differs in the fed and fasted states Digestion and absorption of dietary nutrients are the primary functions of the small intestine, and the motor activity of the small intestine is closely integrated with its digestive

CHAPTER 41  •  Organization of the Gastrointestinal System

861

Beginning of jejunum

Distance (cm) from duodenum

Duodenum

CONTRACTILE ACTIVITY Feeding Migrating motor complex

0 20 70 120 170 220 270 300

Fasting 0

1

2

3 4 Time (hr)

5

6

7

8

Figure 41-6  Mechanical activity in the fasting and fed states. Shown are records of intraluminal pressure along the small intestine of a conscious dog. Before feeding (left side), the pattern is one of MMCs. Feeding triggers a switch to a different pattern, characterized by both segmental contractions that churn the contents and peristaltic contractions that propel the contents along the small intestine. (Data from Itoh Z, Sekiguchi T: Interdigestive motor activity in health and disease. Scand J Gastroenterol Suppl 82:121–134, 1983.)

and absorptive roles. The two classes of small-intestinal motor activity are churning (or mixing) and propulsion of the bolus of luminal contents. Churning—which is accomplished by segmental, nonpropulsive contractions—mixes the luminal contents with pancreatic, biliary, and smallintestinal secretions, thus enhancing the digestion of dietary nutrients in the lumen. These segmental contractions also decrease the unstirred water layer that is adjacent to the apical membranes of the small-intestine cells, thus promoting absorption. Churning or mixing movements occur following eating and are the result of contractions of circular muscle in segments flanked at either end by receiving segments that relax. Churning, however, does not advance the luminal contents along the small intestine. In contrast, propulsion—which is accomplished by propagated, peri­ staltic contractions—results in caudad movement of the intestinal luminal contents, either for absorption at more distal sites of the small or large intestine or for elimination in stool. Peristaltic propulsion occurs as a result of contraction of the circular muscle and relaxation of the longitudinal muscle in the propulsive or upstream segment, together with relaxation of the circular muscle and contraction of the longitudinal muscle in the downstream receiving segment. Thus, circular smooth muscle in the small intestine participates in both churning and propulsion. The Vm changes of intestinal smooth-muscle cells consist of both action potentials (see p. 244) and slow-wave activity (see p. 244). The patterns of electrical and mechanical activ-

ity differ in the fasting and fed states. In the fasting state, the small intestine is relatively quiescent but exhibits synchronized, rhythmic changes in both electrical and motor activity (Fig. 41-6). The interdigestive myoelectric or migrating motor complex (MMC) is the term used to describe these rhythmic contractions of the small intestine that are observed in the fasting state. MMCs in humans occur at intervals of 90 to 120 minutes and consist of four distinct phases: (1) a prolonged quiescent period, (2) a period of increasing action potential frequency and contractility, (3) a period of peak electrical and mechanical activity that lasts a few minutes, and (4) a period of declining activity that merges into the next quiescent period. During the interdigestive period, particles >2 mm in diameter can pass from the stomach into the duodenum, which permits emptying of ingested material from the stomach (e.g., bones, coins) that could not be reduced in size to 0.2 L/24 hr). Diarrhea has many causes and can be classified in various ways. One classification divides diarrheas by the causative factor. The causative factor can be failure to absorb a dietary nutrient, in which case the result is an osmotic diarrhea. An example of osmotic diarrhea is that caused by primary lactase deficiency. Alternatively, the causative factor may not be lack of absorption of a dietary nutrient, but rather endogenous secretion of fluid and electrolytes from the intestine, in which case the result is secretory diarrhea. The leading causes of secretory diarrhea include infections with E. coli (the major cause of traveler’s diarrhea) and cholera (a cause of substantial morbidity and mortality in developing countries). In these and other infectious diarrheas, an enterotoxin produced by one of many bacterial organisms raises intracellular concentrations of cAMP, cGMP, or Ca2+ (see Table 44-2). A second group of secretory diarrheas includes those produced by different, relatively uncommon hormone-producing tumors. Examples of such tumors include those that produce VIP (Verner-Morrison syndrome), glucagon (glucagonomas), and serotonin (carcinoid syndrome). These secretagogues act by raising either [cAMP]i or [Ca2+]i (see Table 44-2). When tumors produce these secretagogues in abundance, the resulting diarrhea can be copious and explosive. As we have seen, the secretory diarrheas have in common their ability to increase [cAMP]i, [cGMP]i, or [Ca2+]i. Table 44-4 summarizes the mechanisms by which these second messengers produce the secretory diarrhea. Because the second messengers do not alter the function of nutrient-coupled Na+ absorption, administration of an oral rehydration solution containing glucose and Na+ is effective in the treatment of enterotoxin-mediated diarrhea (see Box 44-1).

response to the same protein kinases that increase Cl− conductance. The net result of all these changes is the initiation of active Cl− secretion across the epithelial cell. The induction of apical membrane Cl− channels is extremely important in the pathophysiology of many diarrheal disorders. Box 44-3 discusses the changes in ion transport that occur in secretory diarrheas such as that associated with cholera. A central role in cystic fibrosis has been posited for the CFTR Cl− channel in the apical membrane (see p. 122). However, other Cl− channels, including the Ca2+-activated CaCC (see Table 6-2, family No. 17) are likely present in the intestine and may contribute to active Cl− secretion.

CELLULAR MECHANISMS OF K+ ABSORPTION AND SECRETION Overall net transepithelial K+ movement is absorptive in the small intestine and secretory in the colon The gastrointestinal tract participates in overall K+ balance, although compared with the kidneys, the small intestine and

large intestine play relatively modest roles, especially in healthy individuals. The pattern of intestinal K+ movement parallels that of the kidney: (1) the intestines have the capacity for both K+ absorption and secretion, and (2) the intestines absorb K+ in the proximal segments but secrete it in the distal segments. Dietary K+ furnishes 80 to 120 mmol/day, whereas stool K+ output is only ~10 mmol/day. The kidney is responsible for disposal of the remainder of the daily K+ intake (see p. 795). Substantial quantities of K+ are secreted in gastric, pancreatic, and biliary fluid. Therefore, the total K+ load presented to the small intestine is considerably greater than that represented by the diet. The concentration of K+ in stool is frequently >100 mM. This high stool [K+] is the result of several factors, including both colonic K+ secretion and water absorption, especially in the distal part of the colon.

K+ absorption in the small intestine probably occurs via solvent drag Experiments in which a plasma-like solution perfused segments of the intestine have established that K+ is absorbed in the jejunum and ileum of the small intestine and is secreted in the large intestine. Although the small intestine absorbs substantial amounts of K+, no evidence has been presented to suggest that K+ absorption in the jejunum and ileum is an active transport process or even carrier mediated. Thus, K+ absorption in the small intestine is probably passive, most likely a result of solvent drag (i.e., pulled along by bulk water movement; see p. 908), as illustrated in Figure 44-6A. Although changes in dietary Na+ and K+ and alterations in hydration influence K+ movement in the colon, similar physiological events do not appear to affect K+ absorption in the small intestine.

Passive K+ secretion is the primary mechanism for net colonic secretion In contrast to the small intestine, the human colon is a net secretor of K+. This secretion occurs by two mechanisms: a passive transport process that is discussed in this section and an active process that is discussed in the next. Together, these two K+ secretory pathways are greater than the modest component of active K+ absorption in the distal part of the colon and thus account for the overall secretion of K+ by the colon. Passive K+ secretion, which is the pathway that is primarily responsible for overall net colonic K+ secretion, is driven by the lumen-negative Vte of 15 to 25 mV. The route of passive K+ secretion is predominantly paracellular, not transcellular (see Fig. 44-6B). Because Vte is the primary determinant of passive K+ secretion, it is not surprising that passive K+ secretion is greatest in the distal end of the colon, where Vte difference is most negative. Similarly, increases in the lumen-negative Vte that occur as an adaptive response to dehydration—secondary to an elevation in aldosterone secretion (see the next section)—result in an enhanced rate of passive K+ secretion. Information is not available regarding the distribution of passive K+ secretion between surface epithelial and crypt cells.

Chapter 44  •  Intestinal Fluid and Electrolyte Movement

+ PASSIVE K ABSORPTION

A

Active K+ secretion is also present throughout the large intestine and is induced both by aldosterone and by cAMP

3 Na+ Jejunum

+

2K

H2O

Ileum

K+

+ PASSIVE K SECRETION

B

+

3 Na

2 K+

–

K

+

Proximal colon

The lumen potential is –25 mV.

Distal colon

ACTIVE K+ SECRETION

C

K

+

3 Na

+

+

2K

BK +

Na

+

K

Proximal colon Distal colon

2 Cl– NKCC1 + ACTIVE K ABSORPTION

D

3 Na+ H+ +

K

909

+

2K ?

Distal colon High transport

Low transport

Moderate transport

Very low transport

Figure 44-6  Cellular mechanisms of K+ secretion and absorption. A, In

the small intestine, K+ absorption occurs via solvent drag. B, Throughout the colon, passive K+ secretion occurs via tight junctions, driven by a lumen-negative transepithelial voltage. C, Throughout the colon, active K+ secretion is transcellular. D, In the distal colon, active K+ absorption is transcellular. The thickness of the arrows in the insets indicates the relative magnitude of K+ flux in different segments.

In addition to passive K+ secretion, active K+ transport processes—both secretory and absorptive—are also present in the colon. However, active transport of K+ is subject to considerable segmental variation in the colon. Whereas active K+ secretion occurs throughout the colon, active K+ absorption is present only in the distal segments of the large intestine. Thus, in the rectosigmoid colon, active K+ absorption and active K+ secretion are both operative and appear to contribute to total-body homeostasis. The model of active K+ secretion in the colon is quite similar to that of active Cl− secretion (see Fig. 44-5) and is also parallel to that of active K+ secretion in the renal distal nephron (see p. 799). The general paradigm of active K+ transport in the colon is a “pump-leak” model (see Fig. 44-6C). Uptake of K+ across the basolateral membrane is a result of both the Na-K pump and the Na/K/Cl cotransporter (NKCC1), which is energized by the low [Na+]i that is created by the Na-K pump. Once K+ enters the cell across the basolateral membrane, it may exit either across the apical membrane (K+ secretion) or across the basolateral membrane (K+ recycling). The cell controls the extent to which secretion occurs, in part by K+ channels present in both the apical and the basolateral membranes. When apical K+ channel activity is less than basolateral channel activity, K+ recycling dominates. Indeed, in the basal state, the rate of active K+ secretion is low because the apical K+ channel activity is minimal in comparison with the K+ channel activity in the basolateral membrane. It is likely that aldosterone stimulates active K+ secretion in surface epithelial cells of the large intestine, whereas cAMP enhances active K+ secretion in crypt cells. In both cases, the rate-limiting step is the apical BK K+ channel, and both secretagogues act by increasing K+ channel activity. Aldosterone  The mineralocorticoid aldosterone enhances overall net K+ secretion by two mechanisms. First, it increases passive K+ secretion by increasing Na-K pump activity and thus increasing electrogenic Na+ absorption (see Fig. 44-3D). The net effects are to increase the lumen-negative Vte and to enhance passive K+ secretion (see Fig. 44-6B). Second, aldosterone stimulates active K+ secretion by increasing the activity of both apical K+ channels and basolateral Na-K pumps (see Fig. 44-6C). cAMP and Ca2+  VIP and cholera enterotoxin both

increase [cAMP]i and thus stimulate K+ secretion. Increases in [Ca2+]i—induced, for example, by serotonin (or 5hydroxytryptamine [5-HT])—also stimulate active K+ secretion. In contrast to aldosterone, neither of these second messengers has an effect on the Na-K pump; rather, they increase the activity of both the apical and the basolateral K+ channels. Because the stimulation of K+ channels is greater at the apical than at the basolateral membrane, the result is an increase in K+ exit from the epithelial cell across the apical membrane (i.e., secretion). Stimulation of K+ secretion by cAMP and Ca2+, both of which also induce

910

SECTION VII  •  The Gastrointestinal System

active Cl− secretion (see Fig. 44-5), contributes to the significant fecal K+ losses that occur in many diarrheal diseases.

Active K+ absorption takes place only in the distal portion of the colon and is energized by an apical H-K pump As noted above, not only does the distal end of the colon actively secrete K+, it also actively absorbs K+. The balance between the two processes plays a role in overall K+ homeostasis. Increases in dietary K+ enhance both passive and active K+ secretion (see Fig. 44-6B, C). However, dietary K+ depletion enhances active K+ absorption (see Fig. 44-6D). The mechanism of active K+ absorption appears to be an exchange of luminal K+ for intracellular H+ across the apical membrane, mediated by an H-K pump (see pp. 117–118). The colonic H-K pump is ~60% identical at the amino-acid level to both the Na-K pump and the gastric parietal-cell H-K pump. Thus, active colonic K+ absorption occurs via a trans­ cellular route, in contrast to the paracellular route that characterizes K+ absorption in the small intestine (see Fig. 44-6A). The mechanism of K+ exit across the basolateral membrane may involve K/Cl cotransport. Not known is whether active K+ secretion (see Fig. 44-6C) and active K+ absorption (see Fig. 44-6D) occur in the same cell or in different cells.

REGULATION OF INTESTINAL ION TRANSPORT Chemical mediators from the enteric nervous system, endocrine cells, and immune cells in the lamina propria may be either secretagogues or absorptagogues Numerous chemical mediators from several different sources regulate intestinal electrolyte transport. Some of these agonists are important both in health and in diarrheal disorders, and at times only quantitative differences separate normal regulatory control from the pathophysiology of diarrhea. These mediators may function in one or more modes: neural, endocrine, paracrine, and perhaps autocrine (see p. 47). Most of these agonists (i.e., secretagogues) promote secretion, whereas some others (i.e., absorptagogues) enhance absorption. The enteric nervous system (ENS), discussed on pages 339–340 and 855–856, is important in the normal regulation of intestinal epithelial electrolyte transport. Activation of enteric secretomotor neurons results in the release of acetylcholine from mucosal neurons and in the induction of active Cl− secretion (see Fig. 44-5). Additional neurotransmitters, including VIP, 5-HT, and histamine, mediate ENS regulation of epithelial ion transport. An example of regulation mediated by the endocrine system is the release of aldosterone from the adrenal cortex and the subsequent formation of angiotensin II; both dehydration and volume contraction stimulate this reninangiotensin-aldosterone axis (see pp. 841–842). Both angiotensin and aldosterone regulate total-body Na+ homeostasis by stimulating Na+ absorption, angiotensin in the small intestine, and aldosterone in the colon. Their effects on cellular Na+ absorption differ. In the small intestine, angiotensin

TABLE 44-3  Products of Lamina Propria Cells that Affect Intestinal Ion Transport CELL

PRODUCT

Macrophages

Prostaglandins O2 radicals

Mast cells

Histamine

Neutrophils

Eicosanoids Platelet-activating factor

Fibroblasts

Eicosanoids Bradykinin

enhances electroneutral NaCl absorption (see Fig. 44-3C), probably by upregulating apical membrane Na-H exchange. In the colon, aldosterone stimulates electrogenic Na+ absorption (see Fig. 44-3D). The response of the intestine to angiotensin and aldo­ sterone represents a classic endocrine feedback loop: dehydration results in increased levels of angiotensin and aldosterone, the primary effects of which are to stimulate fluid and Na+ absorption by both the renal tubules (see pp. 765–766) and the intestines. The result is restoration of total-body fluid and Na+ content. Regulation of intestinal transport also occurs by paracrine effects. Endocrine cells constitute a small fraction of the total population of mucosal cells in the intestines. These endocrine cells contain several peptides and bioactive amines that are released in response to various stimuli. Relatively little is known about the biology of these cells, but gut distention can induce the release of one or more of these agonists (e.g., 5-HT). The effect of these agonists on adjacent surface epithelial cells represents a paracrine action. Another example of paracrine regulation of intestinal fluid and electrolyte transport is the influence of immune cells in the lamina propria (see Fig. 44-1). Table 44-3 lists these immune cells and some of the agonists that they release. The same agonist may be released from more than one cell, and individual cells produce multiple agonists. These agonists may activate epithelial cells directly or may activate other immune cells or enteric neurons. For example, reactive oxygen radicals released by mast cells affect epithelial-cell function by acting on enteric neurons and fibroblasts, and they also have direct action on surface and crypt epithelial cells. A single agonist usually has multiple sites of action. For example, the histamine released from mast cells can induce fluid secretion as a result of its interaction with receptors on surface epithelial cells (Fig. 44-7). However, histamine can also activate ENS motor neurons, which can in turn alter epithelial-cell ion transport as well as intestinal smoothmuscle tone and blood flow. As a consequence, the effects of histamine on intestinal ion transport are multiple and amplified.

Secretagogues can be classified by their type and by the intracellular second-messenger system that they stimulate Several agonists induce the accumulation of fluid and electrolytes in the intestinal lumen (i.e., net secretion).

Chapter 44  •  Intestinal Fluid and Electrolyte Movement

Exterior milieu

911

Interstitial space Antigen

Epithelial cell

Histamine

–

Receptor

Cl

PGE2

Myofibroblasts

Antibody

IL-1

cAMP

EP2 receptor ACh

Histamine ACh

–

Cl

Enteric neuron

Ca2+

M3 receptor

Intestinal smooth muscle

Mast cell in lamina propria

Figure 44-7  Mast cell activation. Activation of mast cells in the lamina propria triggers the release of histamine, which either directly affects epithelial cells or stimulates an enteric neuron and thus has an indirect effect. The neuron modulates the epithelium (secretion), intestinal smooth muscle (motility), or vascular smooth muscle (blood flow). ACh, acetylcholine; EP2 receptor, prostaglandin E2 receptor; IL-1, interleukin-1; PGE2, prostaglandin E2.

These secretagogues are a diverse, heterogeneous group of compounds, but they can be effectively classified in two different ways: by the type of secretagogue and by the intracellular second messenger that these agonists activate. Grouped according to type, the secretagogues fall into four categories: (1) bacterial exotoxins (i.e., enterotoxins), (2) hormones and neurotransmitters, (3) products of cells of the immune system, and (4) laxatives. Table 44-2 provides a partial list of these secretagogues. A bacterial exotoxin is a peptide that is produced and excreted by bacteria that can produce effects independently of the bacteria. An enterotoxin is an exotoxin that induces changes in intestinal fluid and electrolyte movement. For example, E. coli produces two distinct enterotoxins (the so-called heat-labile and heatstable toxins) that induce fluid and electrolyte secretion via two distinct receptors and second-messenger systems. We can also classify secretagogues according to the signaltransduction system that they activate after binding to a specific membrane receptor. As summarized in Table 44-2, the second messengers of these signal-transduction systems include cAMP, cGMP, and Ca2+. For example, the heat-labile toxin of E. coli binds to apical membrane receptors, becomes internalized, and then activates basolateral adenylyl cyclase. The resulting increase in [cAMP]i activates protein kinase A. VIP also acts by this route (Fig. 44-8). The heat-stable toxin of E. coli binds to and activates an apical receptor guanylyl cyclase, similar to the atrial natriuretic peptide (ANP) receptor (see p. 66). The newly produced cGMP activates protein kinase G and may also activate protein kinase A. The natural agonist for this pathway is guanylin, a 15–amino-acid peptide secreted by mucosal cells of the small and large intestine. Still other secretory agonists (e.g., 5-HT) produce their effects by increasing [Ca2+]i and thus activating protein kinase C or Ca2+-calmodulin–dependent protein kinases. One way that secretagogues can increase [Ca2+]i is by stimulating phospholipase C, which leads to the production of inositol

TABLE 44-4  End Effects of Second Messengers on Intestinal Transport SECOND MESSENGER

INCREASED ANION SECRETION

INHIBITED NaCl ABSORPTION

cAMP

+++

+++

cGMP

+

+++

2+

+++

+++

Ca

1,4,5-trisphosphate (IP3) and the release of Ca2+ from intracellular stores (see p. 60). Secretagogues can also increase [Ca2+]i by activating protein kinases, which may stimulate basolateral Ca2+ channels. Although the secretagogues listed in Table 44-2 stimulate fluid and electrolyte secretion via one of three distinct second messengers (i.e., cAMP, cGMP, and Ca2+), the end effects are quite similar. As summarized in Table 44-4, all three second-messenger systems stimulate active Cl− secretion (see Fig. 44-5) and inhibit electroneutral NaCl absorption (see Fig. 44-3C). The abilities of cAMP and Ca2+ to stimulate Cl− secretion and inhibit electroneutral NaCl absorption are almost identical. In contrast, cGMP’s ability to stimulate Cl− secretion is somewhat less, although its effects on electroneutral NaCl absorption are quantitatively similar to those of cAMP and Ca2+. Both stimulation of Cl− secretion and inhibition of electroneutral NaCl absorption have the same overall effect: net secretion of fluid and electrolytes. It is uncertain whether the observed decrease in electroneutral NaCl absorption is the result of inhibiting Na-H exchange, Cl-HCO3 exchange, or both inasmuch as electroneutral NaCl absorption represents the coupling of separate Na-H and Cl-HCO3 exchange processes via pHi (see Fig. 44-3C).

912

SECTION VII  •  The Gastrointestinal System

PKA catalytic subunits phosphorylate apical membrane proteins.

Epithelial cell

External milieu

Interstitial space PKA regulatory subunit P

Active PKG phosphorylates apical membrane proteins. Receptor guanylyl cyclase Heat-stable toxin (STa)

cAMP

cAMP

cAMP

cAMP

PKA (active)

P

cAMP

PKA catalytic subunit

PKA

cGMP

P

PKG II

CaM kinase

AC

Active PKG type II

PKG II

cGMP

2 Some secretagogues bind to a receptor that generates cGMP.

Gs

Gq

Secretagogue (e.g., VIP, heatlabile toxin) 1 Some secretagogues bind to GPCRs, coupled to Gs, activating adenylyl cyclase. Secretagogue (e.g., serotonin) 3 Other secretagogues bind to GPCRs, coupled to Gq, activating phospholipase C.

Calmodulin

Calcium calmodulin

PLC

Active CaM kinase phosphorylates apical membrane proteins.

PIP2 Active CaM kinase

Ca2+

Ca2+

ER IP3 PKC

PKC

DAG Active PKC

Figure 44-8  Action of secretagogues. Secretagogues (agents that stimulate the net secretion of fluid and electrolytes into the intestinal lumen) act by any of the mechanisms numbered 1, 2, or 3. AC, adenylyl cyclase; CaM, calmodulin; DAG, diacylglycerol; ER, endoplasmic reticulum; Gq and Gs, α-subunit types of G proteins; GPCRs, G protein–coupled receptors; PIP2, phosphatidylinositol 4,5-bisphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PLC, phospholipase C.

Mineralocorticoids, glucocorticoids, and somatostatin are absorptagogues Although multiple secretagogues exist, relatively few agonists can be found that enhance fluid and electrolyte absorption. The cellular effects of these absorptagogues are less well understood than those of the secretagogues. Those few absorptagogues that have been identified increase intestinal fluid and electrolyte absorption by either a paracrine or an endocrine mechanism. Corticosteroids are the primary hormones that enhance intestinal fluid and electrolyte absorption. Mineralocorticoids (e.g., aldosterone) stimulate Na+ absorption and K+ secretion in the distal end of the colon; they do not affect ion transport in the small intestine. Their cellular actions are outlined on page 1027. Aldosterone induces both apical membrane Na+ channels (a process that is inhibited by the diuretic amiloride) and basolateral Na-K pumps; this

action results in substantial enhancement of colonic “electrogenic” Na+ absorption. Although the effects of glucocorticoids on ion transport have most often been considered a result of crossover binding to the mineralocorticoid receptor (see p. 766), it is now evident that glucocorticoids also have potent actions on ion transport via their own receptor and that these changes in ion transport are distinct from those of the mineralocorticoids. Glucocorticoids stimulate electroneutral NaCl absorption (see Fig. 44-3C) throughout the large and small intestine without any effect on either K+ secretion or electrogenic Na+ absorption. Both corticosteroids act, at least in part, by genomic mechanisms (see pp. 71–72). Other agonists appear to stimulate fluid and electrolyte absorption by stimulating electroneutral NaCl absorption and inhibiting electrogenic HCO3− secretion; both these changes enhance fluid absorption. Among these absorptagogues are somatostatin, which is released from endocrine

Chapter 44  •  Intestinal Fluid and Electrolyte Movement

cells in the intestinal mucosa (see pp. 993–994), and the enkephalins and norepinephrine, which are neurotransmitters of enteric neurons. The limited information available suggests that these agonists affect ion transport by decreasing [Ca2+]i, probably by blocking Ca2+ channels. Thus, it appears that fluctuations in [Ca2+]i regulate Na+ and Cl− transport in both the absorptive (low [Ca2+]i) and secretory (high [Ca2+]i)

913

directions. Therefore, Ca2+ is clearly a critical modulator of intestinal ion transport.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 44  •  Intestinal Fluid and Electrolyte Movement

REFERENCES Books and Reviews Alper SL, Sharma AK: The SLC26 gene family of anion transporters and channels. Mol Aspects Med 34:494–515, 2013. Arroyo JP, Kahle KT, Gamba G: The SLC12 family of electroneutral cation-coupled chloride cotransporters. Mol Aspects Med 34:288–298, 2013. Binder HJ, Sandle GI: Electrolyte transport in the mammalian colon. In Johnson LR (ed): Physiology of the Gastrointestinal Tract, 3rd ed. New York, Raven Press, 1994, pp 2133–2172. Donowitz M, Ming Tse C, Fuster D: SLC9/NHE gene family, a plasma membrane and organellar family of Na+/H+ exchangers. Mol Aspects Med 34:236–251, 2013. Farthing MJG: Oral rehydration therapy. Pharmacol Ther 64:477– 492, 1994. Field M, Semrad CE: Toxigenic diarrheas, congenital diarrheas, and cystic fibrosis: Disorders of intestinal ion transport. Annu Rev Physiol 55:631–655, 1993. Greger R, Bleich M, Leipziger J, et al: Regulation of ion transport in colonic crypts. News Physiol Sci 12:62–66, 1997. Kaunitz JD, Barrett KE, McRoberts JA: Electrolyte secretion and absorption: Small intestine and colon. In Yamada T (ed): Textbook of Gastroenterology, vol 1, 2nd ed. Philadelphia, JB Lippincott, 1995, pp 326–361. Montrose MH, Keely SJ, Barrett KE: Electrolyte secretion and absorption. Small intestine and colon. In Yamada T (ed):

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Textbook of Gastroenterology, vol. 1, 4th ed. Philadelphia, Lippincott Williams & Wilkins, 2003, pp 308–340. Palacin M, Estevez R, Bertran J, Zorzano A: Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78:969–1054, 1998. Rao MC: Oral rehydration therapy: New explanations for an old remedy. Annu Rev Physiol 66:385–417, 2004. Zachos NC, Tse M, Donowitz M: Molecular physiology of intestinal Na/H exchange. Annu Rev Physiol 67:411–443, 2005. Journal Articles Canessa CM, Horisberger J-D, Rossier BC: Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361:467–470, 1993. Knickelbein RG, Aronson PS, Schron CM, et al: Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol 249:G236–G245, 1985. Moseley RH, Hoglund P, Wu GD, et al: Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea. Am J Physiol 276:G185–G192, 1999. Schulz S, Green CK, Yuen PST, Garbers DL: Guanylyl cyclase is a heat-stable enterotoxin receptor. Cell 63:941–948, 1990. Singh SK, Binder HJ, Boron WF, Geibel JP: Fluid absorption in isolated perfused colonic crypts. J Clin Invest 96:2373–2379, 1995.

C H A P T E R 45  NUTRIENT DIGESTION AND ABSORPTION Henry J. Binder and Charles M. Mansbach II

In general, the digestive-absorptive processes for most of the constituents of our diet are highly efficient. For example, normal adult intestine absorbs ~95% of dietary lipid. How­ ever, we ingest most of the constituents of dietary food in a form that the intestine cannot readily absorb. Multiple digestive processes convert dietary food to a form that can be absorbed—primarily in the small intestine, but also, to a much smaller extent, in the colon. The digestive process—the enzymatic conversion of complex dietary substances to a form that can be absorbed— is initiated by the sight, smell, and taste of food. Although some digestion (that of carbohydrates) begins in the mouth and additional digestion may occur within the lumen of the stomach, most digestive processes occur in the small intestine. Digestion within the small intestine occurs either in the lumen, mediated by pancreatic enzymes, or at the smallintestinal brush-border membrane (membrane digestion), mediated by brush-border enzymes. Several different patterns of luminal, brush-border, and cytosolic digestion exist (Fig. 45-1). Some of the dietary carbohydrate and protein that escape digestion and absorption in the small intestine are altered in the large intestine by bacterial enzymes to short-chain fatty acids (SCFAs)  N45-1  that are absorbed by the colon. The digestive processes for carbohydrates, proteins, and lipids result in the conversion of dietary nutrients to chemical forms for which intestinal absorptive processes exist. As a consequence, the digestive-absorptive processes for the several dietary constituents are closely integrated and regulated biological events that ensure survival. Multiple diseases can alter these digestive-absorptive processes and can thereby impair nutrient assimilation (i.e., the overall process of digestion and absorption). Because of the substantial segmental distribution of nutrient absorption along the gastrointestinal tract (Fig. 45-2), the clinical manifestations of disease (Table 45-1) often reflect these segmental differences.

CARBOHYDRATE DIGESTION Carbohydrates, providing ~45% of total energy needs of Western diets, require hydrolysis to monosaccharides before absorption We can classify dietary carbohydrates into two major groups: (1) the monosaccharides (monomers), and (2) the oligo­ saccharides (short polymers) and polysaccharides (long 914

polymers). The small intestine can directly absorb the monomers but not the polymers. Some polymers are digestible, that is, the body can digest them to form the monomers that the small intestine can absorb. Other polymers are non­ digestible, or “fiber.” The composition of dietary carbohydrate is quite varied and is a function of culture. The diet of individuals in so-called developed countries contains considerable amounts of “refined” sugar and, compared with individuals in most developing countries, less fiber. Such differences in the fiber content of the Western diet may account for several diseases that are more prevalent in these societies (e.g., colon carcinoma and atherosclerosis). As a consequence, the consumption of fiber by the health-conscious public in the United States has increased during the past 3 decades. In general, increased amounts of fiber in the diet are associated with increased stool weight and frequency. Approximately 45% to 60% of dietary carbohydrate is in the form of starch, which is a polysaccharide. Starch is a storage form for carbohydrates that is primarily found in plants, and it consists of both amylose and amylopectin. In contrast, the storage form of carbohydrates in animal tissues is glycogen, which is consumed in much smaller amounts. Amylose is a straight-chain glucose polymer that typically contains multiple glucose residues, connected by α-1,4 linkages. In contrast, amylopectin is a massive branched glucose polymer that may contain 1 million glucose residues. In addition to the α-1,4 linkages, amylopectin has frequent α-1,6 linkages at the branch points. Amylopectins are usually present in much greater quantities (perhaps 4-fold higher) than amylose. Glycogen—the “animal starch”—has α-1,4 and α-1,6 linkages like amylopectin. However, glycogen is more highly branched (i.e., more α-1,6 linkages). Most dietary oligosaccharides are the disaccharides sucrose and lactose, which represent 30% to 40% of dietary carbohydrates. Sucrose is table sugar, derived from sugar cane and sugar beets, whereas lactose is the sugar found in milk. The remaining carbohydrates are the monosaccharides fructose and glucose, which make up 5% to 10% of total carbohydrate intake. There is no evidence of any intestinal absorption of either starches or disaccharides. Because the small intestine can absorb only monosaccharides, all dietary carbohydrate must be digested to monosaccharides before absorption. The colon cannot absorb monosaccharides. Dietary fiber consists of both soluble and insoluble forms and includes lignins, pectins, and cellulose. These fibers are primarily present in fruits, vegetables, and cereals. Cellulose

Chapter 45  •  Nutrient Digestion and Absorption

N45-1  Fatty Acids: Chain Length Contributed by Emile Boulpaep and Walter Boron Name

Abbreviation

Short-chain fatty acid Medium-chain fatty acid Long-chain fatty acid Very-long-chain fatty acid

SCFA MCFA LCFA VLCFA

Number of Carbon Atoms 21

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Chapter 45  •  Nutrient Digestion and Absorption

Lumen DIGESTION

Epithelium

915

Interstitial space

EXAMPLE

None

Glucose

Luminal hydrolysis of polymer to monomers

Glucose

Protein

Amino acids (AA)

AA

Glucose

Glucose

Sucrose

Brushborder hydrolysis of oligomer to monomers

Fructose

Fructose Intracellular hydrolysis Peptide

Luminal hydrolysis followed by intracellular resynthesis

AA

Glycerol Triacylglycerol

Triacylglycerol Fatty acids

Figure 45-1  General mechanisms of digestion and absorption. Digestion-absorption can follow any of five patterns. First, the substance (e.g., glucose) may not require digestion; the intestinal cells may absorb the nutrient as ingested. Second, a polymer (e.g., protein) may be digested in the lumen to its constituent monomers (e.g., amino acids) by pancreatic enzymes prior to absorption. Third, an oligomer (e.g., sucrose) is digested into its constituent monomers (e.g., monosaccharides) by brush-border enzymes prior to absorption. When in free solution, fructose is present primarily as the pyranose (6-membered ring) form and less so as the furanose (5-membered ring) form. Fourth, an oligomer (e.g., oligopeptide) may be directly absorbed by the cell and then broken down into monomers (e.g., amino acids) inside the cell. Finally, a substance (e.g., TAG) may be broken down into its constituent components prior to absorption; the cell may then resynthesize the original molecule.

TABLE 45-1  Major Gastrointestinal Diseases and Nutritional Deficiencies DISEASE

ORGAN SITE OF PREDOMINANT PATHOLOGY

DEFECTIVE PROCESS

Celiac disease (see Box 45-5)

Duodenum and jejunum

Fat absorption, lactose hydrolysis

Chronic pancreatitis

Exocrine pancreas

Fat digestion

Surgical resection of ileum; Crohn disease of ileum

Ileum

Cobalamin and bile-acid absorption

Primary lactase deficiency

Small intestine

Lactose hydrolysis

is a glucose polymer connected by β-1,4 linkages, which cannot be digested by mammalian enzymes. However, enzymes from colonic bacteria may degrade fiber. This process is carried out with varying efficiency; pectins, gum, and mucilages are metabolized to a much greater degree than either cellulose or hemicellulose. In contrast, lignins, which are aromatic polymers and not carbohydrates, are not altered by microbial enzymes in the colonic lumen and are excreted unaltered in stool.

As we discuss below, the digestive process for dietary carbohydrates has two steps: (1) intraluminal hydrolysis of starch to oligosaccharides by salivary and pancreatic amylases (Fig. 45-3), and (2) so-called membrane digestion of oligosaccharides to monosaccharides by brush-border disaccharidases. The resulting carbohydrates are absorbed by transport processes that are specific for certain monosaccharides. These transport pathways are located in the apical membrane of the small-intestinal villous epithelial cells.

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SECTION VII  •  The Gastrointestinal System

A

CARBOHYDRATES, PROTEINS AND LIPIDS

B

CALCIUM, IRON AND FOLATE Calcium Iron

Carbohydrates, proteins, lipids

Folate

Duodenum Jejunum Ileum

Duodenum Calcium Calcium

High absorption Moderate absorption C

D

BILE ACIDS

Low absorption

COBALAMIN

Very low absorption

Bile acids

Duodenum Jejunum Ileum

Cobalamin

Ileum

Figure 45-2  Sites of nutrient absorption. A, The entire small intestine absorbs carbohydrates, proteins, and lipids. However, the absorption is greatest in the duodenum, somewhat less in the jejunum, and much less in the ileum. The thickness of the arrows indicates the relative magnitude of total absorption at the indicated site in vivo (see inset). The maximal absorptive capacity of a specific segment under optimized experimental conditions (e.g., substrate concentrations) may be greater. B, Some substances are actively absorbed only in the duodenum. C, Bile acids are absorbed along the entire small intestine, but active absorption occurs only in the ileum. D, The vitamin cobalamin is absorbed only in the ileum.

Luminal digestion begins with the action of salivary amylase and finishes with pancreatic amylase Acinar cells from both the salivary glands (see pp. 893–894) and pancreas (see p. 882) synthesize and secrete α-amylases. Salivary and pancreatic amylases, unlike most of the pancreatic proteases that we discuss below, are secreted not in an inactive proenzyme form, but rather in an active form. Salivary and pancreatic α-amylases have similar enzymatic function, and their amino-acid sequences are 94% identical. Salivary α-amylase in the mouth initiates starch digestion; in healthy adults, this step is of relatively limited importance. Salivary amylase is inactivated by gastric acid but can be partially protected by complexing with oligosaccharides. Pancreatic α-amylase completes starch digestion in the lumen of the small intestine. Although amylase binds to the apical membrane of enterocytes, this localization does not provide any kinetic advantage for starch hydrolysis. Cholecystokinin (CCK; see pp. 882–883) stimulates the secretion of pancreatic α-amylase by pancreatic acinar cells. α-amylase is an endoenzyme that hydrolyzes internal α-1,4 linkages (see Fig. 45-3A). α-amylase does not cleave terminal α-1,4 linkages, α-1,6 linkages (i.e., branch points), or α-1,4 linkages that are immediately adjacent to α-1,6 linkages. As a result, starch hydrolysis products are maltose,

maltotriose, and α-limit dextrins. Because α-amylase has no activity against terminal α-1,4 linkages, glucose is not a product of starch digestion. The intestine cannot absorb these products of amylase digestion of starch, and thus further digestion is required to produce substrates (i.e., monosaccharides) that the small intestine can absorb by specific transport mechanisms.

“Membrane digestion” involves hydrolysis of oligosaccharides to monosaccharides by brush-border disaccharidases The human small intestine has three brush-border proteins with oligo­saccharidase activity: lactase, glucoamylase (most often called maltase), and sucrase-isomaltase. These are all integral membrane proteins whose catalytic domains face the intestinal lumen (see Fig. 45-3B). Sucrase-isomaltase is actually two enzymes—sucrase and isomaltase (also known as α-dextrinase or debranching enzyme)—bound together. Thus, four oligosaccharidase entities are present at the brush border. Lactase has only one substrate; it breaks lactose into glucose and galactose. The other three enzymes have more complicated substrate spectra. All will cleave the terminal α-1,4 linkages of maltose, maltotriose, and α-limit dextrins. In addition, each of these three enzymes has at least one

Chapter 45  •  Nutrient Digestion and Absorption

Epithelium

Lumen

A

DIGESTION OF OLIGOSACCHARIDES AT BRUSH BORDER Lumen

α-Amylase

Amylose

Interstitial space

B

DIGESTION OF STARCH IN LUMEN

Lactase Amylopectin

Cytoplasm Lactase splits lactose. Both monomers are transported via SGLT1.

Lactose Terminal α-1,4 link Cannot be cut by amylase

Adjacent α-1,6 linkage Adjacent Terminal α-1,4 link (branching) α-1,4 link α-1,4 link Cannot be cut by amylase

917

SGLT1

+

Glucoamylase (also known as maltase) removes glucose monomers for transport.

2 Na+ +

Glucoamylase

Maltotriose

Maltotriose or maltose

α-Limit dextrins

+

Sucrase-isomaltase is actually two enzymes. The sucrase moiety splits sucrose, as well as maltose and maltotriose.

Maltose

C

SGLT1

+ 2 Na

+

GLUT5

ABSORPTION OF MONOSACCHARIDES Lumen

Epithelium

SGLT1 Galactose Glucose +

2 Na

Glucose 3 Na+

GLUT2

+

Sucrase-isomaltase Sucrose

Sucrase Isomaltase

Maltose

Sucrase Isomaltase

Maltotriose

The isomaltase moiety splits α-limit dextrins, as well as maltose and maltotriose.

2 K+

Fructose GLUT5

Interstitial space

Fructose

GLUT2

α-limit dextrins

+

Maltose Maltotriose

2 Na+

Figure 45-3  Digestion of carbohydrates to monosaccharides. A, Salivary and pancreatic α-amylase are

endoenzymes. They can digest the linear “internal” α-1,4 linkages between glucose residues but cannot break “terminal” α-1,4 linkages between the last two sugars in the chain. They also cannot split the α-1,6 linkages at the branch points of amylopectin or the adjacent α-1,4 linkages. As a result, the products of α-amylase action are linear glucose oligomers, maltotriose (a linear glucose trimer), maltose (a linear glucose dimer), and α-limit dextrins (which contain an α-1,6 branching linkage). B, The brush-border oligosaccharidases are intrinsic membrane proteins with their catalytic domains facing the lumen. Sucrase-isomaltase is actually two enzymes, and therefore, there are a total of four oligosaccharidases that split the oligosaccharides produced by α-amylase into monosaccharides. C, SGLT1 is the Na+-coupled transporter that mediates the uptake of glucose or galactose from the lumen of the small intestine into the enterocyte. GLUT5 mediates the facilitated diffusion of fructose into the enterocyte. Once the monosaccharides are inside the enterocyte, GLUT2 mediates their efflux across the basolateral membrane into the interstitial space.

SGLT1

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SECTION VII  •  The Gastrointestinal System

A PRESENCE OF LACTASE ACTIVITY Plasma glucose rises after glucose or lactose ingestion…

140

Plasma glucose

…and subsequent H2 excreted by lungs is low.

120

Breath H2

Glucose ingested 100

Lactose ingested

Lactose

80 0

Glucose 0

1

2

3

0

1

Hours

2

3

Hours

B LACTASE DEFICIENCY 140

Plasma glucose

…and colonic bacteria metabolize the lactose that enters the colon, resulting in higher H2 excretion.

Lactase-deficient individuals hydrolyze less lactose to glucose… Glucose ingested

120

Lactose

Breath H2

100 Glucose Lactose ingested

80 0 0

1

2

3

Hours

0

1

2

3

Hours

Figure 45-4  Effects of lactase deficiency on levels of glucose in the plasma and H2 in the breath. A, In an individual with normal lactase activity, blood glucose levels rise after the ingestion of either glucose or lactose. Thus, the small intestine can split the lactose into glucose and galactose, and absorb the two monosaccharides. At the same time, H2 in the breath is low. B, In an adult with low lactase activity, the rise in blood levels is less pronounced after ingesting lactose. Because the rise is normal after ingesting glucose, we can conclude that the difference is due to lactase activity. Conversely, the individual with lactase deficiency excretes large amounts of H2 into the breath. This H2 is the product of lactose catabolism by colonic bacteria.

other activity. Maltase can also degrade the α-1,4 linkages in straight-chain oligosaccharides up to nine monomers in length. However, maltase cannot split either sucrose or lactose. The sucrase moiety of sucrase-isomaltase is required to split sucrose into glucose and fructose. The isomaltase moiety of sucrase-isomaltase is critical; it is the only enzyme that can split the branching α-1,6 linkages of α-limit dextrins.  N45-2 The action of the four oligosaccharidases generates several monosaccharides. Whereas the hydrolysis products of maltose are two glucose residues, those of sucrose are glucose and fructose. The hydrolysis of lactose by lactase yields glucose and galactose. The activities of the hydrolysis reactions of sucrase-isomaltase and maltase are considerably greater than the rates at which the various transporters can absorb the resulting monosaccharides. Thus, uptake, not hydrolysis, is the rate-limiting step. In contrast, lactase activity is considerably less than that of the other oligo­ saccharidases and is rate limiting for overall lactose digestion-absorption. The oligosaccharidases have a varying spatial distribution throughout the small intestine. In general, the abundance

and activity of oligosaccharidases peak in the proximal jejunum (i.e., at the ligament of Treitz) and are considerably less in the duodenum and distal ileum. Oligosaccharidases are absent in the large intestine. The distribution of oligosaccharidase activity parallels that of active glucose transport. These oligosaccharidases are affected by developmental and dietary factors in different ways. In many nonwhite ethnic groups, as well as in almost all other mammals, lactase activity markedly decreases after weaning in the postnatal period. The regulation of this decreased lactase activity is genetically determined.  N45-3  The other oligosaccharidases do not decrease in the postnatal period. In addition, long-term feeding of sucrose upregulates sucrase activity. In contrast, fasting reduces sucrase activity much more than it reduces lactase activity. In general, lactase activity is both more susceptible to enterocyte injury (e.g., following viral enteritis) and slower to recover from damage than is other oligosaccharidase activity. Thus, reduced lactase activity (as a consequence of both genetic regulation and environmental effects) has substantial clinical significance in that lactose ingestion may result in a range of symptoms in affected individuals (Fig. 45-4A, Box 45-1).

Chapter 45  •  Nutrient Digestion and Absorption

918.e1

N45-2  Oligosaccharidases Contributed by Emile Boulpaep and Walter Boron The oligosaccharidases are large integral membrane proteins that are anchored to the apical membrane by a transmembrane stalk; >90% of the protein is extracellular. Villous epithelial cells synthe­ size the disaccharidases via the secretory pathway (see pp. 34–35). The proteins undergo extensive N-linked and O-linked glycosylation in the Golgi and then traffic to the apical membrane. Sucrase-isomaltase is a special case. After the insertion of the single sucrase-isomaltase peptide (including its transmembrane stalk) into the brush-border membrane, pancreatic proteases cleave the peptide between the sucrase and isomaltase moieties. After this cleavage, the isomaltase moiety remains

continuous with the transmembrane stalk, and the sucrase moiety remains attached to the isomaltase moiety by van der Waals forces. Thus, sucrase-isomaltase differs from the other two oligosaccharidases in that the mature protein consists of two peptide chains (encoded by the same mRNA nonetheless), each with a distinct catalytic site and distinct substrate specificities. See eFigure 45-1 for a summary of the composition of sugars and oligosaccharides. As we saw in the text, sucrase is unique in splitting sucrose, and the isomaltase is unique in splitting the α-1,6 linkage of α-limit dextrins. The table lists the enzymatic specificities for each of the brush-border oligosaccharidases.

Specificities of Oligosaccharidases SUBSTRATES

TERMINAL α-1,4 LINKAGES

INTERNAL α-1,4 LINKAGES IN OLIGOSACCHARIDES UP TO 9 MONOMERS IN LENGTH

Maltase

✓

✓

Sucrase*

✓

Isomaltase*

✓

ENZYME

LACTOSE (SPLITTING THE β-1,4 LINKAGE BETWEEN D-GALACTOSE AND D-GLUCOSE)

Lactase

✓

SUCRASE (SPLITTING α-1,2 LINKAGES BETWEEN D-GLUCOSE AND D-GALACTOSE)

α-1,6 (BRANCHING) LINKAGES OF α-LIMIT DEXTRINS

✓ ✓

*Sucrase and isomaltase are separate peptides, held together by van der Waals forces and anchored to the membrane via the transmembrane stalk of the isomaltase.

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SECTION VII  •  The Gastrointestinal System

N45-2  Oligosaccharidases—cont’d SUGAR

LINKAGE

COMPONENTS D-Galactose—D-Glucose

β-1,4*

Sucrose

D-Glucose—D-Fructose

α-1,2

Maltose

D-Glucose—D-Glucose

α-1,4

Isomaltose

D-Glucose

α-1,6

Maltotriose

D-Glucose—D-Glucose—D-Glucose

Both α-1,4

-limit dextrins

By definition, these are small D-glucose polymers that have been exhaustively digested by enzymes (e.g., amylase) that cannot attack -1,6 branch points, -1,4 linkages that are adjacent to -1,6 branch points, or terminal -1,4 linkages. According to the rules summarized in the text, these are all the possibilities:

Coupled mainly by 1,4 linkages, but containing one internal 1,6 linkage (highlighted in yellow) from a former branch point. Note: “internal” means that at least one 1,4 linkage separates the 1,6 linkage from an end.

—

Lactose

D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose

—

D-Glucose—D-Glucose—D-Glucose

D-Glucose—D-Glucose—D-Glucose—D-Glucose—D-Glucose

*For a 1,4 linkage, the linkage is between the number 1 carbon atom of the leftmost sugar in the “Components” column and the number 4 carbon atom of the rightmost sugar.

eFigure 45-1  Composition of common oligosaccharide.

Chapter 45  •  Nutrient Digestion and Absorption

N45-3  Lactose Intolerance Contributed by Henry Binder Some authors object to the statement that lactose intolerance in adults is a lactase “deficiency” and instead propose that the normal course of events is for lactase activity to decline after weaning. According to one view, lactase “persistence” evolved in certain human populations after the domestication of herd animals allowed the consumption of nonhuman milk. This hypothesis could account for the geographical distribution of lactose intolerance in humans.

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Chapter 45  •  Nutrient Digestion and Absorption

BOX 45-1  Lactase Deficiency

P

rimary lactase deficiency is most prevalent in nonwhites, and it also occurs in some whites. Primary lactase deficiency represents an isolated deficiency of lactase, with all other brush-border enzymes being at normal levels and without any histological abnormalities. Lactase activity decreases after weaning; the time course of its reduction is determined by hereditary factors. Ingestion of lactose in the form of milk and milk products by individuals with decreased amounts of small-intestinal lactase activity may be associated with a range of gastrointestinal symptoms, including diarrhea, cramps, and flatus, or with no discernible symptoms. Several factors determine whether individuals with lactase deficiency experience symptoms after ingestion of lactose, including rate of gastric emptying, transit time through the small intestine, and, most importantly, the ability of colonic bacteria to metabolize lactose to SCFAs,  N45-1  CO2, and H2. Figure 45-4A shows the rise of plasma [glucose] following the ingestion of either lactose or glucose in adults with normal lactase levels. This figure also shows that the [H2] in the breath rises only slightly following the ingestion of either lactose or glucose in individuals with normal lactase levels. Figure 45-4B shows that in individuals with primary lactase deficiency, the ingestion of lactose leads to a much smaller rise in plasma [glucose], although the ingestion of glucose itself leads to a normal rise in plasma [glucose]. Thus, no defect in glucose absorption per se is present, but simply a markedly reduced capacity to hydrolyze lactose to glucose and galactose. In lactase-deficient individuals, breath H2 is markedly increased after lactose ingestion because nonabsorbed lactose is metabolized by colonic bacteria to H2, which is absorbed into the blood and is subsequently excreted by the lungs. In contrast, the rise in breath H2 after the ingestion of glucose is normal in these individuals. Treatment for symptomatic individuals with primary lactase deficiency is reduction or elimination of consumption of milk and milk products or the use of milk products treated with a commercial lactase preparation.

CARBOHYDRATE ABSORPTION The three monosaccharide products of carbohydrate digestion—glucose, galactose, and fructose—are absorbed by the small intestine in a two-step process involving their uptake across the apical membrane into the epithelial cell and their coordinated exit across the basolateral membrane (see Fig. 45-3C). Na/glucose transporter 1 (SGLT1) is the membrane protein responsible for glucose and galactose uptake at the apical membrane. The exit of all three monosaccharides across the basolateral membrane uses a facilitated sugar transporter (GLUT2). Because SGLT1 cannot carry fructose, the apical step of fructose absorption occurs by the facilitated diffusion of fructose via GLUT5. Thus, although two different apical membrane transport mechanisms exist for glucose and fructose uptake, a single transporter (GLUT2) is responsible for the movement of both monosaccharides across the basolateral membrane.

919

BOX 45-2  Glucose-Galactose Malabsorption

M

olecular studies have been performed on jejunal mucosa from patients with so-called glucose-galactose malabsorption (or monosaccharide malabsorption). These individuals have diarrhea when they ingest dietary sugars that are normally absorbed by SGLT1. This diarrhea results from both reduced small-intestinal Na+ and fluid absorption (as a consequence of the defect in Na+-coupled monosaccharide absorption) and fluid secretion secondary to the osmotic effects of nonabsorbed monosaccharide. Eliminating the monosaccharides glucose and galactose, as well as the disaccharide lactose (i.e., glucose + galactose), from the diet eliminates the diarrhea. The monosaccharide fructose, which crosses the apical membrane via GLUT5, does not induce diarrhea. Early studies identified the abnormality in this hereditary disorder as a defect at the apical membrane that is presumably related to defective or absent SGLT1. Molecular studies of SGLT1 have revealed multiple mutations that result in single amino-acid substitutions in SGLT1, each of which prevents the transport of glucose by SGLT1 in affected individuals. Patients with glucose-galactose malabsorption do not have glycosuria (i.e., glucose in the urine), because glucose reabsorption by the proximal tubule normally occurs via both SGLT1 and SGLT2 (see p. 772).

SGLT1 is responsible for the Na+-coupled uptake of glucose and galactose across the apical membrane The uptake of glucose across the apical membrane via SGLT1 (Fig. 45-5A) represents active transport, because the glucose influx occurs against the glucose concentration gradient (see pp. 121–122). Glucose uptake across the apical membrane is energized by the electrochemical Na+ gradient, which in turn is maintained by the extrusion of Na+ across the basolateral membrane by the Na-K pump. This type of Na+-driven glucose transport is an example of secondary active transport (see p. 115). Inhibition of the Na-K pump reduces active glucose absorption by decreasing the apical membrane Na+ gradient and thus decreasing the driving force for glucose entry. The affinity of SGLT1 for glucose is markedly reduced in the absence of Na+. The varied affinity of SGLT1 for different monosaccharides reflects its preference for specific molecular configurations. SGLT1 has two structural requirements for monosaccharides: (1) a hexose in a D config­ uration, and (2) a hexose that can form a six-membered pyranose ring (see Fig. 45-5B). SGLT1 does not absorb L-glucose, which has the wrong stereochemistry, and it does not absorb D-fructose, which forms a five-membered ring (Box 45-2).  N45-4

The GLUT transporters mediate the facilitated diffusion of fructose at the apical membrane and of all three monosaccharides at the basolateral membrane Early work showed that fructose absorption is independent of Na+ but has characteristics of both a carrier-mediated and a passive process. These observations show that the small intestine has separate transport systems for glucose

Chapter 45  •  Nutrient Digestion and Absorption

N45-4  Na/Glucose Cotransporters Contributed by Emile Boulpaep and Walter Boron Because the membrane potential across the luminal membrane is 40 to 50 mV (cell interior negative), and intracellular [Na+] is far less than luminal [Na+], a “downhill” electrochemical Na+ gradient exists across the apical membrane that is the primary driving force for the uptake of glucose (and other actively transported monosaccharides) by SGLT1 (see pp. 121–122). Glucose uptake at the apical membrane has other characteristics of a carrier-mediated active transport process, including saturation kinetics, competitive inhibition, and energy dependence. SGLT1 belongs to the SLC5 family of transporters that couple Na+ to monosaccharides and other small molecules. These membrane proteins have 14 predicted membranespanning segments. The gene for SGLT1 has been localized to human chromosome 22. Kinetic studies of the SGLT1 expressed in host cells have confirmed many of the characteristics of the Na/glucose cotransport system that had been identified in native tissue. Expression studies have established that the Na+:sugar stoichiometry of SGLT1 is a 2 : 1 ratio. Its cousins SGLT2 and SGLT3 both have an Na+:sugar stoichiometry of 1 : 1. For a discussion of the stereospecificity of sugars, see the biochemistry text by Voet and Voet, page 254 (Fig. 10–4).

REFERENCES Voet D, Voet J: Biochemistry, ed 2. New York, Wiley, 1995. Wright EM, Turk E: The sodium/glucose cotransport family SLC5. Pflugers Arch 447:510–518, 2004.

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A

SECTION VII  •  The Gastrointestinal System

Epithelium

STRUCTURE OF SGLT1 Lumen

Extracellular space N

Interstitial space One of many brush-border peptidases

C

7

2

6 1

3

4

5

7

2

11

9

8

10

13 12

14

(AA)4

Gastric and pancreatic peptidases

Tripeptidase

(AA)3 Oligopeptides (AA)n

(AA)3

Proteins

Cytosol B

AA

H+

AA PepT1

+

Amino acids

AA

AA

(AA)2

(AA)2 H+

STRUCTURAL REQUIREMENTS OF SUGAR

+ AA

Dipeptidase

6

CH2OH

H

5 4

HO

OH 3

H

O H

2

H

AA

+ AA

AA

1

OH

OH

Pyranose ring in D configuration. Figure 45-5  Na+-coupled hexose transporter. A, The SGLT family of proteins has 14 membrane-spanning segments. This diagram represents the structure of the vSGLT Na/galactose cotransporter from the bacterium Vibrio parahaemolyticus. B, SGLT1 transports only hexoses in a D configuration and with a pyranose ring. This figure shows D-glucose; D-galactose is identical, except that the H and OH on carbon 4 are inverted. (A, Data from Faham S, Watanabe A, Besserer GM, et al: The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport. Science 321:810–814, 2008.)

and fructose. Subsequent studies established that facilitated diffusion is responsible for fructose absorption. Fructose uptake across the apical membrane is mediated by GLUT5, a member of the GLUT family of transport proteins (see p. 114). GLUT5 is present mainly in the jejunum.  N45-5 The efflux of glucose, fructose, and galactose across the basolateral membrane also occurs by facilitated diffusion. The characteristics of the basolateral sugar transporter, identified as GLUT2, are similar to those of other sugar transport systems in erythrocytes, fibroblasts, and adipocytes. GLUT2 has no homology to SGLT1 but is 41% identical to GLUT5, which is responsible for the uptake of fructose from the lumen.

PROTEIN DIGESTION Proteins require hydrolysis to oligopeptides or amino acids before absorption in the small intestine With the exception of antigenic amounts of dietary protein that are absorbed intact, proteins must first be digested

One of many AA transporters

Na+

Figure 45-6  Action of luminal, brush-border, and cytosolic peptidases. Pepsin from the stomach and the five pancreatic proteases hydrolyze proteins—both dietary and endogenous—to single amino acids, AA, or to oligopeptides, (AA)n. These reactions occur in the lumen of the stomach or small intestine. A variety of peptidases at the brush borders of enterocytes then progressively hydrolyze oligopeptides to amino acids. The enterocyte directly absorbs some of the small oligopeptides via the action of the H/oligopeptide cotransporter PepT1. These small peptides are digested to amino acids by peptidases in the cytoplasm of the enterocyte.

into their constituent oligopeptides and amino acids before being taken up by the enterocytes. Digestion-absorption occurs through four major pathways. First, several luminal enzymes (i.e., proteases) from the stomach and pancreas may hydrolyze proteins to peptides and then to amino acids, which are then absorbed (Fig. 45-6). Second, lumi­ nal enzymes may digest proteins to peptides, but enzymes present at the brush border digest the peptides to amino acids, which are then absorbed. Third, luminal enzymes may digest proteins to peptides, which are themselves taken up as oligopeptides by the enterocytes. Further digestion of the oligopeptides by cytosolic enzymes yields intracellular amino acids, which are moved by transporters across the basolateral membrane into the blood. Fourth, luminal enzymes may digest dietary proteins to oligopeptides, which are taken up by enterocytes via an endocytotic process (Fig. 45-7) and moved directly into the blood. Overall, protein digestion-absorption is very efficient; 7) do not have increased fecal nitrogen excretion. Five pancreatic enzymes (Table 45-2) participate in protein digestion and are secreted as inactive proenzymes. Trypsinogen is initially activated by a jejunal brush-border enzyme, enterokinase (enteropeptidase), by the cleavage of a hexapeptide, thereby yielding trypsin. Trypsin not only autoactivates trypsinogen but also activates the other pancreatic proteolytic proenzymes. The secretion of proteolytic enzymes as proenzymes, with subsequent luminal activation,

TABLE 45-2  Pancreatic Peptidases PROENZYME

ACTIVATING AGENT

ACTIVE ENZYME

ACTION

PRODUCTS

Trypsinogen

Enteropeptidase (i.e., enterokinase from jejunum) and trypsin

Trypsin

Endopeptidase

Oligopeptides (2–6 amino acids)

Chymotrypsinogen

Trypsin

Chymotrypsin

Endopeptidase

Oligopeptides (2–6 amino acids)

Proelastase

Trypsin

Elastase

Endopeptidase

Oligopeptides (2–6 amino acids)

Procarboxypeptidase A

Trypsin

Carboxypeptidase A

Exopeptidase

Single amino acids

Procarboxypeptidase B

Trypsin

Carboxypeptidase B

Exopeptidase

Single amino acids

Chapter 45  •  Nutrient Digestion and Absorption

N45-6  Pernicious Anemia Contributed by Henry Binder The close relationship between acid and gastrin release is clearly manifested in individuals with impaired acid secretion. In pernicious anemia, atrophy of the gastric mucosa in the corpus and an absence of parietal cells result in a lack in the secretion of both gastric acid and intrinsic factor (IF). Many patients with pernicious anemia exhibit antibody-mediated immunity against their parietal cells, and many of these patients also produce anti-IF autoantibodies. Because IF is required for cobalamin absorption in the ileum, the result is impaired cobalamin absorption. In contrast, the antrum is normal. Moreover, plasma gastrin levels are markedly elevated as a result of the absence of intraluminal acid, which normally triggers gastric D cells to release somatostatin (see pp. 868–870); this, in turn, inhibits antral gastrin release (see Box 42-1). Because parietal cells are absent, the elevated plasma gastrin levels are not associated with enhanced gastric acid secretion. The clinical complications of cobalamin deficiency evolve over a period of years. Patients develop megaloblastic anemia (in which the circulating red blood cells are enlarged), a distinctive form of glossitis, and a neuropathy. The earliest neurological findings are those of peripheral neuropathy, as manifested by paresthesias and slow reflexes, as well as impaired senses of touch, vibration, and temperature. If untreated, the disease will ultimately involve the spinal cord, particularly the dorsal columns, thus producing weakness and ataxia. Memory impairment, depression, and dementia can also result. Parenteral administration of cobalamin reverses and prevents the manifestations of pernicious anemia, but it does not influence parietal cells or restore gastric secretion of either IF or intraluminal acid.

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SECTION VII  •  The Gastrointestinal System

prevents pancreatic autodigestion before enzyme secretion into the intestine. Pancreatic proteolytic enzymes are either exopeptidases or endopeptidases and function in an integrated manner. Trypsin, chymotrypsin, and elastase are endopeptidases with affinity for peptide bonds adjacent to specific amino acids, so that their action results in the production of oligopeptides with two to six amino acids. In contrast, the exopeptidases—carboxypeptidase A and carboxypeptidase B—hydrolyze peptide bonds adjacent to the carboxyl (C) terminus, which results in the release of individual amino acids. The coordinated action of these pancreatic proteases converts ~70% of luminal amino nitrogen to oligopeptides and ~30% to free amino acids.

Brush-border peptidases fully digest some oligopeptides to amino acids, whereas cytosolic peptidases digest oligopeptides that directly enter the enterocyte Small peptides present in the small-intestinal lumen after digestion by gastric and pancreatic proteases undergo further hydrolysis by peptidases at the brush border (see Fig. 45-6). Multiple peptidases are present both on the brush border and in the cytoplasm of villous epithelial cells. This distribution of cell-associated peptidases stands in contrast to that of the oligosaccharidases, which are found only at the brush border. Because each peptidase recognizes only a limited repertoire of peptide bonds, and because the oligopeptides to be digested contain 24 different amino acids, large numbers of peptidases are required to ensure the hydrolysis of peptides. As we discuss below, a transporter on the apical membrane of enterocytes can take up small oligopeptides, primarily dipeptides and tripeptides. Once inside the cell, these oligopeptides may be further digested by cytoplasmic peptidases. The brush-border and cytoplasmic peptidases have substantially different characteristics. For example, the brush-border peptidases have affinity for relatively larger oligopeptides (three to eight amino acids), whereas the cytoplasmic peptidases primarily hydrolyze dipeptides and tripeptides. Because the brush-border and cytoplasmic enzymes often have different biochemical properties (e.g., heat lability and electrophoretic mobility), it is evident that the peptidases in the brush border and cytoplasm are distinct, independently regulated molecules. Like the pancreatic proteases, each of the several brushborder peptidases is an endopeptidase, an exopeptidase, or a dipeptidase with affinity for specific peptide bonds. The exopeptidases are either carboxypeptidases, which release C-terminal amino acids, or aminopeptidases, which hydrolyze the amino acids at the amino (N)–terminal end. Cyto­ plasmic peptidases are relatively less numerous.

PROTEIN, PEPTIDE, AND AMINO-ACID ABSORPTION Absorption of whole protein by apical endocytosis occurs primarily during the neonatal period During the postnatal period, intestinal epithelial cells absorb protein by endocytosis, a process that provides a mechanism

for transfer of passive immunity from mother to child. The uptake of intact protein by the epithelial cell ceases by the sixth month; the cessation of this protein uptake, called closure, is hormonally mediated. For example, administration of corticosteroids during the postnatal period induces closure and reduces the time that the intestine can absorb significant amounts of whole protein. The adult intestine can absorb finite amounts of intact protein and polypeptides. Uncertainty exists regarding the cellular route of absorption, as well as the relationship of the mechanism of protein uptake in adults to that in neonates. Enterocytes can take up by endocytosis a small amount of intact protein, most of which is degraded in lysosomes (see Fig. 45-7). A small amount of intact protein appears in the interstitial space. The uptake of intact protein also occurs through a second, more specialized route. In the small intestine, immediately overlying Peyer’s patches (follicles of lymphoid tissue in the lamina propria), M cells replace the usual enterocytes on the surface of the gut. M cells have few microvilli and are specialized for protein uptake. They have limited ability for lysosomal protein degradation; rather, they package ingested proteins (i.e., antigens) in clathrin-coated vesicles, which they secrete at their basolateral membranes into the lamina propria. There, immunocompetent cells process the target antigens and transfer them to lymphocytes to initiate an immune response. Although protein uptake in adults may not have nutritional value, such uptake is clearly important in mucosal immunity and probably is involved in one or more disease processes.

The apical absorption of dipeptides, tripeptides, and tetrapeptides occurs via an H+-driven cotransporter Virtually all absorbed protein products exit the villous epithelial cell and enter the blood as individual amino acids. Substantial portions of these amino acids are released in the lumen of the small intestine by luminal proteases and brush-border peptidases and, as we discuss below, move across the apical membranes of enterocytes via several amino-acid transport systems (see Fig. 45-6). However, substantial amounts of protein are absorbed from the intestinal lumen as dipeptides, tripeptides, or tetrapeptides and then hydrolyzed to amino acids by intracellular peptidases. The transporter responsible for the uptake of luminal oligopeptides (Fig. 45-8A) is distinct from the various aminoacid transporters. Furthermore, administering an amino acid as a peptide (e.g., the dipeptide glycylglycine) results in a higher blood level of the amino acid than administering an equivalent amount of the same amino acid as a monomer (e.g., glycine; see Fig. 45-8B). One possible explanation for this effect is that the oligopeptide cotransporter, which carries multiple amino acids rather than a single amino acid into the cell, may simply be more effective than amino-acid transporters in transferring amino-acid monomers into the cell. This accelerated peptide absorption has been referred to as a kinetic advantage and raises the question of the usefulness of the enteral administration of crystalline amino acids to patients with impaired intestinal function or catabolic deficiencies. The evidence for a specific transport process for dipeptides, tripeptides, and tetrapeptides comes from direct

Chapter 45  •  Nutrient Digestion and Absorption

A

B OLIGOPEPTIDE ABSORPTION Epithelium Lumen

923

“KINETIC ADVANTAGE” OF PEPTIDE ABSORPTION

Interstitial space PepT1 3 Na

Peptide

+

+

l yc Gl yc

Gl yc

ine

H+

ine

Na+

Peptidases

yl g

H

2K

+

Glycine appearance in blood

Figure 45-8  Absorption of oligopeptides. A, The H/oligopeptide cotransporter PepT1 moves dipeptides, tripeptides, and tetrapeptides into the enterocyte, across the apical membrane. Peptidases in the cytoplasm hydrolyze the oligopeptides into their constituent amino acids, which then exit across the basolateral membrane via one of three Na+-independent amino-acid transporters. B, If glycine is present in the lumen only as a free amino acid, then the enterocyte absorbs it only via apical amino-acid transporters. However, if the same amount of glycine is present in the lumen in the form of the dipeptide glycylglycine, the rate of appearance of glycine in the blood is about twice as high. Thus, PepT1, which moves several amino-acid monomers for each turnover of the transporter, is an effective mechanism for absorbing “amino acids.”

measurements of oligopeptide transport, molecular identification of the transporter, and studies of the hereditary disorders of amino-acid transport, cystinuria, and Hartnup disease. Oligopeptide uptake is an active process driven not by an Na+ gradient, but by a proton gradient. Oligopeptide uptake occurs via an H/oligopeptide cotransporter known as PepT1 (SLC15A1; see p. 123), which is also present in the renal proximal tubule. PepT1 also appears to be responsible for the intestinal uptake of certain dipeptide-like antibiotics (e.g., oral amino-substituted cephalosporins). As noted above, after their uptake, dipeptides, tripeptides, and tetrapeptides are usually hydrolyzed by cytoplasmic peptidases to their constituent amino acids, the forms in which they are transported out of the cell across the basolateral membrane. Because peptides are almost completely hydrolyzed to amino acids intracellularly, few peptides appear in the portal vein. Proline-containing dipeptides, which are relatively resistant to hydrolysis, are the primary peptides present in the circulation.

Amino acids enter enterocytes via one or more group-specific apical transporters Multiple amino-acid transport systems have been identified and characterized in various nonepithelial cells. The absorption of amino acids across the small intestine requires sequential movement across both the apical and basolateral membranes of the villous epithelial cell. Although the amino-acid transport systems have overlapping affinities for various amino acids, the consensus is that at least seven distinct transport systems are present at the apical membrane (see Table 36-1); we discuss the basolateral amino-acid

transporters in the next section. Whereas many apical amino-acid transporters are probably unique to epithelial cells, some of those at the basolateral membrane are probably the same as in nonepithelial cells. The predominant apical amino-acid transport system is system B0 (SLC6A19, SLC6A15; see Table 36-1) and results in Na+-dependent uptake of neutral amino acids. As is the case for glucose uptake (see p. 919), uphill movement of neutral amino acids is driven by an inwardly directed Na+ gradient that is maintained by the basolateral Na-K pump. The uptake of amino acids by system B0 is an electrogenic process and represents another example of secondary active transport. It transports amino acids with an L-stereo configuration and an amino group in the α position. System B0+ (SLC6A14) is similar to system B0 but has broader substrate specificity. System b0+ (SLC7A9/SLC3A1 dimer) differs from B0+ mainly in being independent of Na+. Other apical carrier-mediated transport mechanisms exist for anionic (i.e., acidic) α amino acids, cationic (i.e., basic) α amino acids, β amino acids, and imino acids (see Table 36-1). Because these transporters have overlapping affinities for amino acids, and because of species differences as well as segmental and developmental differences among the transporters, it has been difficult to establish a comprehensive model of apical membrane amino-acid transport in the mammalian small intestine (Box 45-3).

At the basolateral membrane, amino acids exit enterocytes via Na+-independent transporters and enter via Na+-dependent transporters Amino acids appear in the cytosol of intestinal villous cells as the result either of their uptake across the apical

924

SECTION VII  •  The Gastrointestinal System

BOX 45-3  Defects in Apical Amino-Acid Transport: Hartnup Disease and Cystinuria

H

artnup disease and cystinuria are hereditary disorders of amino-acid transport across the apical membrane. These autosomal recessive disorders are associated with both small-intestine and renal-tubule abnormalities (see Box 36-1) in the absorption of neutral amino acids in the case of Hartnup disease and of cationic (i.e., basic) amino acids and cystine in the case of cystinuria. The clinical signs of Hartnup disease are most evident in children and include the skin changes of pellagra, cerebellar ataxia, and psychiatric abnormalities. In Hartnup disease, the absorption of neutral amino acids by system B0 (SLC6A19) in the small intestine is markedly reduced, whereas that of cationic amino acids is intact (Fig. 45-9). The principal manifestation of cystinuria is the formation of kidney stones. In cystinuria, the absorption of cationic amino acids by system b0+ (SLC7A9/SLC3A1 dimer) is abnormal as a result of mutations in SLC7A9 or SLC3A1, but absorption of neutral amino acids is normal. Because neither of these diseases involves the oligopeptide cotransporter, the absorption of oligopeptides containing either

neutral or cationic amino acids is normal in both diseases. Only 10% of patients with Hartnup disease have clinical evidence of protein deficiency (e.g., pellagra) commonly associated with defects in protein or amino-acid absorption. The lack of evidence of protein deficiency is a consequence of the presence of more than one transport system for different amino acids, as well as a separate transporter for oligopeptides. Thus, oligopeptides containing neutral amino acids are absorbed normally in Hartnup disease, and oligopeptides with cationic amino acids are absorbed normally in cystinuria. These two genetic diseases also emphasize the existence of amino-acid transport mechanisms on the basolateral membrane that are distinct and separate from the apical amino-acid transporters. Thus, in both Hartnup disease and cystinuria, oligopeptides are transported normally across the apical membrane and are hydrolyzed to amino acids in the cytosol, and the resulting neutral and cationic amino acids are readily transported out of the cell across the basolateral membrane.

A HARTNUP DISEASE

B CYSTINURIA

L-Phenylalanine

L-Alanine

L-Arginine or cystine

L-Arginine or cystine

Amino acid transporter Oligopeptide cotransporter

L-PhenylalanylL-leucine L-ArginylL-leucine

L-PhenylalanylL-leucine

Oligopeptide cotransporter

L-ArginylL-leucine

Normal subjects Hartnup disease Amino acid absorption

Substrate

Amino acid transporter

Normal subjects Cystinuric patients Amino acid absorption

L-Phe

L-Arg

L-PhenylalanylL-leucine

L-Ala

L-Arg

L-ArginylL-leucine

Figure 45-9  Genetic disorders of apical amino-acid transport. A, In Hartnup disease, an autosomal recessive

disorder, the apical system B0 (SLC6A19) is defective. As a result, the absorption of neutral amino acids, such as L-phenylalanine, is reduced. However, the absorption of L-cystine (i.e., Cys-S-S-Cys) and cationic (i.e., basic) amino acids (e.g., L-arginine) remains intact. The enterocyte can absorb L-phenylalanine normally if the amino acid is present in the form of the dipeptide L-phenylalanyl-L-leucine, inasmuch as the oligopeptide cotransporter PepT1 is normal. B, In cystinuria, an autosomal recessive disorder, the apical system b0+ (SLC7A9/SLC3A1 dimer) is defective. As a result, the absorption of L-cystine and cationic amino acids (e.g., L-arginine) is reduced. However, the absorption of amino acids that use System B0 (e.g., L-alanine) is normal. The enterocyte can absorb L-arginine normally if the amino acid is present in the form of the dipeptide L-arginyl-L-leucine.

Chapter 45  •  Nutrient Digestion and Absorption

BOX 45-4  Defect in Basolateral Amino-Acid Transport:

Lumen

Epithelium

925

Interstitial space

Lysinuric Protein Intolerance

L

ysinuric protein intolerance is a rare autosomal recessive disorder of amino-acid transport across the basolateral membrane (Fig. 45-10). Evidence indicates impaired cationic amino-acid transport, and symptoms of malnutrition are seen. It appears that the defect is in system y+L, which is located solely on the basolateral membrane. System y+L has two subtypes, y+LAT1 (SLC7A7/SLC3A2 dimer) and y+LAT2 (SLC7A6/SLC3A2 dimer). Mutations in the SLC7A7 gene (subtype y+LAT1) cause the disease lysinuric protein intolerance. These patients exhibit normal absorption of cationic amino acids across the apical membrane. Unlike in Hartnup disease or cystinuria, in which the enterocytes can absorb the amino acid normally if it is presented as an oligopeptide, in lysinuric protein intolerance the enterocytes cannot absorb the amino acid regardless of whether the amino acid is “free” or is part of an oligopeptide. These observations are best explained by hypothesizing that the patients hydrolyze intracellular oligopeptides properly but have a defect in the transport of cationic amino acids across the basolateral membrane. This defect is present not only in the small intestine but also in hepatocytes and kidney cells, and perhaps in nonepithelial cells as well.

membrane or of the hydrolysis of oligopeptides that had entered the apical membrane (see Fig. 45-6). The enterocyte subsequently uses ~10% of the absorbed amino acids for intracellular protein synthesis. Movement of amino acids across the basolateral membrane is bidirectional; the movement of any one amino acid can occur via one or more amino-acid transporters. At least six amino-acid transport systems are present in the basolateral membrane (see Table 36-1). Three amino-acid transport processes on the basolateral membrane mediate amino-acid exit from the cell into the blood and thus complete the process of protein assimilation. Two other amino-acid transporters mediate uptake from the blood for the purposes of cell nutrition. The three Na+-independent amino-acid transport systems appear to mediate amino-acid movement out of the epithelial cell into blood. The two Na+dependent processes facilitate their movement into the epithelial cell. Indeed, these two Na+-dependent transporters resemble those that are also present in nonpolar cells. In general, the amino acids incorporated into protein within villous cells are derived more from those that enter across the apical membrane than from those that enter across the basolateral membrane. In contrast, epithelial cells in the intestinal crypt derive almost all their amino acids for protein synthesis from the circulation; crypt cells do not take up amino acids across their apical membrane (Box 45-4).

H

+

Lysine-XX Na+

Lysine-XX PepT1 Lysine

SLC7A7 defective

SLC3A2

Lysine

Figure 45-10  Genetic disorder of basolateral amino-acid transport. Lysinuric protein intolerance is an autosomal recessive defect in which the Na+-independent y+L amino-acid transporter on the apical and basolateral membranes is defective. However, the absence of apical y+L (SLC7A6/ SLC3A2 or SLC7A7/SLC3A2 dimers) does not present a problem because Na+-dependent amino-acid transporters can take up lysine, and PepT1 can take up lysine-containing oligopeptides (Lys-XX). However, there is no other mechanism for moving lysine out of the enterocyte across the basolateral membrane.

of oxygen. Some lipids also contain small but functionally important amounts of nitrogen and phosphorus (Fig. 45-11). Lipids are typified by their preferential solubility in organic solvents compared with water. A widely used indicator of the lipidic nature of a compound is its octanol-water partition coefficient, which for most lipids is between 104 and 107. The biological fate of lipids depends critically on their chemical structure as well as on their interactions with water and other lipids in aqueous body fluids (e.g., intestinal contents and bile). Thus, lipids have been classified according to their physicochemical interactions with water. Lipids may be either nonpolar and thus very insoluble in water (e.g., cholesteryl esters and carotene) or polar and thus interacting with water to some degree. Even polar lipids are only amphiphilic; that is, they have both polar (hydrophilic) and nonpolar (hydrophobic) groups. Polar lipids range from the insoluble, nonswelling amphiphiles (e.g., triacylglycerols) to the soluble amphiphiles (e.g., bile acids). Added in small amounts, insoluble polar lipids form stable monolayers on the surface of water (see Fig. 2-1C), whereas the soluble amphiphiles do not. The physicochemical behavior in bulk solution varies from insolubility—as is the case with triacylglycerols (TAGs) and cholesterol—to the formation of various macroaggregates, such as liquid crystals and micelles. Less-soluble lipids may be incorporated into the macroaggregates of the more polar lipids and are thus stably maintained in aqueous solutions.

Dietary lipids are predominantly TAGs

LIPID DIGESTION Natural lipids of biological origin are sparingly soluble in water Lipids in the diet are derived from animals or plants and are composed of carbon, hydrogen, and a smaller amount

The term fat is generally used to refer to TAGs—formerly called triglycerides—but it is also used loosely to refer to lipids in general. Of the fat in an adult diet, >90% is TAGs, which are commonly long-chain fatty acyl esters of glycerol, a trihydroxyl alcohol. The three esterification (i.e., acylation) positions on the glycerol backbone that are occupied by

926

B

OH C

C

GLYCEROL

H O

H

H

H

C

C

C

H

H

OH OH OH

CH2

CH3

F

PHOSPHATIDYLCHOLINE

G

H3C

N

+

CH3 H3C

O

O

C CH2

O

CH

CH2

C

O

CH2

C

O

O

O

C

O C

O C

CH2

CH2

CH3

CH3

H

H

O

CH2

CH3

H

H

H

H

C

C

C

O

O

C

O C

sn2-MONOACYLGLYCEROL H

H

H

H

H

C

C

C

OH O

OH O

C

CH2

CH2

CH2

CH3

CH3

CH3

I

CHOLESTEROL

CHOLESTERYL ESTER

J

H

OH O

CHOLIC ACID

HO

CH3

OH

+

N

CH3

CH3

CH3

OH HO

P

O C

CH2

CH

CH2 CH2 H3C

CH2

CH3

CH2

CH2

CH2

OH O

CH3

CH3 CH3

–

O

O

CH2

O

C

O

–

O

O CH

C

E

DIACYLGLYCEROL

CH2

O

CH2

H

CH2

CH2

P

H

CH3

CH2

O

H

LYSOPHOSPHATIDYLCHOLINE

CH3

D

TRIACYLGLYCEROL

CH2

CH2

C

O

O

FATTY ACID

O

A

SECTION VII  •  The Gastrointestinal System

C OH

CH CH3 CH3

CH3 CH 3 CH CH2 CH3

CH2

CH3 CH3

CH2 H3C

CH CH3

Figure 45-11  Chemical formulas of some common lipids. The example in A is stearic acid, a fully saturated

fatty acid with 18 carbon atoms. B shows glycerol, a trihydroxy alcohol, with hydroxyl groups in positions sn1, sn2, and sn3. In C, the left sn1– and center sn2–fatty acids are palmitic acid, a fully saturated fatty acid with 16 carbon atoms. The rightmost sn3–fatty acid is palmitoleic acid, which is also a 16-carbon structure, but with a double bond between carbons 9 and 10. In F, the left sn1–fatty acid is palmitic acid, and the right sn2–fatty acid is palmitoleic acid. In I, the example is the result of esterifying cholesterol and palmitic acid.

hydroxyl groups are designated sn1, sn2, and sn3, according to a stereochemical numbering system adopted by an international committee on biochemical nomenclature (see Fig. 45-11C–E). At body temperature, fats are usually liquid droplets. Dietary fat is the body’s only source of essential fatty acids, and its hydrolytic products promote the absorption of fat-soluble vitamins (the handling of which is

discussed on p. 933). Fat is also the major nutrient responsible for postprandial satiety. Typical adult Western diets contain ~140 g of fat per day (providing ~60% of the energy), which is more than the recommended intake of less than ~70 g of fat per day (2 km of bile ductules and ducts, with a volume of ~20 cm3 and a macroscopic surface area of

~400 cm2. Microvilli at the apical surface magnify this area by ~5.5-fold. As noted above, the canaliculi into which bile is secreted form a three-dimensional polygonal meshwork of tubes between hepatocytes, with many anastomotic interconnections (see Fig. 46-1). From the canaliculi, the bile enters the small terminal bile ductules (i.e., canals of Hering), which have a basement membrane and in cross section are surrounded by three to six ductal epithelial cells or hepatocytes (Fig. 46-4A). The canals of Hering then empty into a system of perilobular ducts, which, in turn, drain into interlobular bile ducts. The interlobular bile ducts form a richly anastomosing network that closely surrounds the branches of the portal vein. These bile ducts are lined by a layer of cuboidal or columnar epithelium that has microvillous architecture on its luminal surface. The cells have a prominent Golgi apparatus and numerous vesicles, which probably participate in the exchange of substances among the cytoplasm, bile, and blood plasma through exocytosis and endocytosis. The interlobular bile ducts unite to form larger and larger ducts, first the septal ducts and then the lobar ducts, two hepatic ducts, and finally a common hepatic duct (see Fig. 46-4B). Along the biliary tree, the biliary epithelial cells, or cholangiocytes, are similar in their fine structure except for size and height. However, as discussed below (see pp. 960–961), in terms of their functional properties, cholangiocytes and bile ducts of different sizes are heterogeneous in their expression of enzymes, receptors, and transporters. Increasing emphasis has been placed on the absorptive and secretory properties of the biliary epithelial cells, properties that contribute significantly to the process of bile formation. As with other epithelial cells, cholangiocytes are highly cohesive, with the lateral plasma membranes of contiguous cells

949

Chapter 46  •  Hepatobiliary Function

A

DUCTULES AND SMALL DUCTS Canaliculi

Terminal bile ductules

B

LARGE DUCTS AND GALLBLADDER

Perilobular bile duct

Right hepatic duct

Interlobular bile duct Liver

Left hepatic duct

Cystic duct

Common hepatic duct Common bile duct

Section of liver lobule

Pancreatic duct

Gallbladder Duodenum Ampulla (of Vater)

Hepatocytes

Sphincter of Oddi

Bile flow Figure 46-4  Structure of the biliary tree. A, The bile canaliculi, which are formed by the apical membranes of adjacent hepatocytes, eventually merge with terminal bile ductules (canals of Hering). The ductules eventually merge into perilobular ducts, and then interlobular ducts. B, The interlobular ducts merge into septal ducts and lobar ducts (not shown), and eventually the right and left hepatic ducts, which combine as the common hepatic duct. The confluence of the common hepatic duct and the cystic duct gives rise to the common bile duct. The common bile duct may merge with the pancreatic duct and form the ampulla of Vater before entering the duodenum, as shown in the figure, or have a completely independent lumen. In either case, there is a common sphincter—the sphincter of Oddi—that simultaneously regulates flow out of the common bile duct and the pancreatic duct.

forming tortuous interdigitations. Tight junctions seal contacts between cells that are close to the luminal region and thus limit the exchange of water and solutes between plasma and bile. The common hepatic duct emerges from the porta hepatis after the union of the right and left hepatic ducts. It merges with the cystic duct emanating from the gallbladder to form the common bile duct. In adults, the common bile duct is quite large, ~7 cm in length and ~0.5 to 1.5 cm in diameter. In most individuals, the common bile duct and the pancreatic duct merge before forming a common antrum known as the ampulla of Vater. At the point of transit through the duodenal wall, this common channel is surrounded by a thickening of both the longitudinal and the circular layers of smooth muscle, the so-called sphincter of Oddi. This sphincter constricts the lumen of the bile duct and thus regulates the flow of bile into the duodenum. The hormone cholecystokinin (CCK) relaxes the sphincter of Oddi via a nonadrenergic, noncholinergic neural pathway (see pp. 344–345) involving vasoactive intestinal peptide (VIP). The gallbladder lies in a fossa beneath the right lobe of the liver. This distensible pear-shaped structure has a capacity of 30 to 50 mL in adults. The absorptive surface of the gallbladder is enhanced by numerous prominent folds that

are important for concentrative transport activity, as discussed below. The gallbladder is connected at its neck to the cystic duct, which empties into the common bile duct (see Fig. 46-4B). The cystic duct maintains continuity with the surface columnar epithelium, lamina propria, muscularis, and serosa of the gallbladder. Instead of a sphincter, the gallbladder has, at its neck, a spiral valve—the valve of Heister—formed by the mucous membrane. This valve regulates flow into and out of the gallbladder.

UPTAKE, PROCESSING, AND SECRETION OF COMPOUNDS BY HEPATOCYTES The liver metabolizes an enormous variety of compounds that are brought to it by the portal and systemic circulations. These compounds include endogenous molecules (e.g., bile salts and bilirubin, which are key ingredients of bile) and exogenous molecules (e.g., drugs and toxins). The hepatocyte handles these molecules in four major steps (Fig. 46-5A): (1) the hepatocyte imports the compound from the blood across its basolateral (i.e., sinusoidal) membrane, (2) the hepatocyte transports the material within the cell, (3) the hepatocyte may chemically modify or degrade the

950 A

SECTION VII  •  The Gastrointestinal System

C

HEPATOCYTE Space of Disse

SECRETION OF BILE ACIDS AND SALTS Conjugated and unconjugated bile salts BA– H+ + BA– Charged Unconjugated (salt) BA-Z– + Na BA– Neutral OATP1B1, H.BA (acid) 1B3 NTCP MRP4 (ABCC4) Pool of BA– and BA-Z– HCO3– . H BA

Sinusoid endothelium 4

Bile canaliculus 2

1

3 Chemical modification

Binding protein

BA-Z– BP

Blood in sinusoid

+

BA-Y– BA-Z–

H+

Y–

BP.BA-Z– BA-Y– Hepatocyte

Bile canaliculus

BA– H2O

AA

MRP2 (ABCC2)

Z

B HOUSEKEEPING TRANSPORTERS Na+

BA–

+

Na Glucose

2 HCO3–

Na CO2

BSEP (ABCB11)

BA– or BA-Z–

+

BP GLUT2 H+

H2O CO2 OH–

HCO3–

–

SO4–

D

SECRETION OF OTHER ORGANIC ANIONS

Carbonic anhydrase

Other organic acid OA–

OATP1B1, 1B3

HCO3–

Bile salts Cholesterol Phospholipids Bilirubin Cl – HCO3–

OA–

OATP

MRP4 (ABCC4)

–

GSH–

HCO3

Y

Cl –

OA–

GSH–

Glutathione

OA-Y

GSH– 3 Na+

Ca++

+

Cl –

K

MRP2 (ABCC2)

GSH-Y

Bile canaliculus

H+

2 K+

BCRP (ABCG2)

Y +

Na

GSH–

S-Y

Y

S

Figure 46-5  Transporters in hepatocyte. A, The hepatocyte can process compounds in four steps: (1) uptake from blood across the basolateral (i.e., sinusoidal) membrane; (2) transport within the cell; (3) control chemical modification or degradation; and (4) export into the bile across the apical (i.e., canalicular) membrane. B, The hepatocyte has a full complement of housekeeping transporters. C, Bile acids can enter the hepatocyte in any of several forms: the unconjugated salt (BA−); the neutral, protonated bile acid (H ⋅ BA); or the bile salt conjugated to taurine or glycine (BA-Z−, where Z represents taurine or glycine). The three pathways for bile acid entry across the basolateral membrane are the Na+-driven transporter NTCP, which prefers BA-Z− but also carries BA−; nonionic diffusion of H ⋅ BA; and an OATP. Binding proteins (BPs) may ferry conjugated bile acids across the cytoplasm. Some bile acids are conjugated to sulfate or glucuronate (Y); these exit the cell across the canalicular membrane via the MRP2 (multidrug resistance–associated protein 2) transporter. Most bile acids are conjugated to glycine or taurine (Z) prior to their extrusion into the bile via BSEP. D, Organic anions (OA), including bile acids and bilirubin, may enter across the basolateral membrane via an OATP. After conjugation with sulfate or glucuronate (Y), these compounds may be extruded into the bile by MRP2. GSH synthesized in the hepatocyte, after conjugation to Y, can enter the canaliculus via MRP2. Unconjugated GSH can enter the canaliculus via an unidentified transporter. GSH can exit the hepatocyte across the basolateral membrane via an OATP. AA, amino acid.

Chapter 46  •  Hepatobiliary Function

compound intracellularly, and (4) the hepatocyte excretes the molecule or its product or products into the bile across the apical (i.e., canalicular) membrane. Thus, compounds are secreted in a vectorial manner through the hepatocyte.

An Na-K pump at the basolateral membranes of hepatocytes provides the energy for transporting a wide variety of solutes via channels and transporters Like other epithelial cells, the hepatocyte is endowed with a host of transporters that are necessary for basic housekeeping functions.  N46-2  To the extent that these transporters are restricted to either the apical or basolateral membrane, they have the potential of participating in net transepithelial transport. For example, the Na-K pump (see pp. 115–117) at the basolateral membrane of hepatocytes maintains a low [Na+]i and high [K+]i (see Fig. 46-5B). A basolateral Ca pump (see p. 118) maintains [Ca2+]i at an extremely low level, ~100 nM, as in other cells. The hepatocyte uses the inwardly directed Na+ gradient to fuel numerous active transporters, such as the Na-H exchanger, Na/HCO3 cotransporter, and Na+-driven amino-acid transporters. As discussed below, the Na+ gradient also drives one of the bile acid transporters. The hepatocyte takes up glucose via the GLUT2 facilitateddiffusion mechanism (see p. 114), which is insensitive to regulation by insulin. The basolateral membrane has both K+ and Cl− channels. The resting membrane potential (Vm) of −30 to −40 mV is considerably more positive than the equilibrium potential for K+ (EK) because of the presence of numerous “leak” pathways, such as the aforementioned electrogenic Na+-driven transporters as well as Cl− channels (ECl = Vm).

Hepatocytes take up bile acids, other organic anions, and organic cations across their basolateral (sinusoidal) membranes Bile Acids and Salts  The primary bile acids are cholic acid and chenodeoxycholic acid, both of which are synthesized by hepatocytes (see p. 959, below). Other “secondary” bile acids form in the intestinal tract as bacteria dehydroxylate the primary bile acids. Because the pK values of the primary bile acids are near neutrality, most of the bile acid molecules are neutral; that is, they are bile acids (H ⋅ BA) and thus are not very water soluble. Of course, some of these molecules are deprotonated and hence are bile salts (BA−). The liver may conjugate the primary bile acids and salts to glycine or taurine (Z in Fig. 46-5C), as well as to sulfate or glucuronate (Y in Fig. 46-5C). Most of the bile acids that the liver secretes into the bile are conjugated, such as taurocholate (the result of conjugating cholic acid to taurine). These conjugated derivatives have a negative charge and hence they, too, are bile salts (BA-Z− and BA-Y−). Bile salts are far more water soluble than the corresponding bile acids. Because the small intestine absorbs some bile acids and salts, they appear in the blood plasma, mainly bound to albumin, and are presented to the hepatocytes for re-uptake. This recycling of bile acids, an example of enterohepatic circulation (see p. 962 below). Dissociation from albumin occurs before uptake. Surprisingly, the presence of albumin actually stimulates Na+-dependent taurocholate uptake,

951

perhaps by increasing the affinity of the transporter for taurocholate. Uptake of bile acids has been studied extensively and is mediated predominantly by an Na+-coupled transporter known as Na/taurocholate cotransporting polypeptide or NTCP (a member of the SLC10A1 family; see Fig. 46-5C). This transporter is a 50-kDa glycosylated protein, and it appears to have seven membrane-spanning segments. NTCP handles unconjugated bile acids, but it has a particularly high affinity for conjugated bile acids. In addition, NTCP can also transport other compounds, including neutral steroids (e.g., progesterone, 17β-estradiol sulfate), cyclic oligopeptides (e.g., amantadine and phalloidin), and a wide variety of drugs (e.g., verapamil, furosemide).  N46-3 Although NTCP also carries unconjugated bile acids, as much as 50% of these unconjugated bile acids may enter the hepatocyte by passive nonionic diffusion (see Fig. 46-5B). Because unconjugated bile acids are weak acids of the form

H ⋅ BA  H + + BA −

(46-1)

the neutral H ⋅ ΒΑ form can diffuse into the cell. Conjugation of bile acids enhances their hydrophilicity (taurine more so than glycine) and promotes dissociation of the proton from the side chain (i.e., lowers the pKa), thus raising the concentration of BA−. Both properties decrease the ability of bile acid to traverse membranes via passive nonionic diffusion. Organic Anions  The organic anion–transporting polypeptides (OATPs) are members of the SLC21 family (see p. 125)  N46-4  and mediate the Na+-independent uptake of a wide spectrum of endogenous and exogenous amphipathic compounds—including bile acids, bilirubin, eicosanoids, steroid and thyroid hormones, prostaglandins, statin drugs, methotrexate, bromosulfophthalein, and many xenobiotics. Individual OATPs share considerable overlap in substrate specificity and can substantially influence the pharma­ cokinetics and pharmacological efficacy of drugs they carry. OATPs—predicted to have 12 membrane-spanning segments, with intracellular amino and carboxy termini— appear to exchange organic anions for intracellular HCO3− (see Fig. 46-5C, D). Expression of OATPs is under the control, in a cell- and tissue-specific way, of nuclear receptors (FXR, LXR, SXR, CAR; see Table 3-6) and hepatocyte nuclear factor 4 (HNF4). OATP1B1, OATP1B3, and OATP2B1 are liver specific and are located on the sinusoidal (basolateral) membrane of hepatocytes. Thus, the basolateral uptake of bile acids into the hepatocyte is a complex process that involves both an Na+dependent transporter (NTCP) and Na+-independent transporters (OATPs), as well as nonionic diffusion of unconjugated bile acids. Bilirubin  Senescent erythrocytes are taken up by macrophages in the reticuloendothelial system, where the degradation of hemoglobin leads to the release of bilirubin into the blood (Fig. 46-6A and Box 46-1). The mechanism by which hepatocytes take up unconjugated bilirubin remains con­ troversial. As evidenced by yellow staining of the sclerae and skin in the jaundiced patient, bilirubin can leave the

Chapter 46  •  Hepatobiliary Function

N46-2  Hepatocyte Housekeeping Functions

951.e1

N46-3  Regulation of Na/Taurocholate Cotransport

Contributed by Fred Suchy

Contributed by Emile Boulpaep and Walter Boron

As noted in the text, the basolateral membrane of the hepatocyte has both K+ and Cl− channels. The basolateral K+ conductance is high and is regulated by cAMP, [Ca2+]i, cell volume, and temperature. The basolateral Cl− conductance is under the regulation of hormones and cell volume. As is the case for most cells, hepatocytes actively regulate their intracellular pH (see pp. 644–645) using two acid extruders, the basolateral (i.e., sinusoidal) Na-H exchanger and an electrogenic Na/HCO3 cotransporter. The apical (i.e., canalicular) Cl-HCO3 exchanger may contribute as an acid loader. The pH gradient across the canalicular membrane also drives the transport of inorganic solutes (e.g., HCO3-SO4 exchange) and maintains the transmembrane gradients of weak acids and bases that cross the membrane by nonionic diffusion (see p. 784).

Bile acid uptake via the Na/taurocholate cotransporting polypeptide (NTCP) is under the regulation of several second messengers. For example, cAMP stimulates taurocholate uptake, whereas this effect is blocked by inhibitors of protein kinase A. This direct stimulation presumably reflects the phosphorylation of the transporter or an essential activator. cAMP also stimulates uptake indirectly by increasing translocation of the transport protein to the membrane. Certain hormones, such as prolactin, also stimulate bile acid uptake directly. As is the case for many other transporters, NTCP activity is low in the fetus and neonate and increases with development. NTCP has now been classified as a member of the SLC10 gene family (see Table 5-4) of Na/bile-salt cotransporters. For a detailed discussion of the family members, consult the review by Hagenbuch and Dawson listed below.

REFERENCE

N46-4  Organic Anion Transporters Contributed by Emile Boulpaep and Walter Boron The organic-anion transporting proteins (OATPs) have now been classified as members of the SLC21 gene family (see Table 5-4). For a detailed discussion of the family mem­ bers, consult the review by Hagenbuch and Meier listed below. Note that some have inappropriately stated that because this family has an immense number of genes, it really ought to be treated as a superfamily, with the designation SLCO (here the O refers to “organic”). However, the superfamily is large only when one includes genes from all known organisms. The actual number of OATP genes in any given vertebrate organism (humans have 11 such genes) is about the same as for other SLC families. Thus, the OATPs are appropriately described as a “family.”

REFERENCE Hagenbuch B, Meier PJ: Organic anion transporting polypeptides of the OATP/SLC21 family: Phylogenetic classification as OATP/SLC0 superfamily, new nomenclature and molecular/functional properties. Pflugers Arch 447:653– 665, 2004.

Hagenbuch B, Dawson P: The sodium bile salt cotransport family SLC10. Pflugers Arch 447:566–570, 2004.

952 A

SECTION VII  •  The Gastrointestinal System

B BILIRUBIN SECRETION

HEME METABOLISM

Systemic circulation Bilirubin-albumin complex

A N

D

Fe

N

2+

N

B (Electrogenic)

N

?

Bilirubin Heme

NADPH + H+ 2O2

ER

Heme oxygenase

NADP

2+

+

+

CO

O

+ H2O

OATP1B1, 1B3

Bilirubin HCO3–

?

+

Fe

Conjugated bilirubin

Bilirubin

C

UGT

B

C

D

A

N H

N H

N

N H

O

Glucuronic acid

Bilirubinmono- and diglucuronide

Biliverdin

(Electroneutral)

Bilirubinglucuronide and urobilinogen

+

NADPH + H

Biliverdin reductase

MRP2 (ABCC2)

NADP+

Hepatocyte

Bile O

B

C

N H

N H H

D

A

N H H

N H

Bilirubin 8H•

Gallbladder

O

Bilirubinglucuronide

Microbial enzymes

Bilirubin Small intestine B O

C

H

N H H

N H H

D C H2

H

N H H

Urobilinogen

A N H H

B

H

C

C H2

N H

Kidney

Kidney

2H•

N H

Excretion in feces

O

Urobilinogen

O

Stercobilin

C H

D

H

A

N

C H2

N H

Urobilin in urine

O

Urine Urobilin

Figure 46-6  Excretion of bilirubin. A, Macrophages phagocytose senescent red blood cells and break the heme down to bilirubin, which travels in the blood, linked to albumin, to the liver. The conversion to the colorless urobilinogen occurs in the terminal ileum and colon, whereas the oxidation to the yellowish urobilin occurs in the urine. B, The hepatocyte takes up bilirubin across its basolateral membrane via an OATP and other unidentified mechanisms. The hepatocyte then conjugates the bilirubin with one or two glucuronic acid residues and exports this conjugated form of bilirubin into the bile. Bacteria in the terminal ileum and colon convert some of this bilirubin glucuronide back to bilirubin. This bilirubin is further converted to the colorless urobilinogen. If it remains in the colon, the compound is further converted to stercobilin, which is the main pigment of feces. If the urobilinogen enters the plasma and is filtered by the kidney, it is converted to urobilin and gives urine its characteristic yellow color. NADP+, oxidized form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.

Chapter 46  •  Hepatobiliary Function

953

BOX 46-1  Jaundice

J

aundice denotes a yellowish discoloration of body tissues, most notable in the skin and sclera of the eyes. The condition is caused by an accumulation of bilirubin in extracellular fluid, either in free form or after conjugation. Bilirubin is a yellowgreen pigment that is the principal degradation product of heme (see Fig. 46-6A), a prosthetic group in several proteins, including hemoglobin. The metabolism of hemoglobin (Hb) of senescent red cells accounts for 65% to 80% of total bilirubin production. Hb released into the circulation is phagocytized by macrophages throughout the body, which split Hb into globin and heme. Cleavage of the heme ring releases both free iron, which travels in the blood by transferrin, and a straight chain of 4-pyrrole nuclei called biliverdin (see Fig. 46-6A), which the cell rapidly reduces to free bilirubin. This lipophilic form of bilirubin is often referred to as unconjugated bilirubin. After it enters the circulation, unconjugated bilirubin binds reversibly to albumin and travels to the liver, which avidly removes it from the plasma (see Fig. 46-6B). After hepatocytes take up bilirubin, they use UGT1A1 to convert the bilirubin to monoglucuronide and diglucuronide conjugates. These two forms of water-soluble, conjugated bilirubin—which make up the direct bilirubin measured in clinical laboratories— enter the bile canaliculus via MRP2 (see Table 5-6). Although suitable for excretion into bile, conjugated bilirubin cannot be absorbed by the biliary or intestinal epithelia. Because of avid extraction and conjugation of bilirubin by the liver, the normal plasma concentration of bilirubin, which is mostly of the unconjugated variety, is ~0.5 mg/dL or lower. The skin or eyes may begin to appear jaundiced when the bilirubin level rises to 1.5 to 3 mg/dL. Jaundice occurs under several circumstances. Increased destruction of red blood cells or hemolysis may cause unconjugated hyperbilirubinemia. Transient physiological neonatal jaundice results from an increased turnover of red blood cells combined with the immaturity of the pathways for conjugation of bilirubin (exacerbated in premature infants) Pathological conditions that can increase bilirubin production in neonates include isoimmunization, heritable hemolytic disorders, and extravasated blood (e.g., from bruises and cephalhematomas). Genetic disorders of bilirubin conjugation include the mild deficiency of UGT1A1 seen in the common Gilbert syndrome and

circulation and enter tissues by diffusion. However, uptake of albumin-bound bilirubin by the isolated, perfused rat liver and isolated rat hepatocytes is faster than can occur by diffusion and is consistent with a carrier-mediated process. Electroneutral, electrogenic, and Cl−-dependent transport have been proposed (see Fig. 46-6B). OATP1B1 and OATP1B3 can transport conjugated, and possibly unconjugated, bilirubin in vitro. Indeed, human mutations resulting in the complete deficiency of OATP1B1 or OATP1B3 cause Rotor syndrome, a relatively benign autosomal recessive disorder characterized by conjugated— not unconjugated—hyperbilirubinemia. How did this con­ jugated bilirubin—made only in hepatocytes—get into the blood? It is now clear that hepatocytes secrete substantial amounts of glucuronidated bilirubin across the sinusoidal membrane into the space of Disse and that OATP1B1/ OATP1B3 is responsible for the reuptake of this conjugated bilirubin under physiological conditions. Other hepatic

the near-complete or complete deficiency of UGT1A1 seen in the rare Crigler-Najjar syndrome. Extreme unconjugated hyperbilirubinemia can lead to a form of brain damage called kernicterus (from the Dutch kern [nucleus, as in a brain nucleus] + the Greek icteros [jaundice]). Neonatal hyperbilirubinemia is often treated with phototherapy, which converts bilirubin to photoisomers and colorless oxidation products that are less lipophilic than bilirubin and do not require hepatic conjugation for excretion. Photoisomers are excreted mainly in the bile, and oxidation products, predominantly in the urine. Jaundice can also result from defects in the secretion of conjugated bilirubin from hepatocytes into bile canaliculi (as with certain types of liver damage) or from defects in transiting the bilirubin to the small intestine (as with obstruction of the bile ducts). In either case, conjugated bilirubin refluxes back into the systemic circulation, where it now accounts for most of the bilirubin in plasma. Because the kidneys can filter the highly soluble conjugated bilirubin—in contrast to the poorly soluble free form of bilirubin mostly bound to albumin—it appears in the urine. Thus, in obstructive jaundice, conjugated bilirubin imparts a dark yellow color to the urine. Measurement of free and conjugated bilirubin in serum serves as a sensitive test for detecting liver disease. Under normal conditions, approximately half of the bilirubin reaching the intestinal lumen is metabolized by bacteria into the colorless urobilinogen (see Fig. 46-6A). The intestinal mucosa reabsorbs ~20% of this soluble compound into the portal circulation. The liver then extracts most of the urobilinogen and re-excretes it into the gastrointestinal tract. The kidneys excrete a small fraction (~20% of daily urobilinogen production) into the urine. Urobilinogen may be detected in urine by using a clinical dipstick test. Oxidation of urobilinogen yields urobilin, which gives urine its yellow color. In the feces, metabolism of urobilinogen yields stercobilin, which contributes to the color of feces. In obstructive jaundice, no bilirubin reaches the intestine for conversion into urobilinogen, and therefore no urobilinogen appears in the blood for excretion by the kidney. As a result, tests for urobilinogen in urine are negative in obstructive jaundice. Because of the lack of stercobilin and other bile pigments in obstructive jaundice, the stool becomes clay colored.

mechanisms may mediate the uptake of unconjugated bilirubin. Organic Cations  The major organic cations transported by the liver are aromatic and aliphatic amines, including important drugs such as cholinergics, local anesthetics, and antibiotics, as well as endogenous solutes such as choline, thiamine, and nicotinamide (Fig. 46-7). At physiological pH, ~40% of drugs are organic cations, in equilibrium with their respective conjugate weak bases (see p. 628). Members of the organic cation transporter (OCT) family mediate the uptake of a variety of structurally diverse lipophilic organic cations of endogenous or xenobiotic origin (see p. 115). OCT-mediated transport is electrogenic, independent of an Na+ ion or proton gradient, and may occur in either direction across the plasma membrane. Human hepatocytes express only OCT1 (SLC22A1) and OCT3 (SLC22A3), localized to the sinusoidal membrane. OCT1 and OCT3 have

954

SECTION VII  •  The Gastrointestinal System

Sinusoid

Hepatocyte MDR3 (ABCB4)

APL

PL MATE1

H+

OC+

ATP8B1

OC+ MDR1 (ABCB1) OATP1A2

H+

Organic cation

OCT1 and OCT3

OC+

Bulky OC+ Small OC+

ABCG5/ABCG8 Cholesterol Figure 46-7  Excretion of organic cations and lipids. APL, aminophospholipid; C, cholesterol; PL, phospholipid.

partly overlapping substrate specificities. OCT1 is also present in the plasma membrane of cholangiocytes. Acyclovir and lidocaine are examples of OCT1 substrates. The neurotransmitters epinephrine, norepinephrine, and histamine are exclusive OCT3 substrates. In addition to the OCTs, members of the OATP family as well as an electroneutral proton-cation exchanger may contribute to organic cation uptake across the basolateral membrane. Neutral Organic Compounds  This group of molecules is also taken up by an Na+-independent, energy-dependent process, although the nature of the driving force is not known. The best-characterized substrate is ouabain, uptake of which is inhibited by other neutral steroids, such as cortisol, aldosterone, estradiol, and testosterone. OATP1B1 transports some of these compounds. We return to Figures 46-5 through 46-7 below, when we discuss the movement of solutes into the bile canaliculus.

Inside the hepatocyte, the basolateral-to-apical movement of many compounds occurs by protein-bound or vesicular routes Bile Salts  Some compounds traverse the cell while bound to intracellular “binding” proteins (see Fig. 46-5C). The binding may serve to trap the molecule within the cell, or it may be involved in intracellular transport. For bile salts, three such proteins have been identified. In humans, the main bile acid–binding protein appears to be the hepatic dihydrodiol dehydrogenase, one of a large family of dehydrogenases, the catalytic and binding properties of which are organ and species specific. The two others are glutathioneS-transferase B and fatty acid–binding protein. Intracellular sequestration of bile salts by these proteins may serve an important role in bile acid transport or regulation of bile acid synthesis. Transcellular diffusion of bile salts bound to

proteins can be detected within seconds after bile salts are applied to hepatocytes; this mechanism may be the primary mode of cytoplasmic transport under basal conditions. Free, unbound bile acids may also traverse the hepatocyte by rapid diffusion. At high sinusoidal concentrations, hydrophobic bile acids may partition into membranes of intracellular vesicles. These conditions may also cause increased targeting of the vesicles to the canalicular membrane—that is, transcellular bile acid transport by a vesicular pathway. Whether transcellular transport occurs by protein-binding or vesicular pathways, it is unknown how bile acids are so efficiently targeted to the canalicular membrane for excretion into bile. Bilirubin  After uptake at the basolateral membrane, unconjugated bilirubin is transported to the endoplasmic reticulum (ER), where it is conjugated to glucuronic acid (see Fig. 46-6). Because the resulting bilirubin glucuronide is markedly hydrophobic, it was thought that intracellular transport was mediated by binding proteins such as glutathione-S-transferase B. However, spontaneous transfer of bilirubin between phospholipid vesicles occurs by rapid movement through the aqueous phase, in the absence of soluble proteins. Thus, direct membrane-to-membrane transfer may be the principal mode of bilirubin transport within the hepatocyte. In addition, the membrane-tomembrane flux of bilirubin is biased toward the membrane with the higher cholesterol/phospholipid ratio. Hence, the inherent gradient for cholesterol from the basolateral membrane to the ER membrane may direct the flux of bilirubin to the ER.

In phase I of the biotransformation of organic anions and other compounds, hepatocytes use mainly cytochrome P-450 enzymes The liver is responsible for the metabolism and detoxification of many endogenous and exogenous compounds. Some compounds taken up by hepatocytes (e.g., proteins and other ligands) are completely digested within lysosomes. Specific carriers exist for the lysosomal uptake of sialic acid, cysteine, and vitamin B12. Clinical syndromes resulting from an absence of these carriers have also been identified. The lysosomal acid hydrolases cleave sulfates, fatty acids, and sugar moieties from larger molecules. Hepatocytes handle other compounds by biotransformation reactions that usually occur in three phases. Phase I reactions represent oxidation or reduction reactions in large part catalyzed by the P-450 cytochromes. The diverse array of phase I reactions includes hydroxylation, dealkylation, and dehalogenation, among others. The common feature of all of these reactions is that one atom of oxygen is inserted into the substrate. Hence, these monooxygenases make the substrate (RH) a more polar compound, poised for further modification by a phase II reaction. For example, when the phase I reaction creates a hydroxyl group (ROH), the phase II reaction may increase the water solubility of ROH by conjugating it to a highly hydrophilic compound such as glucuronate, sulfate, or glutathione: Phase I

Phase II

RH → ROH  → RO-Conjugate

(46-2) 

Chapter 46  •  Hepatobiliary Function

Finally, in phase III, the conjugated compound moves out of the liver via transporters on the sinusoidal and canalicular membranes. The P-450 cytochromes are the major enzymes involved in phase I reactions. Cytochromes are colored proteins that contain heme for use in the transfer of electrons. Some cytochromes—not the P-450 system—are essential for the electron transport events that culminate in oxidative phosphorylation in the mitochondria. The P-450 cytochromes, so named because they absorb light at 450 nm when bound to CO, are a diverse but related group of enzymes that reside mainly in the ER and typically catalyze hydroxylation reactions. Fifty-seven human CYP genes encode hundreds of variants of cytochrome P-450 enzymes (see Table 50-2). Genetic polymorphisms exist in the genes encoding all the main P-450 enzymes that contribute to drug and other xenobiotic metabolism, and the distribution and frequency of variant alleles can vary markedly among populations. In this text, we encounter P-450 oxidases in two sets of organs. In cells that synthesize steroid hormones—the adrenal cortex (see p. 1021), testes (see p. 1097), and ovary (see p. 1117) and placenta (see Table 56-5)—the P-450 oxidases are localized either in the mitochondria or in the ER, where they catalyze various steps in steroidogenesis. In the liver, these enzymes are located in the ER, where they catalyze a vast array of hydroxylation reactions involving the metabolism of drugs and chemical carcinogens, bile acid synthesis, and the activation and inactivation of vitamins. The same reactions occur in other tissues, such as the intestines and the lungs. Hepatic microsomal P-450 enzymes have similar molecular weights (48 to 56 kDa). The functional protein is a holoenzyme that consists of an apoprotein and a heme prosthetic group. The apoprotein region confers substrate specificity, which differs among the many P-450 enzymes. These substrates include RH moieties that are as wide ranging as the terminal methyl group of fatty acids, carbons in the rings of steroid molecules, complex heterocyclic compounds, and phenobarbital. In general, phase I processes add or expose a functional group, a hydroxyl group in the case of the P-450 oxidases, which renders the molecule reactive with phase II enzymes. The metabolic products of phase I may be directly excreted, but more commonly, because of only a modest increment in solubility, further metabolism by phase II reactions is required.

In phase II of biotransformation, conjugation of phase I products makes them more water soluble for secretion into blood or bile In phase II, the hepatocyte conjugates the metabolites generated in phase I to produce more hydrophilic compounds, such as glucuronides, sulfates, and mercapturic acids. These phase II products are readily secreted into the blood or bile. Conjugation reactions are generally considered to be the critical step in detoxification. Either a defect in a particular enzyme, which may result from a genetic defect, or saturation of the enzyme with excess substrate may result in a decrease in the overall elimination of a compound. One example is gray syndrome, a potentially fatal condition that occurs after the administration of chloramphenicol to

955

newborns who have low glucuronidation capacity. Infants have an ashen gray appearance and become weak and apathetic, and complete circulatory collapse may ensue. Hepatocytes use three major conjugation reactions: 1. Conjugation to glucuronate. The uridine diphosphate– glucuronosyltransferases (UGTs), which reside in the smooth endoplasmic reticulum (SER) of the liver, are divided into two families based on their substrate specificity. The UGT1 family consists of at least nine members encoded by genes located on chromosome 2. These UGTs catalyze the conjugation of glucuronic acid with phenols or bilirubin (see Fig. 46-6B). The UGT2 family contains at least nine UGTs encoded by genes on chromosome 4. These UGTs catalyze the glucuronidation of steroids or bile acids. The two members of the UGT3 family reside on chromosome 5. Because UGT1s are essential for the dual conjugation of bilirubin (see Fig. 46-6B) and because only conjugated bilirubin can be excreted in bile, congenital absence of UGT1A1 activity results in jaundice from birth and bilirubin encephalopathy, as seen in patients with Crigler-Najjar syndrome type I. 2. Conjugation to sulfate. The sulfotransferases—which are located in the cytosol rather than in the SER—catalyze the sulfation of steroids, catechols, and foreign compounds such as alcohol and metabolites of carcinogenic hydrocarbons. Their substrate specificity is greater than that of the UGTs. The different cellular localization of these two groups of enzymes suggests that they act cooperatively rather than competitively. In general, sulfates are not toxic and are readily eliminated, with the exception of sulfate esters of certain carcinogens. 3. Conjugation to glutathione. Hepatocytes also conjugate a range of compounds to reduced glutathione (GSH) for excretion and later processing in either the bile ducts or kidney (Fig. 46-8). Glutathione is a tripeptide composed of glutamate γ-linked to cysteine, which, in turn, is α-linked to glycine. The liver has the highest concentration of glutathione (~5 mM), with ~90% found in the cytoplasm and 10% in the mitochondria. Glutathione-S-transferases, which are mainly cytosolic, catalyze the conjugation of certain substrates to the cysteine moiety of GSH. Substrates include the electrophilic metabolites of lipophilic compounds (e.g., epoxides of polycyclic aromatic hydrocarbons), products of lipid peroxidation, and alkyl and aryl halides. In some cases, the conjugates are then secreted into bile and are further modified by removing the glutamyl residue from the glutathione by γ-glutamyl transpeptidase on the bile duct epithelial cell. The fate of glutathione-S-conjugates in bile is largely unknown. Some (e.g., the leukotrienes) undergo enterohepatic circulation. In other cases, the glutathione conjugates are secreted into plasma and are filtered by the kidney, where a γ-glutamyl transpeptidase on the proximal tubule brush border again removes the glutamyl residue. Next, a dipeptidase removes the glycine residue to produce a cysteineS-conjugate. The cysteine-S-conjugate is either excreted in the urine or is acetylated in the kidney or liver to form a mercapturic acid derivative, which is also excreted in the urine. Although glutathione conjugation is generally considered a detoxification reaction, several such conjugates undergo activation into highly reactive intermediates.

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SECTION VII  •  The Gastrointestinal System

Extracellular space

Bile duct

S

–

S

R

Cys –

Enterohepatic circulation

–

Dipeptidase

R

–

R

R

Glu Cys–Gly

Cys

S

Brush-border γ-glutamyl transpeptidase

–

S

–

– R

–

– S

Cys–Gly

Proximal tubule brush border

Urine

CH2

–

Glu

γ Glu–Cys–Gly GSH-R

γ-glutamyl transpeptidase

GSH-R

R

–

–

GSH SH

R

–

γGlu–Cys–Gly

NH – CH – COO–

–

Hepatocyte

S

Blood

– GSH-R

R

C– –O

–

S

R

CH3

γ Glu–Cys–Gly

γGlu–Cys–Gly –

Transporter

Mercapturic acid derivative –

R-group

Kidney filtration

Gly

Figure 46-8  Conjugation to GSH and formation of mercapturic acids. The first step is for glutathione-Stransferase to couple the target compound (R) to the S on the cysteine residue of GSH. After MRP2 transports this GSH conjugate into the canalicular lumen (see Fig. 46-5D), a γ-glutamyl transpeptidase may remove the terminal glutamate residue. Alternatively, the conjugate may reach the blood and be filtered by the kidney where a γ-glutamyl transpeptidase at the brush border and a dipeptidase generate a cysteine derivative of R. Acetylation yields the mercapturic acid derivative, which appears in the urine.

Other forms of conjugation include methylation (e.g., catechols, amines, and thiols), acetylation (e.g., amines and hydrazines), and conjugation (e.g., bile acids) with amino acids such as taurine, glycine, or glutamine. The involvement of multiple enzyme systems in these detoxification reactions facilitates the rapid removal of toxic species and provides alternative pathways in the event of failure of the preferred detoxification mechanism.

In phase III of biotransformation, hepatocytes excrete products of phase I and II into bile or sinusoidal blood Phase III involves multidrug transporters of the ATP-binding cassette (ABC) family (see Table 5-6)—such as MDR1 (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2)—located on the canalicular membrane. These transporters have broad substrate specificity and play an important role in protecting tissues from toxic xenobiotics and endogenous metabolites. However, their overexpression often leads to the development of resistance to anticancer drugs and can adversely affect therapy with other drugs, such as antibiotics. MRP2, which transports conjugated bilirubin, can also transport other conjugated substrates, including drugs and xenobiotics conjugated to glutathione. ABC proteins of broad substrate specificity—such as MRP4 (ABCC4) and MRP6 (ABCC6)—are also expressed on the basolateral or sinusoidal membrane of hepatocytes. MRP4, which has been studied best, facilitates the efflux of bile-salt conjugates (see Fig. 46-5C), conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs into sinusoidal blood. These sinusoidal efflux pumps are upregulated in cholestasis, which enables renal elimination of substances with compromised canalicular transport.

The interactions of xenobiotics with nuclear receptors control phase I, II, and III The nuclear receptors (see Table 3-6) for xenobiotics, the steroid and xenobiotic receptor (SXR, also known as the pregnane X receptor, or PXR), the constitutive androstane receptor (CAR), and the aryl hydrocarbon receptor (AhR) coordinately induce genes involved in the three phases of xenobiotic biotransformation. Many xenobiotics are ligands for orphan NRs, CAR, and SXR, which heterodimerize with the retinoid X receptor (RXR) and transcriptionally activate the promoters of many genes (see pp. 90–92) involved in drug metabolism. Similarly, many polycyclic aromatic hydrocarbons bind to AhR, which then dimerizes with the AhR nuclear translocator (ARNT), inducing cytochrome P-450 genes. Enzymes upregulated by SXR include the phase I drugmetabolizing enzymes of the P-450 family, such as CYP3A, which metabolizes >50% of all drugs in humans. SXR also activates the phase II enzyme glutathione-S-transferase, which is critical for catalyzing conjugation of many substrates to glutathione. SXR also upregulates MDR1 (see Table 5-6). Although these pathways are for the most part hepatoprotective, a particular compound may elicit SXR-mediated alterations in CYP3A activity that may profoundly influence the metabolism of another drug—perhaps thereby compromising the therapeutic efficacy of that drug or enhancing the production of a toxic metabolite. The constitutive androstane receptor (CAR) is also an important regulator of drug metabolism. CAR regulates all the components of bilirubin metabolism, including uptake (possibly through OATP), conjugation (UTG1A1), and excretion (MRP2).

Chapter 46  •  Hepatobiliary Function

Hepatocytes secrete bile acids, organic anions, organic cations, and lipids across their apical (canalicular) membranes At the apical membrane, the transport of compounds is generally unidirectional, from cell to canalicular lumen. An exception is certain precious solutes, such as amino acids and adenosine, which are reabsorbed from bile by Na+dependent secondary active transport systems. Bile Salts  Bile-salt transport from hepatocyte to canalicular lumen (see Fig. 46-5C) occurs via an ATP-dependent transporter called the bile-salt export pump (BSEP or ABCB11; see Table 5-6). BSEP has a very high affinity for bile salts (taurochenodeoxycholate > taurocholate > tau­ roursodeoxycholate > glycocholate). The electrical charge of the side chain is an important determinant of canalicular transport inasmuch as only negatively charged bile salts are effectively excreted. Secretion of bile salts occurs against a significant cell-to-canaliculus concentration gradient, which may range from 1 : 100 to 1 : 1000. Mutations in the BSEP gene can, in children, cause a form of progressive intrahepatic cholestasis that is characterized by extremely low bile acid concentrations in the bile. Organic Anions  Organic anions that are not bile salts move from the cytoplasm of the hepatocyte to the canalicular lumen largely via MRP2 (ABCC2, see Table 5-6 and Fig. 46-5D). MRP2 is electrogenic, ATP dependent, and has a broad substrate specificity  N46-5  —particularly for divalent, amphipathic, phase II conjugates with glutathione, glucuronide, glucuronate, and sulfates. Its substrates include bilirubin diglucuronide, sulfated bile acids, glucuronidated bile acids, and several xenobiotics. In general, transported substrates must have a hydrophobic core and at least two negative charges separated by a specific distance. MRP2 is critical for the transport of GSH conjugates across the canalicular membrane into bile. Although MRP2 has a low affinity for GSH, functional studies suggest that other mechanisms for GSH transport exist. Animal models of defective MRP2 exhibit conjugated hyperbilirubinemia, which corresponds phenotypically to Dubin-Johnson syndrome in humans. Another canalicular efflux pump for sulfated conjugates is breast cancer resistance protein (BCRP or ABCG2), which transports estrone-3-sulfate (see Fig. 55-8) and dehydroepiandrosterone sulfate (see Fig. 54-6)— breakdown products of sex steroids. Other anions, such as HCO3− and SO2− 4 , are excreted by anion exchangers. Organic Cations  Biliary excretion of organic cations is poorly understood. MDR1 (ABCB1; see Table 5-6) is present in the canalicular membrane, where it secretes into the bile canaliculus (see Fig. 46-7) bulky organic cations, including xenobiotics, cytotoxins, anticancer drugs, and other drugs (e.g., colchicine, quinidine, verapamil, cyclosporine). Other organic cations move into the canaliculus via the multidrug and toxin extrusion 1 (MATE1) transporters, which are driven by a pH gradient (see Fig. 46-7). The MATEs are one of the most highly conserved transporter families in nature, and MATE1 is highly expressed in many tissues. Thus, transcellular cation movement in liver is

957

mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux systems. In some cases, organic cations appear to move passively across the apical membrane into the canaliculus, sequestered by biliary micelles. Biliary Lipids  Phospholipid is a major component of bile. MDR3 (ABCB4; see Table 5-6) is a flippase that promotes the active translocation of phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane. Bile salts then extract the PC from the outer leaflet so that the PC becomes a component of bile, where it participates in micelle formation. Indeed, in humans with an inherited deficiency of MDR3, progressive liver disease develops, characterized by extremely low concentrations of phospholipids in the bile. Lipid asymmetry in the canalicular membrane is essential for protection against the detergent properties of bile salts. The P-type ATPase ATP8B1 in the canalicular membrane translocates aminophospholipids—such as phosphatidyl­ serine (PS) and phosphatidylethanolamine (PE)—from the outer to the inner leaflet of the bilayer, thereby leaving behind an outer leaflet that is depleted of PC, PS, and PE but enriched in sphingomyelin and cholesterol. The resulting lipid asymmetry renders the membrane virtually detergent insoluble and helps to maintain the functional complement of enzymes and transporters within the lipid bilayer. ATP8B1 is also present in the apical membranes of several other epithelia, including cholangiocytes and the epithelia of gallbladder, pancreas, and intestine. Mutations in ATP8B1 produce a chronic, progressive cholestatic liver disease (progressive familial intrahepatic cholestasis type 1). Bile is also the main pathway for elimination of cholesterol. A heterodimer composed of the “half ” ABC transporters ABCG5 and ABCG8 (see Table 5-6) is located on the canalicular membrane. This transporter is responsible for the secretion of cholesterol into bile. Although the mechanism is uncertain, the ABCG5/ABCG8 complex may form a channel for cholesterol translocation or alternatively may undergo a conformational change following ATP hydrolysis, thereby flipping a cholesterol molecule into the outer membrane leaflet in a configuration favoring release into the canalicular lumen. Mutations in the genes encoding either of the two ABC monomers lead to sitosterolemia, a disorder associated with defective secretion of dietary sterols into the bile, increased intestinal absorption of plant and dietary sterols, hypercholesterolemia, and early-onset atherosclerosis.

Hepatocytes take up proteins across their basolateral membranes by receptor-mediated endocytosis and fluid-phase endocytosis The hepatocyte takes up macromolecules, such as plasma proteins, from the blood plasma through endocytosis, transports these molecules across the cytoplasm, and then secretes them into the bile through exocytosis. Three forms of endocytosis have been identified in the basolateral (sinusoidal) membrane: fluid-phase endocytosis (nonspecific), adsorptive endocytosis (nonspecific), and receptor-mediated endocytosis (specific).  N46-6

Chapter 46  •  Hepatobiliary Function

957.e1

N46-5  MRP2 (ABCC2) Contributed by Emile Boulpaep and Walter Boron A member of the ABC family (see Table 5-6), MRP2 has a broad substrate specificity but has the highest affinities for the bilirubin conjugated to monoglucuronide or diglucuronide. The affinity is also high for leukotriene C4. The congenital deficiency of MRP2 causes Dubin-Johnson syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia (i.e., high levels of conjugated bilirubin in the blood).

REFERENCES Dubin IN, Johnson FB: Chronic idiopathic jaundice with unidentified pigment in liver cells: A new clinicopathological entity with a report of 12 cases. Medicine (Baltimore) 33:155–197, 1954. Nies AT, Keppler D: The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch 453:643–659, 2006.

N46-6  Protein Transport by Hepatocytes Contributed by Fred Suchy As noted in the text, hepatocytes take up proteins across their basolateral (i.e., sinusoidal) membranes via three forms of endocytosis. We discuss the fate of these endocytosed proteins in the next two paragraphs.

the process of protein sorting is disturbed, and newly synthesized apical membrane proteins may accumulate in a subapical vesicular compartment or may be missorted to the basolateral domain.

Intracellular Transport

Apical Exocytosis

Once proteins move into the hepatocyte by basolateral endocytosis, they can be transported across the cytoplasm within vesicles. This process, known as transcytosis, requires microtubules and is blocked by microtubule inhibitors, such as colchicine. Vesicular carriers transport the endocytosed proteins from the basolateral to the apical (i.e., canalicular) plasma membrane, where they exit via exocytosis. These same transcytotic vesicular carriers also ferry newly synthesized apical-membrane and secretory proteins. In the liver, most proteins destined for the apical membrane are initially transported from the trans-Golgi network to the basolateral membrane and subsequently transcytosed to the apical surface. It is believed that certain signal sequences on the protein (see p. 28) designate it as an apical membrane protein and are responsible for its correct targeting. The constitutive expression, rapid transport, and slow turnover of apical proteins mean that few are in the biosynthetic pipeline at steady state. During perturbations of liver function, such as cholestasis,

The principal pathway for secretion of high-molecular-weight proteins, whether they originate de novo from within the hepatocyte or come from the plasma, is exocytosis at the apical membrane. Exocytosis may also be used to recruit transport proteins to the plasma membrane. For example, cAMP stimulates sorting of MRP2 (multidrug resistance–associated protein 2, ABCC2) to the canalicular membrane, a process that is accompanied by increases in canalicular membrane area and that can be inhibited by nocodazole, a microtubule inhibitor. Thus, the targeting of vesicles to the apical membrane may be a highly regulated (rather than constitutive) process and may be an important determinant of organic anion excretion into bile. A similar mechanism of both exocytosis and endocytosis at the apical membrane may be involved in the cell volume-regulatory responses to hyperosmotic stress (see p. 131) and hypo-osmotic stress (see pp. 131–132).

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SECTION VII  •  The Gastrointestinal System

Fluid-phase endocytosis involves the uptake of a small amount of extracellular fluid with its solutes and is a result of the constitutive process of membrane invagination and internalization (see pp. 41–42). The process is nondiscriminatory and inefficient. Adsorptive endocytosis involves nonspecific binding of the protein to the plasma membrane before endocytosis, and it results in more efficient protein uptake. Receptor-mediated endocytosis is quantitatively the most important mechanism for the uptake of macromolecules (see p. 42). After endocytosis, the receptor recycles to the plasma membrane, and the ligand may be excreted directly into bile by exocytosis or delivered to lysosomes for degradation. Receptor-mediated endocytosis is involved in the hepatic removal from the blood of proteins such as insulin, polymeric immunoglobulin A (IgA), asialoglycoproteins, and epidermal growth factor.

BILE FORMATION The secretion of canalicular bile is active and isotonic The formation of bile occurs in three discrete steps. First, the hepatocytes actively secrete bile into the bile canaliculi. Second, intrahepatic and extrahepatic bile ducts not only transport this bile but also secrete into it a watery, HCO3− rich fluid. These first two steps may produce ~900 mL/day of so-called hepatic bile (Table 46-2). Third, between meals, approximately half the hepatic bile—perhaps 450 mL/day— is diverted to the gallbladder, which stores the bile and isosmotically removes salts and water. The result is that the gallbladder concentrates the key remaining solutes in bile fluid—bile salts, bilirubin, cholesterol, and lecithin—by 10- to 20-fold. The 500 mL/day of bile that reaches the duodenum through the ampulla of Vater is thus a mixture

TABLE 46-2  Composition of Bile PARAMETER pH

HEPATIC BILE 7.5

+

Na (mM) +

141–165

GALLBLADDER BILE 6.0 220

K (mM)

2.7–6.7

14

Ca2+ (mM)

1.2–3.2

15

Cl− (mM)

77–117

31

12–55

19

− 3

HCO (mM) Total phosphorus (g/L)

0.15

Bile acids (g/L)

3–45

32

Total fatty acids (g/L)

2.7

24

Bilirubin (g/L)

1–2

3

1.4–8.1

34

Phospholipids (g/L)

1.4

Cholesterol (g/L)

1–3.2

6.3

Proteins (g/L)

2–20

4.5

Data from Boyer JL: Mechanisms of bile secretion and hepatic transport. In Andreoli TE, Hoffman JF, Fanestil DD, Schultz SG (eds): Physiology of Membrane Disorders. New York, Plenum, 1986.

of relatively “dilute” hepatic bile and “concentrated” gallbladder bile. The first step in bile formation cannot be ultrafiltration because the hydrostatic pressure in the canaliculi is significantly higher than the sinusoidal perfusion pressure. This situation is in marked contrast to glomerular filtration by the kidney (see pp. 743–745), which relies predominantly on passive hydrostatic forces for producing the fluid in Bowman’s space. Instead, bile formation is an active process. It is sensitive to changes in temperature and to metabolic inhibitors. Bile formation by hepatocytes requires the active, energy-dependent secretion of inorganic and organic solutes into the canalicular lumen, followed by the passive movement of water. Canalicular bile is an isosmotic fluid. Water movement into the bile canaliculus can follow both paracellular and transcellular pathways. As far as the paracellular pathway is concerned, the movement of water through the tight junctions between hepatocytes carries with it solutes by solvent drag (see p. 467). Further down the biliary tree (i.e., ducts and gallbladder), where the pore size of paracellular junctions is significantly smaller, solvent drag is not as important. Organic solutes do not readily enter bile distal to the canaliculi. As far as the transcellular pathway is concerned, water enters hepatocytes via aquaporin 9 (AQP9), found exclusively on the sinusoidal membrane. AQP9 also allows the passage of a wide variety of neutral solutes such as urea, glycerol, purines, and pyrimidines. The canalicular membrane expresses AQP8. Under basal conditions, AQP8 is predominantly localized to intracellular vesicles so that water permeability in the canalicular membrane is lower than that in the sinusoidal membrane and is rate limiting for transcellular water transport. However, upon cAMP stimulation, AQP8 from the intracellular pool inserts into the canalicular membrane, which substantially increased the water permeability of this membrane. With cAMP stimulation, transcellular water permeability in the hepatocyte is similar to that in the renal proximal tubule, where water flow is largely transcellular. Indeed, the transcellular pathway accounts for most of the water entering the bile canaliculus during choleresis.

Major organic molecules in bile include bile acids, cholesterol, and phospholipids Bile has two important functions: (1) bile provides the sole excretory route for many solutes that are not excreted by the kidney, and (2) secreted bile salts and acids are required for normal lipid digestion (see pp. 925–929) and absorption (see pp. 929–933). Both hepatic bile and gallbladder bile are complex fluids that are isosmotic with plasma (~300 mOsm) and consist of water, inorganic electrolytes, and a variety of organic solutes, including bilirubin, cholesterol, fatty acids, and phospholipid (see Table 46-2). The predominant cation in bile is Na+, and the major inorganic anions are Cl− and HCO3− . Solutes whose presence in bile is functionally important include micelleforming bile acids, phospholipids, and IgA. Bile acids promote dietary lipid absorption through their micelle-forming properties (see p. 929). As shown in

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Chapter 46  •  Hepatobiliary Function

CHOLESTEROL

PRIMARY BILE ACIDS OH

O C

12

3

7

HO

HO 7α-hydroxylase

SECONDARY BILE ACIDS

OH

OH

BILE SALTS O

Bacteria

C

7α-dehydroxylase

OH H

COO–

pKa~3.7

HO

Deoxycholic acid

O C

OH

Glycine

O

Bacteria C

7α-dehydroxylase

OH H 7

CH2

Conjugation OH

Cholic acid

HO

NH

NH

CH2

CH2

SO3–

Conjugation OH

pKa~1.5

HO

Lithocholic acid

Chenodeoxycholic acid

Taurine

Figure 46-9  Synthesis of bile acids. The liver converts cholesterol to the primary bile acids—cholic acid and chenodeoxycholic acid—in a series of 14 reactions occurring in four different cellular organelles. Bacteria in the terminal ileum and colon may dehydroxylate bile acids, yielding the secondary bile acids deoxycholic acid and lithocholic acid. The hepatocytes conjugate most of the primary bile acids to small molecules such as glycine and taurine before secreting them into the bile. In addition, those secondary bile acids that return to the liver via the enterohepatic circulation may also be conjugated to glycine or taurine

Figure 46-9, hepatocytes synthesize the so-called primary bile acids—cholic acid and chenodeoxycholic acid—from cholesterol. Indeed, biliary excretion of cholesterol and conversion of cholesterol to bile acids are the principal routes of cholesterol excretion and catabolism, so that bile formation is pivotal for total-body cholesterol balance. The first step in this conversion is catalyzed by cholesN46-7  a specific cytoterol 7α-hydroxylase (CYP7A1),  chrome P-450 enzyme located in the SER. As we see below, secondary bile acids are the products of bacterial dehydroxylation in the terminal ileum and colon. After being absorbed and returning to the liver (enterohepatic circulation, discussed below), these secondary bile acids may also undergo conjugation. Figure 46-9 shows typical examples of conjugation reactions. Phospholipids in bile help to solubilize cholesterol as well as diminish the cytotoxic effects of other bile acids on hepatocytes and bile duct cells. IgA inhibits bacterial growth in bile. Excretory or waste products found in bile include cholesterol, bile pigments, trace minerals, plant sterols, lipophilic drugs and metabolites, antigen-antibody complexes, and oxidized glutathione. Bile is also the excretory route for compounds that do not readily enter the renal glomerular filtrate, either because they are associated with proteins such as albumin or because they are associated with formed elements in blood. Although these compounds are generally lipophilic, they also include the heavy metals. Some bile acids (e.g., the trihydroxy bile acid cholic acid) are only partly bound to serum albumin and may therefore enter the glomerular filtrate. However, they are actively reabsorbed by the renal tubule. In health, bile acids are virtually absent from the urine.

l Tota

Bile flow

bile

flow

n retio sec e l i b Bile acid– ular alic dependent Can flow

Bile acid–independent flow

Ductular secretion

Canalicular bile secretion

Bile acid excretion rate Figure 46-10  Components of bile flow.

Canalicular bile flow has a constant component driven by the secretion of small organic molecules and a variable component driven by the secretion of bile acids Total bile flow is the sum of the bile flow from hepatocytes into the canaliculi (canalicular flow) and the additional flow from cholangiocytes into the bile ducts (ductular flow). In most species, the rate of canalicular bile secretion (i.e., milliliters per minute) increases more or less linearly with the rate of bile acid secretion (i.e., moles per minute). Canalicular bile flow is the sum of two components (Fig. 46-10): (1) a “constant” component that is independent of bile acid secretion (bile acid–independent flow) and (2) a rising component that increases linearly with bile acid secretion (bile acid–dependent flow). In humans, most of the canalicular bile flow is bile acid dependent. If we now add the ductular

Chapter 46  •  Hepatobiliary Function

N46-7  Cholesterol 7α-Hydroxylase Contributed by Fred Suchy As noted in the text, the first step in the conversion of cholesterol to bile acids is the hydroxylation of cholesterol at position 7 by cholesterol 7α-hydroxylase. Bile acid levels regulate the activity of this enzyme, probably by both positive and negative feedback. Negative feedback in a cultured rat hepatocyte model occurs partly at the transcriptional level.

959.e1

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SECTION VII  •  The Gastrointestinal System

secretion, which is also “constant,” we have the total bile flow in Figure 46-10. We discuss the canalicular secretion in the remainder of this section and ductular secretion in the following section.

Lumen

As discussed in the previous section, biliary epithelial cells, or cholangiocytes, are the second major source of the fluid in hepatic bile. Experimentally, one can isolate cholangiocytes from normal liver or from the liver of experimental animals in which ductular hyperplasia has been induced by ligating the bile duct. These cholangiocytes (Fig. 46-11) have 6 of the 13 known human aquaporins, an apical Cl-HCO3 exchanger AE2, and several apical Cl− channels, including the cystic fibrosis transmembrane conductance regulator (CFTR; see p. 120). In a mechanism that may be similar to that in pancreatic duct cells (see Fig. 43-6), the Cl-HCO3 exchanger, in parallel with the Cl− channels for Cl− recycling,

Interstitial space

H2O

Na+ – 2 HCO3

HCO3–

tion of organic compounds probably provides the major driving force for bile acid–independent flow. For example, glutathione, present in bile in high concentrations, may generate a potent osmotic driving force for canalicular bile formation.  N46-8

Secretin stimulates the cholangiocytes of ductules and ducts to secrete a watery, HCO3- -rich fluid

+

Cholangiocyte

Cl –

Bile Acid–Independent Flow in the Canaliculi  The secre-

Bile Acid–Dependent Flow in the Canaliculi  The negatively charged bile salts in bile are in a micellar form and are—in a sense—large polyanions. Thus, they are effectively out of solution and have a low osmotic activity coefficient. However, the positively charged counterions accompanying these micellar bile acids are still in aqueous solution and may thus represent the predominant osmotic driving force for water movement in bile acid–dependent flow. If one infuses an animal with a nonphysiological bile acid that does not form micelles or one that forms micelles only at a rather high concentration, the osmotic activity will be higher, and thus the exogenous bile acid will be more effective in producing bile acid–dependent flow. In other words, the slope of the blue bile acid–dependent line in Figure 46-10 would be steeper than for physiological bile acids. Bile flow does not always correlate with the osmotic activity of the bile acid. In some cases, bile acids increase electrolyte and water flux by other mechanisms, such as by stimulating Na+-coupled cotransport mechanisms or by modulating the activity of other solute transporters. For example, the bile acid ursodeoxycholic acid produces a substantial increase in bile flow by markedly stimulating biliary HCO3− excretion. Bile acids in the lumen may also stimulate the secretion of other solutes by trapping them in the lumen. These solutes include bilirubin and other organic anions, as well as lipids such as cholesterol and phospholipids. The mixed micelles formed by the bile acids apparently sequester these other solutes, thus lowering their effective luminal concentration and favoring their entry. Therefore, excretion of cholesterol and phospholipid is negligible when bile acid output is low, but it increases and approaches maximum values as bile acid output increases.

Na+

CO2

CO2

CA

H2O

OH– +

Na+

H Cl –

Na+

Other Cl– channels

K+ K+

Cl – [cAMP]

Cystic fibrosis transmembrane conductance regulator

Secretin Glucagon VIP

Na

+

+

H2O

Somatostatin (inhibitory)

Figure 46-11  Secretion of an HCO3− -rich fluid by cholangiocytes. Secretin, glucagon, VIP, and gastrin-releasing peptide (GRP) all are choleretics. Somatostatin either enhances fluid absorption or inhibits secretion. CA, carbonic anhydrase.

can secrete an HCO3− -rich fluid.  N46-9  AQP1, CFTR, and AE2 colocalize to intracellular vesicles in cholangiocytes; secretory agonists cause all three to co-redistribute to the apical membrane. A complex network of hormones, mainly acting via cAMP, regulates cholangiocyte secretory function. Secretin receptors (see pp. 886–887) are present on the cholangiocyte basolateral membrane, a fact that explains why secretin produces a watery choleresis—that is, a bile rich in HCO3− (i.e., alkaline) but poor in bile acids. The hormones glucagon (see pp. 1050–1053) and vasoactive intestinal peptide (VIP; see Fig. 13-9) have similar actions.  N46-10  These hormones raise [cAMP]i and thus stimulate apical Cl− channels and the Cl-HCO3 exchanger. A Ca2+-activated Cl− channel is also present in the apical membrane.  N46-11 Cholangiocytes are also capable of reabsorbing fluid and electrolytes, as suggested by the adaptation that occurs after removal of the gallbladder (i.e., cholecystectomy). Bile found within the common bile duct of fasting cholecystectomized animals is similar in composition to the concentrated bile typically found in the gallbladder. Thus, the ducts have partially taken over the function of the gallbladder (see below). The hormone somatostatin inhibits bile flow by lowering [cAMP]i, an effect opposite that of secretin. This inhibition may be caused by enhancing fluid reabsorption by bile ducts or by inhibiting ductular secretion of the HCO3− -rich fluid discussed above.

Chapter 46  •  Hepatobiliary Function

N46-8  Contribution of Inorganic Solutes to Bile Acid–Independent Flow

960.e1

N46-9  HCO3- Secretion by Cholangiocytes Contributed by Fred Suchy

Contributed by Fred Suchy

As noted in the text, the mechanism of HCO3− secretion by cholangiocytes may be similar to that of secretion by pancreatic duct cells. For a discussion of the latter process, see  N46-20. In addition, cholangiocytes transport water into bile via an aquaporin water channel. Na-H exchangers are expressed on both the basolateral membrane (NHE1) and the apical membrane (NHE2) of cholangiocytes.

In addition to organic solutes, inorganic solutes also contribute to bile acid–independent flow of bile. The secretion of these inorganic electrolytes occurs primarily by solvent drag (see p. 467) and passive diffusion (e.g., through canalicular Cl− channels; see Fig. 46-5B). To the extent that these inorganic electrolytes enter the canalicular lumen by passive diffusion, they pull in water osmotically and thus contribute to bile acid– independent flow. However, this is not a major effect.

N46-20  HCO3- Secretion by the Pancreatic Duct Contributed by Emile Boulpaep and Walter Boron The current model for HCO3− secretion by the pancreatic duct is very similar to that outlined in Figure 43-6. However, we can now add some important details about the apical step of HCO3− secretion. The Cl-HCO3 exchanger at the apical membrane is a member of the SLC26 family (Mount and Romero, 2004)—previously known as the SAT family—specifically, SLC26A6 (also known as CFEX). We now appreciate that SLC26A6, which is capable of exchanging several different anions (e.g., Cl−, HCO3− , oxalate), is electrogenic (Jiang et al, 2002). When mediating Cl-HCO3 exchange, it appears that SLC26A6 exchanges two HCO3− ions for every Cl− ion. This stoichiometry would strongly favor the efflux of HCO3− across the apical membrane of the pancreatic duct cell. As noted in the text, the Cl− that enters the cell via SLC26A exits the cell via apical Cl− channels, principally CFTR. Interestingly, it appears that an interaction between the SLC26A6 protein and CFTR greatly increases the open probability of CFTR (Ko et al, 2004).

N46-10  Regulation of Cholangiocyte Secretion

Another member of the SLC26 family—SLC26A3—also is present in the apical membrane of pancreatic duct cells. SLC26A3 is also electrogenic but has a stoichiometry opposite to that of SLCA6, two Cl− ions for every HCO3− . This transporter would extrude Cl− (and take up HCO3− ) from the duct cell across the apical membrane. Its physiological function might be to reabsorb HCO3− at times when the duct is not secreting HCO3− or to contribute to the recycling of Cl− when the duct is secreting HCO3− .

REFERENCES Jiang Z, Grichtchenko II, Boron WF, Aronson PS: Specificity of anion exchange mediated by mouse Slc26a6. J Biol Chem 277:33963–33967, 2002. Ko SB, Zeng W, Dorwart MR, et al: Gating of CFTR by the STAS domain of SLC26 transporters. Nat Cell Biol 6:343–350, 2004. Mount DB, Romero MF: The SLC26 gene family of multifunctional anion exchangers. Pflugers Arch 447:710–721, 2004.

N46-11  Ca2+-Activated Cl− Channels Contributed by Emile Boulpaep and Walter Boron

Contributed by Fred Suchy In addition to the hormones mentioned on pages 960–961, gastrin-releasing peptide (GRP) also stimulates fluid and HCO3− secretion from cholangiocytes, but through mechanisms other than cAMP, cGMP, and Ca2+. Exposure of polarized cholangiocytes to ATP results in luminal secretion through activation of P2µ receptors on the apical membrane. Release of ATP into bile appears to serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

Ca2+-activated Cl− channels (CaCCs) play important physiological roles. One group of CaCCs are encoded by the ANO genes (see Table 6-2, family No. 17). The bestrophins, encoded by at least four BEST genes, constitute another family of CaCCs. The molecular identity of CaCCs in the liver is unknown.

REFERENCES Hartzell HC, Putzier I, Arreola J: Calcium-activated chloride channels. Annu Rev Physiol 67:719–758, 2005. Hartzell HC, Qu Z, Yu K, et al: Molecular physiology of bestrophins: Multifunctional membrane proteins linked to Best disease and other retinopathies. Physiol Rev 88:639–672, 2008. Koumi S, Sato R, Aramaki T: Characterization of the calciumactivated chloride channel in isolated guinea-pig hepatocytes. J Gen Physiol 104:357–373, 1994.

Chapter 46  •  Hepatobiliary Function

Solutes reabsorbed from bile by cholangiocytes are recycled. As shown in Figure 46-2, the intralobular bile ducts are endowed with a rich peribiliary vascular plexus that is supplied by the hepatic artery. The blood draining this plexus finds its way into the hepatic sinusoids. This plexus is analogous to the capillaries of the gut, which, via the portal vein, also find their way into the hepatic sinusoids. Thus, some solutes, such as the hydrophilic bile acid ursodeoxycholic acid, may be absorbed by the cholangiocytes from bile and returned to the hepatocytes for repeat secretion, a process that induces significant choleresis.

The gallbladder stores bile and delivers it to the duodenum during a meal The gallbladder is not an essential structure of bile secretion. Tonic contraction of the sphincter of Oddi facilitates gallbladder filling by maintaining a positive pressure within the common bile duct. As we noted above, up to 50% of hepatic bile—or ~450 mL/day—is diverted to the gallbladder during fasting. The remaining ~450 mL/day passes directly into the duodenum. Periods of gallbladder filling between meals are interrupted by brief periods of partial emptying of concentrated bile and probably aspiration of dilute hepatic bile in a process analogous to the function of a bellows. Gallbladder emptying and filling is under feedback control. During feeding, CCK secreted by duodenal I cells (see Table 41-1) causes gallbladder contraction and the release of bile into the duodenum, where the bile promotes fat digestion and suppresses further CCK secretion. On reaching the ileum, bile acids induce synthesis of fibroblast growth factor 19 (FGF19); FGF19, after transit in portal blood, causes relaxation of gallbladder smooth muscle, which allows gallbladder refilling. Thus, CCK and FGF19 control the periodicity of gallbladder emptying and filling. During the interdigestive period, the gallbladder concentrates bile acids—and certain other components of bile—up to 10- or even 20-fold within the gallbladder lumen because they are left behind during the isotonic reabsorption of NaCl and NaHCO3 by the leaky gallbladder epithelium (Fig. 46-12). The apical step of NaCl uptake and transport is electroneutral and is mediated by parallel Na-H and Cl-HCO3

Lumen

Epithelial cell

Interstitial space

+

2 K+

H+

+

HCO3– Cl– H2O

K+

K

+

Na

3 Na Cl–

Na+ Cl– H2O

Figure 46-12  Isotonic fluid reabsorption by the gallbladder epithelium.

961

exchangers. At the basolateral membrane, Na+ exits through the Na-K pump, whereas Cl− most likely exits by Cl− channels. Both water and HCO3− move passively from lumen to blood through the tight junctions. Water can also move through the cell via AQP1 (expressed on apical and basolateral membranes) and AQP8 (found only apically). The net transport is isotonic, which leaves behind gallbladder bile that is also isotonic but has a higher concentration of bile salts, K+, and Ca2+. Net fluid and electrolyte transport across the gallbladder epithelium is under hormonal regulation. Both VIP (released from neurons innervating the gallbladder) and serotonin inhibit net fluid and electrolyte absorption. Conversely, α-adrenergic blockade of neuronal VIP release increases fluid absorption. Although the gallbladder reabsorbs NaCl by parallel Na-H and Cl-HCO3 exchange at the apical membrane, Na-H exchange outstrips Cl-HCO3 exchange; the end result is net secretion of H+ ions. This action neutralizes the HCO3− and acidifies the bile. The H+ secreted by the gallbladder protonates the intraluminal contents. This action greatly increases the solubility of calcium salts in bile and reduces the likelihood of calcium salt precipitation and gallstone formation. Common “pigment gallstones” contain one or more of several calcium salts, including carbonate, biliru­ binate, phosphate, and fatty acids. The solubility of each of these compounds is significantly increased by the acidification of bile. Mucus secretion by gallbladder epithelial cells results in the formation of a polymeric gel that protects the apical surface of the gallbladder epithelium from the potentially toxic effects of bile salts. However, excessive mucin synthesis can be deleterious. For example, in animal models of cholesterol cholelithiasis (i.e., formation of gallstones made of cholesterol), a marked increase in mucin release precedes crystal and stone formation.

The relative tones of the gallbladder and sphincter of Oddi determine whether bile flows from the common hepatic duct into the gallbladder or into the duodenum Bile exiting the liver and flowing down the common hepatic duct reaches a bifurcation that permits flow either into the cystic duct and then into the gallbladder or into the common bile duct, through the sphincter of Oddi, and into the duodenum (see Fig. 46-4). The extent to which bile takes either path depends on the relative resistances of the two pathways. The sphincter of Oddi—which also controls the flow of pancreatic secretions into the duodenum—corresponds functionally to a short (4- to 6-mm) zone within the wall of the duodenum. The basal pressure within the lumen of the duct at the level of the sphincter is 5 to 10 mm Hg. The pressure in the lumen of the resting common bile duct is also 5 to 10 mm Hg, compared with a pressure of ~0 mm Hg inside the duodenum. The basal contraction of the sphincter prevents reflux of the duodenal contents into the common bile duct. In its basal state, the sphincter exhibits high-pressure, phasic contractions several times per minute. These contractions are primarily peristaltic and directed in antegrade fashion to provide a motive force toward the duodenum. Thus, the

962

SECTION VII  •  The Gastrointestinal System

BOX 46-2  Cholestasis

T

he term cholestasis refers to the suppression of bile secretion. Biliary constituents may therefore be retained within the hepatocyte and regurgitated into the systemic circulation. Cholestasis causes three major groups of negative effects: first, regurgitation of bile components (bile acids, bilirubin) into the systemic circulation gives rise to the symptoms of jaundice and pruritus (itching). Second, cholestasis damages hepatocytes, as evidenced by the release of clinically important liver enzymes (e.g., alkaline phosphatase) into the plasma. Third, because the bile acids do not arrive in the duodenum, lipid digestion and absorption may be impaired. Many acute and chronic liver diseases produce cholestasis by mechanically obstructing the extrahepatic bile ducts or by impairing bile flow at the level of the hepatocytes or intrahepatic bile ducts. The mechanisms underlying the obstructive and functional forms of cholestasis are complex and have not been completely defined. Experimental modeling of cholestasis has produced multiple abnormalities: (1) altered plasma-membrane composition and fluidity; (2) inhibition of membrane proteins, including the Na-K pump and aquaporins; (3) reduced expression of genes encoding transporters for bile acids and other organic anions; (4) altered expression of nuclear receptors and associated epigenetic modifications that regulate transporters; (5) increased permeability of the paracellular pathway, with backdiffusion of biliary solutes into the plasma; (6) altered function of microfilaments, with decreased contractions of bile canaliculi; and (7) loss of the polarized distribution of some plasma-membrane proteins. Cholestatic conditions, such as bile duct obstruction, markedly increase the basolateral expression of MRP4 and MRP6 as well as OSTα-OSTβ—which normally are expressed only minimally. The induction of these transporters allows the efflux of bile acids and other cholephilic anions from the hepatocyte into sinusoidal blood.

sphincter of Oddi acts principally as an adjustable occluding mechanism and a regulator of bile flow. Both hormonal and cholinergic mechanisms appear to be involved in gallbladder emptying. Dietary lipid stimulates the release of CCK from duodenal I cells (see pp. 889–890). This CCK not only stimulates pancreatic secretion but also causes smooth-muscle contraction and evacuation of the gallbladder. The coordinated response to CCK also includes relaxation of the sphincter of Oddi, which enhances bile flow into the duodenum (Box 46-2).

ENTEROHEPATIC CIRCULATION OF BILE ACIDS The enterohepatic circulation of bile acids is a loop consisting of secretion by the liver, reabsorption by the intestine, and return to the liver in portal blood for repeat secretion into bile Bile acids are important for promoting the absorption of dietary lipids in the intestine. The quantity of bile acid that the liver normally secretes in a day varies with the number of meals and the fat content of these meals, but it typically ranges between 12 and 36 g. The liver’s basal rate of synthesis of bile acids from cholesterol (see Fig. 46-9) is only

~600 mg/day in healthy humans, sufficient to replace the equivalent losses of bile acid in the feces. Obviously, the gastrointestinal tract must have an extremely efficient mechanism for recycling the bile acids secreted by the liver (Fig. 46-13). This recycling, known as the enterohepatic circulation, occurs as the terminal ileum and colon reabsorb bile acids and return them to the liver in the portal blood. The total pool of bile acids in the gastrointestinal tract is ~3 g. This pool must recirculate ~4 to 12 times per day, or as many as 5 or more times for a single fat-rich meal. If reabsorption of bile acids is defective, as can happen after resection of the ileum, de novo synthesis of bile acids by the liver can be as high as 4 to 6 g/day.

Efficient intestinal conservation of bile acids depends on active apical absorption in the terminal ileum and passive absorption throughout the intestinal tract Most of the bile secreted into the duodenum is in the conjugated form. Very little of these bile salts is reabsorbed into the intestinal tract until they reach the terminal ileum, an arrangement that allows the bile salts to remain at high levels throughout most of the small intestine, where they can participate in lipid digestion (see pp. 925–929) and absorption (see pp. 929–933). However, the enterohepatic circulation must eventually reclaim 95% or more of these secreted bile salts. Some of the absorption of bile acids by the intestines is passive and occurs along the entire small intestine and colon. Nevertheless, the major component of bile acid absorption is active and occurs only in the terminal ileum (see Fig. 46-13). Passive absorption of bile acids occurs along the entire small intestine and colon (see Fig. 46-13), but it is less intensive than active absorption. The mechanism of bile acid uptake across the apical membrane may consist of either ionic or nonionic diffusion (see pp. 784–785). Nonionic diffusion—or passive diffusion of the protonated or neutral form of the bile acid—is 10-fold greater than ionic diffusion. The extent of nonionic diffusion for a given bile acid depends on the concentration of its neutral, protonated form, which is maximized when the luminal pH is low and the pK of the bile acid is high. At the normal intestinal pH of 5.5 to 6.5, few of the taurine-conjugated bile salts are protonated, a small amount of the glycine-conjugated bile salts are protonated, and ~50% of unconjugated bile acids are protonated. Thus, the unconjugated bile acids are in the best position to be reabsorbed by nonionic diffusion, followed by the glycineconjugated bile acids and then finally by the taurineconjugated bile acids. Among these unconjugated bile acids, more lipophilic bile acids, such as chenodeoxycholate and deoxycholate, diffuse more readily through the apical membrane than do hydrophilic bile acids such as cholic acid. Nonionic diffusion also depends on the total concentration of the bile acid (i.e., neutral plus charged form), which, in turn, depends on the maximum solubilizing capacity of bilesalt micelles for that bile acid. Active absorption of bile acids in the intestine is restricted to the terminal ileum (see Fig. 46-13). This active process preferentially absorbs the negatively charged conjugated bile salts—the form not well absorbed by the passive

Chapter 46  •  Hepatobiliary Function

FGF19

963

Unconjugated BA–

Synthesis of ~600 mg/day of “primary bile acids”

BA-Z– Conjugated

BA–

Bile salts

Z BA-Z–

LIVER

BA-Z–

Bile storage

H.BA

GALLBLADDER

Passive absorption H.BA

BA– + H+

SMALL INTESTINE

PORTAL BLOOD

BA-Z– Passive absorption

H+ Bacteria in the terminal ileum and the colon deconjugate bile salts (BA-Z–) to form bile acids (H.BA) and also dehydroxylate primary bile acids to form secondary bile acids. TERMINAL ILEUM

Deoxycholic acid

+

BA– Lumen H.BA

Interstitial space ASBT

BA-Z–

OSTα-OSTβ

BA-Z– +

H.BA

Lithocholic acid

(Conjugated bile acid) BA-Z–

Enterocyte in terminal ileum Epithelium

Na

SHP COLON

FXR FGF19

H + BA– CECUM

H.BA

Passive absorption

~600 mg bile acids lost daily in feces Figure 46-13  Enterohepatic circulation of bile acids. The bile acids that the liver delivers to the duodenum

in the bile are primarily conjugated to taurine or glycine (BA-Z−), and these conjugates enter the portal blood in the terminal ileum to return to the liver. Some unconjugated bile acids and secondary bile acids also return to the hepatocyte for resecretion.

mechanisms. Active uptake of bile salts involves saturation kinet­ics, competitive inhibition, and a requirement for Na+. The Na+-dependent transporter responsible for the apical step of active absorption is known as the apical Na/bile-salt transporter (ASBT or SLC10A2), a close relative of the hepatocyte transporter NTCP (see Fig. 46-5C). After bile salts enter ileal enterocytes across the apical membrane, they exit across the basolateral membrane via the heteromeric organic solute transporter OSTα-OSTβ. Because the most polar bile salts are poorly absorbed by nonionic diffusion, it is not surprising that the ASBT in the apical membrane of the enterocytes of the terminal ileum has the highest affinity and maximal transport rates for these salts. For example, ASBT is primarily responsible

for absorbing the ionized, taurine-conjugated bile salts in the ileum. Conversely, ASBT in the ileum is relatively poor at absorbing the more lipophilic bile acids, which tend to be absorbed passively in the upper intestine. On their entry into portal blood, the bile acids are predominantly bound to albumin and, to a lesser extent, lipoproteins. The liver removes or clears these bile acids from portal blood by the transport mechanisms outlined above in Figure 46-5C. Hepatic clearance of bile acids is often expressed as the percentage of bile acids removed during a single pass through the liver. The hepatic extraction of bile acids is related to bile acid structure and the degree of albumin binding. It is greatest for hydrophilic bile acids and lowest for protein-bound, hydrophobic bile acids.

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SECTION VII  •  The Gastrointestinal System

The small fraction of bile acids that escapes active or passive absorption in the small intestine is subject to bacterial modification in the colon. This bacterial modification takes two forms. First, the bacteria deconjugate the bile. Second, the bacteria perform a 7α-dehydroxylation reaction with the formation of secondary bile acids. These secondary bile acids include deoxycholate and lithocholate (see Fig. 46-9). The deconjugated secondary bile acids may then be either absorbed passively in the colon or excreted in the feces; their fate depends on their physicochemical properties and their binding to luminal contents. Up to one third of the deoxycholate formed in the colon may be reabsorbed by nonionic diffusion. Lithocholate, which is relatively insoluble, is absorbed to a much lesser extent. The secondary bile acids formed by colonic bacteria and recycled back to the liver may undergo biotransformation through conjugation to glycine and taurine. Thus, the enterohepatic circulation of bile acids is driven by two mechanical pumps: (1) the motor activity of the gallbladder, and (2) peristalsis of the intestines to propel the bile acids to the terminal ileum and colon. It is also driven by two chemical pumps: (1) energy-dependent transporters located in the terminal ileum, and (2) energy-dependent transporters in the hepatocyte. The bile acid receptor FXR, a member of the nuclear receptor family (see Table 3-6), controls multiple components of the enterohepatic circulation of bile acids. Primary bile acids are potent agonists of FXR, which transcriptionally regulates several genes involved in bile acid homeostasis, producing negative feedback by four mechanisms: 1. FXR in hepatocytes induces expression of a transcrip­ tion factor called the small heterodimer partner (SHP); SHP, in turn, inhibits another nuclear receptor, the liver receptor homolog 1 (LRH-1), which is required for CYP7A1 expression. The result is that FXR inhibits the expression of cholesterol 7α-hydroxylase (CYP7A1)— the rate-limiting enzyme for bile acid synthesis (see Fig. 46-9). 2. FXR in the ileum increases the synthesis and secretion into portal blood of FGF19, which then activates the FGF receptor 4 signaling pathway in the liver, repressing CYP7A1. 3. FXR in hepatocytes upregulates BSEP (which increases bile acid secretion; see Fig. 46-5C) and downregulates NTCP (which decreases bile acid uptake; see Fig. 46-5C) by SHP-dependent mechanisms. The net result is a reduction of intracellular bile acids. 4. FXR in the ileum, via SHP, downregulates ASBT (see Fig. 46-13, inset), thereby reducing bile acid uptake. FXR also induces the expression of basolateral OSTα-OSTβ, thereby increasing bile acid efflux. The net result is a reduction of intracellular bile acids. Thus, FXR coordinates bile acid synthesis and transport by the liver and intestine. The bile acid signaling network also includes the G protein–coupled receptor TGR5, which is highly expressed in the apical membrane and primary cilium of cholangiocytes and gallbladder epithelial cells, but minimally in hepatocytes. Activation of TGR5 by bile acids in the biliary tract leads to a rise in [cAMP]i, which causes Cl− secretion to increase (Box 46-3).

BOX 46-3  Gallstones

D

uring hemolytic anemias, small, dark pigment gallstones may form secondary to the excess production and excretion of bilirubin. However, most gallstones (~80%) consist mainly of cholesterol. Thus, cholelithiasis is largely a disturbance of bile secretion and cholesterol elimination. When cholesterol and phospholipids are secreted together into the bile, they form unilamellar bilayered vesicles. These vesicles become incorporated into mixed micelles that form because of the amphiphilic properties of bile acids. Micellation allows cholesterol to remain in solution in its passage through the biliary tree. However, if the concentration of bile acids is insufficient to maintain all the cholesterol in the form of mixed micelles, the excess cholesterol is left behind as vesicles in the aqueous phase. These cholesterol-enriched vesicles are relatively unstable and are prone to aggregate and form large multilamellar vesicles, from which cholesterol crystals nucleate. Growth of crystals may result in the formation of gallstones. An excess of biliary cholesterol in relation to the amount of phospholipids and bile acids can result from hypersecretion of cholesterol, inadequate secretion of bile acids, or both. Cholelithiasis may be further promoted by other factors, such as gallbladder mucin and other nonmucous glycoproteins, as well as by stasis of bile in the gallbladder. Polymorphisms in the hepatic cholesterol transporter ABCG5/G8 and the bilirubin-conjugating enzyme UGT1A1 (see p. 955) contribute to the formation of gallstones in humans.

THE LIVER AS A METABOLIC ORGAN The liver is a metabolically active and highly aerobic organ. It receives ~28% of the total blood flow and extracts ~20% of the oxygen used by the body. The liver is responsible for the synthesis and degradation of carbohydrates, proteins, and lipids. The small molecules that are products of digestion are efficiently sorted in the liver for metabolism, storage, or distribution to extrahepatic tissues for energy. The liver provides energy to other tissues mainly by exporting two substrates that are critical for oxidization in the peripheral tissues, glucose and ketone bodies (e.g., acetoacetate).

The liver can serve as either a source or a sink for glucose The liver is one of the key organs that maintain blood glucose concentrations within a narrow range, in a dynamic process involving endogenous glucose production and glucose utilization. The fasting blood [glucose] is normally 4 to 5 mM. Between meals, when levels of insulin are relatively low (see p. 1036) and levels of glucagon are high (see p. 1052), the liver serves as a source of plasma glucose, both by synthesizing glucose and by generating it from the breakdown of glycogen. The de novo synthesis of glucose from lactate, pyruvate, and amino acids—gluconeogenesis (see p. 1176)— is one of the liver’s most important functions; it is essential for maintaining a normal plasma concentration of glucose, which is the primary energy source for most tissues. The second way in which the liver delivers glucose to blood plasma is by glycogenolysis (see p. 1182). Stored glycogen may account for as much as 7% to 10% of the total

Chapter 46  •  Hepatobiliary Function

weight of the liver. Glycogenolysis in the liver yields glucose as its major product, whereas glycogen breakdown in muscle produces lactic acid (see Fig. 58-9). After a meal, when levels of insulin are relatively high, the liver does just the opposite: it acts as a sink for glucose by taking it up from the portal blood and either breaking it down to pyruvate or using it to synthesize glycogen (see Fig. 51-8 and pp. 1179–1181). Glucose oxidation has two phases. In the anaerobic phase, glucose is broken down to pyruvic acid (glycolysis). In the aerobic phase, pyruvic acid is completely oxidized to H2O and CO2 through the citric acid cycle. The liver also consumes glucose by using it for glycogen synthesis. Carbohydrate that is not stored as glycogen or oxidized is metabolized to fat. All the aforementioned processes are regulated by hormones such as insulin (see Fig. 51-8) and glucagon (see Fig. 51-12), which enable rapid responses to changes in the metabolic requirements of the body.

The liver synthesizes a variety of important plasma proteins (e.g., albumin, coagulation factors, and carriage proteins) and metabolizes dietary amino acids Protein Synthesis  One of the major functions of the liver is to produce a wide array of proteins for export to the blood plasma (Table 46-3). These products include major plasma proteins that are important for maintaining the colloid osmotic pressure of plasma (see p. 470). Other products include factors involved in hemostasis (blood clotting) and fibrinolysis (breakdown of blood clots), carriage proteins that bind and transport hormones and other substances in the blood, prohormones, and lipoproteins (Table 46-4). The liver synthesizes plasma proteins at a maximum rate of 15 to 50 g/day.  N46-12 Amino-Acid Uptake  A major role of the liver is to take up and metabolize dietary amino acids that are absorbed by the gastrointestinal tract (see p. 923) and are transported to the liver in portal blood. These amino acids are taken up by both Na+-dependent and Na+-independent transporters that are identical to some of the amino-acid transporters in the kidney, small intestine, and other tissues (see Table 36-1). An unusual feature of the liver is that, with few exceptions, the same amino-acid transporter may be located on both the basolateral and apical membranes. For example, Na+dependent glutamate uptake by the excitatory amino-acid transporters SLC1A1 (EAAT3) and SLC1A2 (EAAT2) occurs primarily at the apical membrane, but dexamethasone (corticosteroid) treatment can induce their expression at the basolateral membrane. In general, hepatic amino-acid transporters are highly regulated at the transcriptional and posttranslational levels.  N46-13 Amino-Acid Metabolism  Under physiological conditions, total and individual plasma concentrations of amino acids are tightly regulated. The liver controls the availability of amino acids in the systemic blood, activating ureagenesis after a high-protein meal and repressing it during fasting or low protein intake. Unlike glucose, which can be stored, amino acids must either be used immediately (e.g., for the

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TABLE 46-3  Proteins Made by the Liver for Export Major Plasma Proteins Albumin α1-fetoprotein Plasma fibronectin (an α2-glycoprotein) C-reactive protein α2-microglobulin Various other globulins

Factors Involved in Hemostasis/Fibrinolysis Coagulation: fibrinogen and all others except for factor VIII Inhibitors of coagulation: α1-antitrypsin and antithrombin III, α2-macroglobulin, protein S, protein C Fibrinolysis: plasminogen Inhibitors of fibrinolysis: α2-antiplasmin Complement C3

Carriage Proteins (Binding Proteins) Ceruloplasmin (see pp. 970–971) Corticosteroid-binding globulin (CBG, also called transcortin; see p. 1021) Growth hormone–binding protein (low-affinity form; see p. 994) Haptoglobin Hemopexin Insulin-like growth factor 1–binding proteins (see p. 996) Retinol-binding protein (RBP; see p. 970) Sex hormone–binding globulin (SHBG; see p. 1099) Thyroxine-binding globulin (TBG; see pp. 1008–1009) Transferrin (see p. 941) Transthyretin (see pp. 1008–1009) Vitamin D–binding protein (see p. 1064)

Prohormones Angiotensinogen (see p. 1028)

Apolipoproteins Apo Apo Apo Apo Apo Apo Apo

A-I A-II A-IV B-100 C-II D E

synthesis of proteins) or broken down. The breakdown of α-amino acids occurs by deamination to α-keto acids and NH +4 (Fig. 46-14). The α-keto acids (“carbon skeleton”), depending on the structure of the parent amino acid, are metabolized to pyruvate, various intermediates of the citric acid cycle (see Fig. 58-11), acetyl coenzyme A (acetyl CoA), or acetoacetyl CoA. The liver detoxifies ~95% of the NH +4 through a series of reactions known as the urea cycle (see Fig. 46-14); the liver can also use NH +4 —together with glutamate—to generate glutamine.  N46-14  Individual deficiencies in each of the enzymes involved in the urea cycle have been described and result in life-threatening hyperammonemia. The urea generated by the urea cycle exits the hepatocyte via a urea channel, which is, in fact, AQP9. The urea then enters the blood and is ultimately excreted by the kidneys (see pp. 770–772). The glutamine synthesized by the liver also enters the blood. Some of this glutamine is metabolized by the kidney to yield glutamate and NH +4 , which is exported in the urine (see pp. 829–831).

Chapter 46  •  Hepatobiliary Function

N46-12  Synthesis of Plasma Proteins by the Liver

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N46-13  Glutamate Transporters Contributed by Emile Boulpaep and Walter Boron

Contributed by Fred Suchy The synthesis of the hepatic proteins for secretion into the blood plasma occurs via the secretory pathway (see pp. 34–35). The synthesis begins in the rough endoplasmic reticulum (RER). Nearly all proteins secreted by the liver are glycosylated. N-linked glycosylation occurs in the RER (see p. 32), and further remodeling occurs in the Golgi (see pp. 37–38). O-linked glycosylation also occurs in the Golgi (see pp. 38–39). The ER can also conjugate proteins with lipid.

The high-affinity glutamate transporters are classified as members of the SLC1 gene family (see Table 5-4).

Gene Name

Transporter Names

SLC1A1 SLC1A2

EAAT3 or EAAC1 EAAT2 or GLT-1

For a detailed discussion of the family members, consult the review by Kanai and Hediger. listed below

REFERENCE Kanai Y, Hediger MA: The glutamate/neutral amino acid transporter family SLC1: Molecular, physiological and pharmacological aspects. Pflugers Arch 447:467–479, 2004.

N46-14  Hepatic Detoxification of NH+4 by Formation of Glutamine Contributed by Emile Boulpaep and Walter Boron Each day, as part of protein catabolism, the liver detoxifies ~940 mmol of amino groups that are derived from the breakdown of amino acids. The liver detoxifies NH+4 by converting it to urea (95% of the total) and glutamine (the remaining 5%). Both products leave the liver and reach the kidney, which disposes of them—directly or indirectly—in the urine. As indicated in Figure 39-6, the liver consumes ~40 mmol of these amino groups in the reaction

Glutamate + NH+4 → Glutamine + H2O

(NE 46-1)

Glutamine is the most prevalent amino acid in the body. The liver detoxifies the remainder via the urea cycle.

The enzyme required for the conversion in Equation NE 46-1 is glutamine synthetase (see p. 290). In the liver, this enzyme is restricted to the last one or two hepatocytes contiguous with the hepatic venule (zone III; see Fig. 46-3). This strict localization is thought to play an important role in glutamine metabolism. The uptake of an individual amino acid into the hepatocyte may represent the rate-limiting step in its own metabolism and may therefore be an important target for regulation. This type of regulation of uptake occurs for alanine, a critical substrate of gluconeogenesis, and also for glutamine.

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SECTION VII  •  The Gastrointestinal System

TABLE 46-4  Major Classes of Lipoproteins VLDL

IDL

LDL

Density (g/cm3)

CHYLOMICRONS 80% of hepatic vitamin A under normal conditions. Retinol may also undergo oxidation to retinal and conversion to retinoic acid, which plays a key role in phototransduc­ tion (see p. 367). Retinoic acid is conjugated to glucuronide and is secreted into bile, where it undergoes enterohepatic circulation and excretion. Liver disease resulting in cholestasis may lead to a secondary vitamin A deficiency by inter­ fering with absorption in the intestine (lack of the bile needed for digestion/absorption of vitamin A) or by impairing delivery to target tissues because of reduced hepatic synthesis of RBP. Vitamin D  Skin cells—under the influence of ultraviolet light—synthesize vitamin D3 (see p. 1064). Dietary vitamin D can come from either animal sources (D3) or plant sources (D2). In either case, the first step in activation of vitamin D is the 25-hydroxylation of vitamin D, catalyzed by a hepatic cytochrome P-450 enzyme. This hydroxylation is followed by 1-hydroxylation in the kidney to yield a product (1,25dihydroxyvitamin D) with full biological activity. Termination of the activity of 1,25-dihydroxyvitamin D also occurs in the liver by hydroxylation at carbon 24, mediated by another cytochrome P-450 enzyme. Vitamin E  The fat-soluble vitamin E is absorbed from the intestine primarily in the form of α- and γ-tocopherol. It is incorporated into chylomicrons and VLDLs with other products of dietary lipid digestion. As noted above, these particles reach the systemic circulation via the lymphatics and undergo some triacylglycerol hydrolysis. In the process, some vitamin E is transferred to other tissues. The αand γ-tocopherol remaining in the remnant chylomicrons is transported into the liver, which is the major site of dis-

crimination between the two forms. The α-tocopherol is secreted again as a component of hepatically derived VLDL and perhaps HDL. The γ-tocopherol appears to be metabolized or excreted by the liver. A hepatic tocopherolbinding protein may play a role in this discriminatory process. Vitamin K  Vitamin K is a fat-soluble vitamin produced by intestinal bacteria. This vitamin is essential for the γ-carboxylation—by the ER enzyme γ-glutamyl carboxylase— of certain glutamate residues in coagulation factors II, VII, IX, and X as well as anticoagulants protein C and protein S (see Table 18-4) and certain other proteins. Intestinal absorption and handling of vitamin K—which is present in two forms, K1 and K2—are similar to those of the other fatsoluble vitamins, A, D, and E. Common causes of vitamin K deficiency, which can lead to a serious bleeding disorder, include extrahepatic or intrahepatic cholestasis, fat malabsorption, biliary fistulas, and dietary deficiency, particularly in association with antibiotic therapy.

The liver stores copper and iron Copper  The trace element copper is essential for the function of cuproenzymes such as cytochrome C oxidase and superoxide dismutase (see p. 1238). Approximately half the copper in the diet (recommended dietary allowance, 1.5 to 3 mg/day) is absorbed in the jejunum and reaches the liver in the portal blood, mostly bound to albumin. A small fraction is also bound to amino acids, especially histidine. High-affinity copper import across the hepatocyte basolateral membrane is mediated by the copper transport protein CTR1 (SLC31A1). Copper then binds to members of a family of intracellular metallochaperones that direct the metal to the appropriate pathway for incorporation into cuproenzymes or for biliary excretion. It is unknown how hepatocytes distribute copper to the different intracellular routes. The copper chaperone Atox1 ferries the copper through the cytosol to the Wilson disease P-type ATPase ATP7B (Box 46-5; see also p. 118), which is located predominantly in the trans-Golgi network and late endosomes. Intracellular copper levels modulate the activity, post-translational modification, and intracellular localization of ATP7B. Once inside the vesicular lumen, copper can couple with apoceruloplasmin (apo-Cp) to form holo-ceruloplasmin holoCp, which the hepatocyte secretes across the sinusoidal membrane into the blood. Alternatively, the hepatocyte can secrete the copper—perhaps with hepatic copper-binding proteins such as COMMD1 (copper metabolism MURR1 domain)—across the canalicular membrane into the bile. More than 80% of the copper absorbed each day is excreted in bile, for a total of 1.2 to 2.4 mg/day. The small intestine cannot reabsorb the secreted Cu-protein complexes. Processes that impair the biliary excretion of copper result in the accumulation of copper, initially in the lysosomal fraction of hepatocytes, with subsequent elevation of plasma copper levels. Ceruloplasmin, an α2-globulin synthesized by the liver, binds 95% of copper present in the systemic circulation. Ceruloplasmin has ferroxidase activity but has no critical role in the membrane transport or metabolism of copper.

Chapter 46  •  Hepatobiliary Function

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BOX 46-5  Wilson Disease

W

ilson disease is inherited as an autosomal recessive illness caused by a mutation in ATP7B, the pump responsible for copper accumulation in the trans-Golgi network. The impaired biliary excretion of copper causes a buildup of copper in cells, which produces toxic effects in the liver, brain, kidney, cornea, and other tissues. The disease is rare, but it must be considered in the differential diagnosis of anyone younger than 30 years with evidence of significant liver disease. Patients most often have neuropsychiatric complications, including ataxia, tremors, increased salivation, and behavioral changes. Slit-lamp examination of the cornea reveals the diagnostic KayserFleischer rings at the limbus of the cornea.

Iron  Dietary iron is absorbed by the duodenal mucosa and then transported through the blood bound to transferrin (see p. 941), a protein synthesized in the liver. The liver also takes up, secretes, and stores iron. Entry of iron into hepatocytes is mediated through specific cell-surface transferrin receptors (see p. 42). Within the cell, a small pool of soluble iron is maintained for intracellular enzymatic reactions, primarily for those involved in electron transport. However, iron is also toxic to the cell. Hence, most intracellular iron is complexed to ferritin (see p. 941). The toxicity of iron is clearly evident when normal storage mechanisms become overwhelmed, as occurs in hemochromatosis (see Box 45-6), an autosomal recessive disease in which regulation of iron absorption is uncoupled from total-body storage levels. Hepatocytes also play a critical role in iron homeostasis by synthesizing hepcidin (see p. 941), which lowers plasma

Because of the lack of functional ATP7B, the apocerulo­ plasmin in the trans-Golgi network cannot bind copper to form ceruloplasmin. As a result, the hepatocytes secrete apoceruloplasmin, which lacks the ferroxidase activity of ceruloplasmin. Moreover, the serum concentrations of ceruloplasmin are low. Indeed, the best way to confirm the diagnosis of Wilson disease is the detection of a low serum ceruloplasmin level and elevated urinary copper excretion. A few affected patients have normal ceruloplasmin levels, and the diagnosis must then be sought through liver biopsy. The disease can be treated by chelating the excess copper with penicillamine.

iron levels by downregulating the iron-efflux pump FPN1 (see p. 941) in the intestine and macrophages, thereby blocking the release of iron into the circulation. The consequent iron retention in duodenal enterocytes effectively blocks dietary iron absorption and leads to iron retention in reticuloendothelial macrophages. The expression of the HAMP gene, which encodes hepcidin, increases with iron loading and inflammatory cytokines, and decreases with anemia and hypoxia (consistent with enhanced erythropoiesis; see pp. 440–442).

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 46  •  Hepatobiliary Function

REFERENCES Books and Reviews Alpini G, McGill JM Larusso NF: The pathobiology of biliary epithelia. Hepatology 35:1256–1268, 2002. Anderson CM, Stahl A: SLC27 fatty acid transport proteins. Mol Aspects Med 34:516–528, 2013. Ballatori N, Li N, Fang F, et al: OST alpha-OST beta: A key membrane transporter of bile acids and conjugated steroids. Front Biosci 14:2829–2844, 2009. Chiang JY: Bile acids: Regulation of synthesis. J Lipid Res 50(10): 1955–1966, 2009. Claro da Silva T, Polli JE, Swaan PW: The solute carrier family 10 (SLC10): Beyond bile acid transport. Mol Aspects Med 34:252– 269, 2013. Davit-Spraul A, Gonzales E, Baussan C, Jacquemin E: The spectrum of liver diseases related to ABCB4 gene mutations: Pathophysiology and clinical aspects. Semin Liver Dis 30(2):134–146, 2010. Epub April 26, 2010. Ferrier B, Conjard A, Martin M, Baverel G: Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule. Biochem J 337:543–550, 1999. Firrincieli D, Zuniga S, Poupon R, Housset C, Chignard N: Role of nuclear receptors in the biliary epithelium. Dig Dis 29(1):52–57, 2011. Hagenbuch B, Stieger B: The SLCO (former SLC21) superfamily of transporters. Mol Aspects Med 34:396–412, 2013. Kanai Y, Clémençon B, Simonin A, et al: The SLC1 high-affinity glutamate and neutral amino acid transporter family. Mol Aspects Med 34:108–120, 2013. Kim H, Wu X, Lee J: SLC31 (CTR) family of copper transporters in health and disease. Mol Aspects Med 34:561–570, 2013. Kipp H, Arias IM: Trafficking of canalicular ABC transporters in hepatocytes. Annu Rev Physiol 64:595–608, 2002. Koepsell H: The SLC22 family with transporters of organic cations, anions and zwitterions. Mol Aspects Med 34:413–435, 2013. Kullak-Ublick GA, Hagenbuch B, Stieger B: Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. Gastroenterology 109: 1274–1282, 1995. Kullak-Ublick GA, Stieger B, Meier PJ: Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126:322–342, 2004. Palacin M, Estevez R, Bertran J, Zorzano A: Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78:969–1054, 1998. Rhainds D, Brissette L: The role of scavenger receptor class B type I (SR-BI) in lipid trafficking. Defining the rules for lipid traders. Int J Biochem Cell Biol 36:39–77, 2004. Sato R: Sterol metabolism and SREBP activation. Arch Biochem Biophys 15;501(2):177–181, 2010. Slot AJ, Molinski SV, Cole SP: Mammalian multidrug-resistance proteins (MRPs). Essays Biochem 50(1):179–207, 2011. Small DM: Role of ABC transporters in secretion of cholesterol from liver into bile. Proc Natl Acad Sci U S A 100:4–6, 2003. Stieger B: The role of the sodium-taurocholate cotransporting polypeptide (NTCP) and of the bile salt export pump (BSEP) in physiology and pathophysiology of bile formation. Handb Exp Pharmacol 201:205–259, 2011. Svoboda M, Riha J, Wlcek K, et al: Organic anion transporting polypeptides (OATPs): Regulation of expression and function. Curr Drug Metab 2(2):139–153, 2011. Tao TY, Gitlin JD: Hepatic copper metabolism: Insights from genetic disease. Hepatology 37:1241–1247, 2003.

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Theurl M, Theurl I, Hochegger K, et al: Kupffer cells modulate iron homeostasis in mice via regulation of hepcidin expression. J Mol Med (Berl). 86(7):825–835, 2008. Wang JF, Chou KC: Molecular modeling of cytochrome P450 and drug metabolism. Curr Drug Metab 11(4):342–346, 2010. Xiao C, Lewis GF: Regulation of chylomicron production in humans. Biochim Biophys Acta 1821(5):736–746, 2012. Epub October 6, 2011. Zhao C, Dahlman-Wright K: Liver X receptor in cholesterol metabolism. J Endocrinol 204(3):233–240, 2010. Journal Articles Bull LN, van Eijk MJT, Pawlikowska L: A gene encoding a P-type ATPase mutated in two forms of hereditary cholestasis. Nat Genet 18:219–224, 1998. Chan LM, Lowes S, Hirst BH: The ABCs of drug transport in intestine and liver: Efflux proteins limiting drug absorption and bioavailability. Eur J Pharm Sci 21:25–51, 2004. Doege H, Baillie RA, Ortegon AM, et al: Targeted deletion of FATP5 reveals multiple functions in liver metabolism: Alterations in hepatic lipid homeostasis. Gastroenterology 130(4): 1245–1258, 2006. Gibbons GF: Regulation of fatty acid and cholesterol synthesis: Cooperation or competition? Prog Lipid Res 42:479–497, 2003. Groen A, Romero MR, Kunne C, et al: Complementary functions of the flippase ATP8B1 and the floppase ABCB4 in maintaining canalicular membrane integrity. Gastroenterology 141(5):1927– 1937.e1-4, 2011. Hagenbuch B, Meier PJ: Molecular cloning, chromosomal localization and functional characterization of a human liver Na+/bile acid cotransporter. J Clin Invest 93:1326–1331, 1994. Hagenbuch B, Meier PJ: Organic anion transporting polypeptides of the OATP/SLC21 family: Phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/ functional properties. Pflugers Arch 447:653–665, 2004. Hagenbuch B, Meier PJ: The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609:1–18, 2003. Havel RJ, Hamilton RL: Hepatic catabolism of remnant lipoproteins: Where the action is. Arterioscler Thromb Vasc Biol 24: 213–215, 2004. Jonker JW, Schinkel AH: Pharmacological and physiological functions of the polyspecific organic cation transporters: OCT1, 2, and 3 (SLC22A1-3). J Pharmacol Exp Ther 308:2–9, 2004. Oude Elferink RPJ, Meijer DKF, Kuipers F, et al: Hepatobiliary secretion of organic compounds: Molecular mechanisms of membrane transport. Biochim Biophys Acta 1241:215–268, 1995. Roach PJ: Glycogen and its metabolism. Curr Mol Med 2:101–120, 2002. Roden M, Bernroider E: Hepatic glucose metabolism in humans— its role in health and disease. Best Pract Res Clin Endocrinol Metab 17:365–383, 2003. Stanford KI, Bishop JR, Foley EM, et al: Syndecan-1 is the primary heparan sulfate proteoglycan mediating hepatic clearance of triglyceride-rich lipoproteins in mice. J Clin Invest 119(11): 3236–3245, 2009. van de Steeg E, Stránecký V, Hartmannová H, Nosková L, et al: Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J Clin Invest 122(2):519–528, 2012. Wu AL, Coulter S, Liddle C, et al: FGF19 regulates cell proliferation, glucose and bile acid metabolism via FGFR4-dependent and independent pathways. PLoS One 6(3):e17868, 2011.

C H A P T E R 47  ORGANIZATION OF ENDOCRINE CONTROL Eugene J. Barrett

With the development of multicellular organisms that have specialized tissues and organs, two major systems evolved to communicate and coordinate body functions: 1. The nervous system integrates tissue functions by a network of cells and cell processes that constitute the nervous system and all subdivisions, as discussed in Chapters 10 through 16. 2. The endocrine system integrates organ function via chemicals that are secreted from endocrine tissues or “glands” into the extracellular fluid. These chemicals, called hormones, are then carried through the blood to distant target tissues where they are recognized by specific high-affinity receptors. As discussed in Chapter 3, these receptors may be located either on the surface of the target tissue, within the cytosol, or in the target cell’s nucleus. These receptor molecules allow the target cell to recognize a unique hormonal signal from among the numerous chemicals that are carried through the blood and bathe the body’s tissues. The accuracy and sensitivity of this recognition are remarkable in view of the very low concentration (10−9 to 10−12 M) at which many hormones circulate. Once a hormone is recognized by its target tissue or tissues, it can exert its biological action by a process known as signal transduction (see Chapter 3). Here in Chapter 47, we discuss how the signal-transduction cascades couple the hormone to its appropriate end responses. Some hormones elicit responses within seconds (e.g., the increased heart rate provoked by epinephrine or the stimulation of hepatic glycogen breakdown caused by glucagon), whereas others may require many hours or days (e.g., the changes in salt retention elicited by aldosterone or the increases in protein synthesis caused by growth hormone [GH]). We also examine the principles underlying the feedback mechanisms that control endocrine function. In Chapters 48 through 52, we see how the principles introduced in this chapter apply to some specific endocrine systems.

PRINCIPLES OF ENDOCRINE FUNCTION Chemical signaling can occur through endocrine, paracrine, or autocrine pathways As shown in Figure 3-1A, in classic endocrine signaling, a hormone carries a signal from a secretory gland across a 974

large distance to a target tissue. Hormones secreted into the extracellular space can also regulate nearby cells without ever passing through the systemic circulation. This regulation is referred to as paracrine action of a hormone (see Fig. 3-1B). Finally, chemicals can also bind to receptors on or in the cell that is actually secreting the hormone and thus affect the function of the hormone-secreting cell itself. This action is referred to as autocrine regulation (see Fig. 3-1C). All three mechanisms are illustrated for individual endocrine systems in subsequent chapters. At the outset, it can be appreciated that summation of the endocrine, paracrine, and autocrine actions of a hormone can provide the framework for a complex regulatory system. Endocrine Glands  The major hormones of the human body are produced by one of seven classic endocrine glands or gland pairs: the pituitary, the thyroid, the parathyroids, the testes, the ovaries, the adrenals (cortex and medulla), and the endocrine pancreas. In addition, other tissues that are not classically recognized as part of the endocrine system produce hormones and play a vital role in endocrine regulation. These tissues include the central nervous system (CNS), particularly the hypothalamus, as well as the gastrointestinal tract, adipose tissue, liver, heart, and kidney. In some circumstances, particularly with certain neoplasms, nonendocrine tissues can produce hormones that are usually thought to be made only by endocrine glands (Box 47-1). Paracrine Factors  Numerous specialized tissues that are not part of the classic endocrine system release “factors” into the extracellular fluid that can signal neighboring cells to effect a biological response. The interleukins, or lymphokines, are an example of such paracrine factors, as are several of the growth factors, such as platelet-derived growth factor (PDGF), fibroblast growth factor, and others. These factors are not hormones in the usual sense. They are not secreted by glandular tissue, and their sites of action are usually (but not always) within the local environment. However, these signaling molecules share many properties of the classic peptide and amine hormones in that they bind to surface receptors and regulate one or more of the specific intracellular signaling mechanisms described in Chapter 3. The distinction between the hormones of the classic endocrine systems and other biologically active secreted peptides blurs even further in the case of neuropeptides. For example, the hormone somatostatin, a 28–amino-acid

Chapter 47  •  Organization of Endocrine Control

BOX 47-1  Neoplastic Hormone Production

T

he ability of nonendocrine tissue to produce hormones first became apparent with the description of clinical syndromes in which some patients with lung cancer were found to make excessive amounts of AVP, a hormone usually made by the hypothalamus. Shortly afterward, people with other lung or gastrointestinal tumors were found to make ACTH, which is normally made only in the pituitary. Subsequently, many hormone-secreting neoplastic tissues were described. As the ability to measure hormones in tissues has improved and, in particular, as the capability of measuring mRNA that codes for specific peptide hormones has developed, it has become clear that hormone production by neoplastic tissue is quite common, although most tumors produce only small amounts that may have no clinical consequence. The production of hormones by nonendocrine neoplastic cells has been most clearly defined for cancers of the lung. Several different types of lung cancer occur, each deriving from a different cell line, and yet each is capable of producing one or several hormones. The clinical syndromes that result from secretion of these hormones are often called paraneoplastic syndromes. Thus, lung cancers arising from squamous cells are sometimes associated with hypercalcemia, which results from the secretion of a protein—parathyroid hormone–related peptide—that can mimic the activity of PTH (see p. 1069). Small-cell lung cancers are notorious for their ability to secrete numerous hormones, including AVP (with resultant hyponatremia; see Box 38-3), ACTH (with resultant Cushing syndrome; see Box 50-1), and many others. Still other types of lung cancer produce other paraneoplastic syndromes. Nearly all these ectopic, neoplastic sources of hormone produce peptide hormones. Other sources of hormone production, in addition to lung cancer, include gastrointestinal tumors, renal and bladder cancer, neural tumors, unique tumors called carcinoid tumors that can arise almost anywhere in the body, and even lymphomas and melanomas. In some patients, the symptoms and signs resulting from ectopic hormone production may appear before any other reason exists to suspect an underlying neoplasm, and these symptoms may be the key clues to the correct diagnosis.

peptide secreted by the δ cells of the pancreatic islet, acts in paracrine fashion on other islet cells to regulate insulin and glucagon secretion (see p. 1053). However, somatostatin is also made by hypothalamic neurons. Nerve terminals in the hypothalamus release somatostatin into the pituitary portal bloodstream (see pp. 993–994). This specialized segment of the circulatory system then carries the somatostatin from the hypothalamus to the anterior pituitary, where it inhibits the secretion of GH. Somatostatin in the hypothalamus is one of several neuropeptides that bridge the body’s two major communication systems.

Hormones may be peptides, metabolites of single amino acids, or metabolites of cholesterol Although the chemical nature of hormones is diverse, most commonly recognized mammalian hormones can be grouped into one of several classes. Table 47-1 is a list of many of the recognized classic mammalian hormones, which

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TABLE 47-1  Chemical Classification of Selected Hormones Peptide Hormones Adrenocorticotropic hormone (ACTH) Atrial natriuretic peptide (ANP) Arginine vasopressin (AVP), also known as antidiuretic hormone (ADH) Calcitonin Cholecystokinin (CCK) Corticotropin-releasing hormone (CRH) Follicle-stimulating hormone (FSH) Glucagon Gonadotropin-releasing hormone (GnRH) Growth hormone (GH) Growth hormone–releasing hormone (GHRH) Inhibin Insulin Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) Luteinizing hormone (LH) Oxytocin (OT) Parathyroid hormone (PTH) Prolactin (PRL) Secretin Somatostatin Thyrotropin (TSH) Thyrotropin-releasing hormone (TRH) Vasoactive intestinal peptide (VIP)

Amino Acid–Derived Hormones Dopamine (DA) Epinephrine (Epi), also known as adrenaline Norepinephrine (NE), also known as noradrenaline Serotonin, also known as 5-hydroxytryptamine (5-HT) Thyroxine (T4) Triiodothyronine (T3)

Steroid Hormones Aldosterone Cortisol Estradiol (E2) Progesterone Testosterone

are divided into three groups based on their chemical structure and how they are made in the body. Peptide hormones include a large group of hormones made by a variety of endocrine tissues. Insulin, glucagon, and somatostatin are made in the pancreas. The pituitary gland makes GH; the two gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH); adrenocorticotropic hormone (ACTH); thyrotropin (also called thyroid-stimulating hormone or TSH); and prolactin (PRL). The parathyroid glands make parathyroid hormone (PTH), and the thyroid gland make calcitonin. In addition, other peptide hormones, such as somatostatin and several releasing hormones (e.g., growth hormone– releasing hormone [GHRH]), are made by the hypothalamus. Secretin, cholecystokinin, glucagon-like peptide 1 (GLP-1) and other hormones are made by the gastrointestinal tract, which is not considered a classic endocrine gland. The synthesis of catecholamines (from tyrosine) and steroid hormones (from cholesterol) requires a number of

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SECTION VIII  •  The Endocrine System

enzymes present in only very specialized tissues. Synthesis of thyroid hormone is even more complex and is essentially restricted to the thyroid gland. Several glands make two or more hormones. Examples are the pituitary, the pancreatic islets, and the adrenal glands. However, for the most part, individual cells within these glands are specialized to secrete a single hormone. One exception is the gonadotropin-producing cells of the pituitary, which secrete both FSH and LH.

Hormones can circulate either free or bound to carrier proteins

of autoimmune hyperthyroidism), and Addison disease (one form of adrenal insufficiency). Before the description of insulin autoantibodies, it was thought that the immune system simply did not react to self-antigens. 2. Because antibodies with a high affinity for insulin were induced in patients who were treated with insulin, Berson and Yalow reasoned that these antibodies could be used to measure the amount of insulin in serum. Figure 47-1 illustrates the principle of a radioimmunoassay and how it is used to measure the concentration of a hormone (or other chemicals). If we incubate increasing amounts of a radiolabeled hormone with an antibody to that hormone, the quantity of labeled hormone that is bound to the antibody yields a saturation plot (see Fig. 47-1A). If we now add unlabeled hormone to the incubation mixture, less radioactively labeled hormone remains complexed to the antibody as unlabeled hormone takes its place. The more unlabeled hormone we add, the less labeled hormone is bound to the antibody (see Fig. 47-1B). A displacement curve is created by plotting the amount of radioactively labeled hormone complexed to the antibody as a function of the concentration of unlabeled hormone that is added (see Fig. 47-1C). This displacement curve can then be used as a standard curve to estimate the amount of hormone present in unknown samples. This estimate is accurate only if two assumptions hold true: first, that nothing else in the unknown mixture binds with the antibody other than the hormone under study, and second, that nothing in the unknown sample interferes with normal binding of the hormone to the antibody.

Once secreted, many hormones circulate freely in the blood until they reach their target tissue. Others form complexes with circulating binding protein; this use of binding proteins is particularly applicable for thyroid hormones (thy­ roxine [T4] and triiodothyronine [T3]), steroid hormones, insulin-like growth factor types 1 and 2 (IGF-1 and IGF-2), and GH. Formation of a complex between a hormone and a circulating binding protein serves several functions. First, it provides the blood with a reservoir or pool of the hormone and thus minimizes minute-to-minute fluctuations in hormone concentration. Second, it extends the half-life of the hormone in the circulation. For example, >99.99% of T4 circulates bound to one of three binding proteins (see pp. 1008–1009); the half-life of circulating bound T4 is 7 to 8 days, whereas the half-life of free T4 is only several minutes. The hormones bound to plasma binding proteins appear to be those whose actions are long term—in particular, those involving induction of the synthesis of new protein in target tissues. Hormones that play a major short-term role in the regulation of body metabolism (e.g., catecholamines, many peptide hormones) circulate freely without associated bind­ ing proteins. The presence of plasma binding proteins can affect the total circulating concentration of a hormone without necessarily affecting the concentration of unbound or free hormone in the blood. For example, during pregnancy the liver’s synthesis of T4-binding globulin increases. Because this protein avidly binds T4, the free T4 concentration ordinarily would fall. However, the pituitary senses the small decline in free T4 levels and secretes more TSH. As a result, the thyroid makes more T4, so plasma levels of total T4 rise. However, the free T4 level does not rise.

Antibodies that are highly specific for the chemical structure of interest can frequently be obtained. Moreover, these antibodies are of sufficiently high affinity to bind even the often minute amounts of hormone that is circulating in blood. Thus, radioimmunoassays—and recent adaptations that substitute chemiluminescent or enzymatic detection for radioactivity—have emerged as a potent and popular tool. Immunoassays are now used for the measurement of virtually all hormones, as well as many drugs, viruses, and toxins. Much of our understanding of the physiology of hormone secretion and action has been gained by the use of immunoassay methodology. Yalow shared the 1977 Nobel Prize in Medicine or Physiology for the discovery of the radioimmunoassay (Berson died before the honor was bestowed).  N47-1

Immunoassays allow measurement of circulating hormones

Hormones can have complementary and antagonistic actions

In the late 1950s, Solomon Berson and Rosalyn Yalow demonstrated that patients who receive insulin form antibodies directed against the insulin molecule. This observation was important in two respects: 1. It advanced the principle that the body’s immune system can react to endogenous compounds; therefore, auto­ immunity or reaction to self-antigens does occur. This notion is a fundamental tenet of our current understanding of many autoimmune diseases, among which are endocrine diseases such as type 1 diabetes mellitus, autoimmune hypothyroidism, Graves disease (a common form

Regulation of many complex physiological functions necessitates the complementary action of several hormones. This principle is true both for minute-to-minute homeostasis and for more long-term processes. For example, epinephrine (adrenaline), cortisol, and glucagon each contribute to the body’s response to a short-term bout of exercise (e.g., swimming the 50-m butterfly or running the 100-m dash). If any of these hormones is missing, exercise performance is adversely affected, and even more seriously, severe hypoglycemia and hyperkalemia (elevated plasma [K+]) may develop. On a longer time scale, GH, insulin, IGF-1, thyroid hormone,

Chapter 47  •  Organization of Endocrine Control

N47-1  Rosalyn Yalow For more information about Rosalyn Yalow and the work that led to her Nobel Prize, visit http://www.nobelprize.org/ nobel_prizes/medicine/laureates/1977/# (accessed September 2014).

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A

C

SATURATION PLOT

Increasing the amount of unlabeled hormone gradually decreases the amount of labeled antibody.

50

If increasing amounts of radiolabeled hormone are incubated with a fixed amount of an antibody to that hormone, the quantity of the hormone that binds to the antibody saturates.

[Hormoneantibody complex]

DISPLACEMENT CURVE 70 Dish 1 in panel B 60

Labeled 40 hormone bound to antibody 30 20

Dish 2 in panel B

10

[Total labeled hormone]

977

0

Dish 3 in panel B 1

2

3

4

5

6

[Total unlabeled hormone] B

DISPLACEMENT OF A LABELED HORMONE BY AN UNLABELED HORMONE 1

+ +

+ +

+

+

2

+

+

+

= Antibody = Labeled hormone = Unlabeled hormone

+ +

+ +

+

+

+

+ +

+

3

+

+

+

Equilibrium exists between the labeled hormone, the antibody, and the hormone-antibody complex.

+

+ + +

+ +

+

With the addition of a small amount of the unlabeled hormone, some of which binds to the antibody, fewer antibodies are labeled.

+ +

+

+ + +

+

+

+

+ +

Further addition of unlabeled hormone leads to further displacement of the labeled hormone from the antibody. The number of labeled antibodies is thus less than that in 2 .

Figure 47-1  Principles of the radioimmunoassay.

and sex steroids are all needed for normal growth. Deficiency of GH, IGF-1, or thyroid hormone results in dwarfism. Deficiency of sex steroids, cortisol, or insulin produces less severe disturbances of growth. Integration of hormone action can also involve hormones that exert antagonistic actions. In this case, the overall effect on an end organ depends on the balance between opposing influences. One example is the counterpoised effects of insulin and glucagon on blood glucose levels. Insulin lowers glucose levels by inhibiting glycogenolysis and gluconeogenesis in the liver and by stimulating glucose uptake into muscle and adipose tissue. Glucagon, in contrast, stimulates hepatic glycogenolysis and gluconeogenesis. Whereas glucagon does not appear to directly antagonize glucose uptake by muscle or fat, epinephrine (which, like glucagon, is released in response to hypoglycemia) does. Balancing of tissue function by opposing humoral effector mechanisms appears to be an important regulatory strategy for refining the control of many cellular functions.

Endocrine regulation occurs through feedback control The key to any regulatory system is its ability to sense when it should increase or decrease its activity. For the endocrine system, this function is accomplished by feedback control of hormone secretion (Fig. 47-2A). The hormone-secreting cell functions as a “sensor” that continually monitors the circulating concentration of some regulated variable. This variable may be a metabolic factor (e.g., glucose concentration) or the activity of another hormone. When the endocrine gland senses that too much (or too little) of the regulated variable is circulating in blood, it responds by decreasing (or increasing) the rate of hormone secretion. This response in turn affects the metabolic or secretory behavior of the target tissue, which may either directly feed back to the sensing cell or stimulate some other cell that eventually signals the sensor regarding whether the altered function of the endocrine gland has been effective.

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SECTION VIII  •  The Endocrine System

A

SIMPLE FEEDBACK LOOP Sensor

B

HIERARCHICAL CONTROL Cerebral cortex

Hormone

Target 1 Other hormone or metabolite

Hypothalamus CRH

Anterior pituitary Target 2

ACTH

Adrenal cortex

Cortisol

Target tissue Figure 47-2  Feedback control of hormone secretion. A, A sensor (e.g., a β cell in a pancreatic islet) detects some regulated variable (e.g., plasma [glucose]) and responds by modulating its secretion of a hormone (e.g., insulin). This hormone, in turn, acts on target 1 (e.g., liver or muscle) to modulate its production of another hormone or a metabolite (e.g., reducing [glucose]), which may affect target 2 (e.g., making less glucose available to the brain). In addition, the other hormone or metabolite feeds back on the original sensor cell. B, Under the influence of the cerebral cortex, the hypothalamus releases CRH, which stimulates the anterior pituitary to release ACTH, which in turn stimulates the adrenal cortex to release cortisol. The cortisol acts on a number of effector organs. In addition, the cortisol feeds back on both the anterior pituitary and the hypothalamus.

A simple example is insulin secretion by the β cells of the pancreas. Increases in plasma [glucose] are sensed by the β cell, which secretes insulin in response. The rise in plasma [insulin] acts on the liver to decrease the synthesis of glucose and on the muscle to promote the storage of glucose. As a result, plasma [glucose] falls, and this decrease is sensed by the β cell, which reduces the rate of insulin secretion. This arrangement represents a very simple feedback system. Other systems can be quite complex; however, even this simple system involves the recognition of two circulating signals. The liver and muscle recognize the increase in plasma [insulin] as one signal, and the pancreatic β cell (the cell responsible for insulin secretion) recognizes the signal of a rise or decline in blood [glucose] as the other signal. In each case, the sensing system within a particular tissue is linked to an effector system that transduces the signal to the appropriate biological response.

Endocrine regulation can involve hierarchic levels of control Faced with a stress (e.g., a severe infection or extensive blood loss), the cerebral cortex stimulates the hypothalamus to release a neuropeptide called corticotropin-releasing hormone (CRH; see Fig. 47-2B). Carried by the pituitary portal system (blood vessels that connect the hypothalamus to the anterior pituitary), CRH stimulates the anterior pituitary to release another hormone, ACTH, which in turn stimulates the adrenal cortical cells to synthesize cortisol. Cortisol regulates vascular tone as well as metabolic and growth functions in a variety of tissues. This stress response therefore involves the cerebral cortex, specialized neuroendocrine tissue in the hypothalamus, as well as two glands, the pituitary and the adrenal cortex. This hierarchic control is regulated by feedback, just as in the simple feedback between plasma [glucose] and insulin. Within this CRH-ACTH-cortisol axis, feedback can occur at several levels. Cortisol inhibits the production of CRH by the hypothalamus as well as the sensitivity of the pituitary to a standard dose of CRH, which directly reduces ACTH release. Feedback in hierarchic endocrine control systems can be quite complex and frequently involves interaction between the CNS and the endocrine system. Other examples are regulation of the female menstrual cycle (see pp.1110–1116) and regulation of GH secretion (see pp. 992–994). Among the classic endocrine tissues, the pituitary (also known as the hypophysis) plays a special role (Fig. 47-3). Located at the base of the brain, just below the hypothalamus, the pituitary resides within a saddle-shaped cavity called the sella turcica (from the Latin sella [saddle] + turcica [Turkish]), which has bony anterior, posterior, and inferior borders and fibrous tissue that separate it from venous sinuses on either side. The human pituitary is composed of both an anterior lobe and a posterior lobe. Through vascular and neural connections, the pituitary bridges and integrates neural and endocrine mechanisms of homeostasis. The pituitary is a highly vascular tissue. The posterior pituitary receives arterial blood, whereas the anterior pituitary receives only portal venous inflow from the median eminence. The pituitary portal system is particularly important in carrying neuropeptides from the hypothalamus and pituitary stalk to the anterior pituitary.

The anterior pituitary regulates reproduction, growth, energy metabolism, and stress responses Glandular tissue in the anterior lobe of the pituitary synthesizes and secretes six peptide hormones: GH, TSH, ACTH, LH, FSH, and PRL. In each case, secretion of these hormones is under the control of hypothalamic releasing hormones (Table 47-2). The sources of these releasing hormones are small-diameter neurons located mainly in the “periventricular” portion of the hypothalamus that surrounds the third ventricle (see pp. 275–277). These small-diameter neurons synthesize the releasing hormones and discharge them into the median eminence and neural stalk, where they enter leaky capillaries—which are not part of the blood-brain barrier (see pp. 284–287). The releasing hormones then travel via the pituitary portal veins to the anterior pituitary.

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TABLE 47-2  Hypothalamic and Pituitary Hormones ANTERIOR PITUITARY RELEASING (INHIBITORY) FACTOR MADE BY HYPOTHALAMUS

TARGET CELL IN ANTERIOR PITUITARY

HORMONE RELEASED BY ANTERIOR PITUITARY

TARGET OF ANTERIOR PITUITARY HORMONE

GHRH (inhibited by somatostatin)

Somatotroph

GH

Stimulates IGF-1 production by multiple somatic tissues, especially liver

TRH

Thyrotroph

TSH

Thyroid follicular cells, stimulated to make thyroid hormone

CRH

Corticotroph

ACTH

Fasciculata and reticularis cells of the adrenal cortex, to make corticosteroids

GnRH

Gonadotroph

FSH

Ovarian follicular cells, to make estrogens and progestins Sertoli cells, to initiate spermatogenesis

GnRH

Gonadotroph

LH

Leydig cells, to make testosterone

(inhibited by dopamine)

Lactotroph

PRL

Mammary glands, initiates and maintains milk production

POSTERIOR PITUITARY HORMONE SYNTHESIZED IN HYPOTHALAMUS

HORMONE RELEASED INTO POSTERIOR PITUITARY

TARGET OF POSTERIOR PITUITARY HORMONE

AVP

AVP

Collecting duct, to increase water permeability

OT

OT

Uterus, to contract Mammary gland, to eject milk

GnRH, gonadotropin-releasing hormone; OT, oxytocin.

Once in the anterior pituitary, a releasing factor (e.g., GHRH) stimulates specialized cells to release a particular peptide hormone (e.g., GH) into the systemic bloodstream. The integrative function of the anterior pituitary can be appreciated by realizing that the main target for four of the anterior pituitary hormones (i.e., TSH, ACTH, and LH/FSH) is other endocrine tissue. Thus, these four anterior pituitary hormones are themselves “releasing hormones” that trigger the secretion of specific hormones. For example, TSH causes the follicular cells in the thyroid gland to synthesize and release thyroid hormones. The mechanism by which the pituitary regulates these endocrine glands is discussed in detail in Chapters 48 through 50. GH also acts as a releasing factor in that it regulates the production of another hormone, IGF-1. IGF-1 is made in principally nonendocrine tissues (e.g., liver, kidney, muscle, and cartilage). Nevertheless, IGF-1 in the circulation feeds back on the hypothalamus to decrease GHRH level and on the pituitary to inhibit GH secretion. In this respect, the GH–IGF-1 axis is similar to axes involving classic pituitary pathways, such as the thyrotropin-releasing hormone (TRH)–TSH axis. Regulation of PRL secretion differs from that of other anterior pituitary hormones in that no endocrine feedback mechanism has yet been identified. In humans, the pituitary secretes PRL at relatively low levels throughout life. However, its major biological action is important only in women during lactation. Although PRL is not part of an identified feedback system, its release is controlled. Left to its own devices, the anterior pituitary would secrete high levels of PRL. However, secretion of PRL is normally inhibited by the release of dopamine (DA) from the hypothalamus (see

pp. 993–994). During breast stimulation, neural afferents inhibit hypothalamic DA release, thus inhibiting release of the inhibitor and permitting lactation to proceed. PRL receptors are present on multiple tissues other than the breast. However, other physiological actions beyond lactation have not been well characterized.

The posterior pituitary regulates water balance and uterine contraction Unlike the anterior pituitary, the posterior lobe of the pituitary is actually part of the brain. The posterior pituitary (or neurohypophysis) contains the nerve endings of largediameter neurons whose cell bodies are in the supraoptic and paraventricular nuclei of the hypothalamus (see Fig. 47-3). Recall that the hypothalamic neurons that produce releasing factors, which act on “troph” cells in the anterior pituitary, are small-diameter neurons. The large-diameter hypothalamic neurons synthesize arginine vasopressin (AVP) and oxytocin and then transport these hormones along their axons to the site of release in the posterior pituitary. Thus, like the anterior pituitary, the posterior pituitary releases peptide hormones. Also as in the anterior pituitary, release of these hormones is under ultimate control of the hypothalamus. However, the hypothalamic axons traveling to the posterior pituitary replace both the transport of releasing factors by the portal system of the anterior pituitary and the synthesis of hormones by the anterior pituitary “troph” cells. Although the posterior pituitary is part of the brain, it is one of the so-called circumventricular organs (see pp. 284–285) whose vessels breach the blood-brain barrier and allow the secreted AVP and oxytocin to reach the systemic circulation.

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SECTION VIII  •  The Endocrine System

Large-bodied neurons make AVP and OT, and transport these hormones down their axons to the posterior pituitary for release.

Neurosecretory cells

Paraventricular nucleus Dorsomedial nucleus

Posterior nucleus Lateral hypothalamic area

Preoptic nuclei Small-bodied neurons make releasing hormones, transport these down their axons, and secrete them into primary capillary plexuses.

Ventromedial nucleus Supraoptic nucleus Arcuate nucleus

Suprachiasmatic nucleus

Infundibular nucleus

Hypothalamus

Median eminence

Optic chiasm Superior hypophyseal artery Hypothalamohypophyseal tract

Long portal veins

Anterior pituitary hormones

Primary plexus of hypophyseal portal system

Short portal veins Trabecula (fibrous tissue)

Pituitary stalk

Posterior pituitary hormones

“Troph” cells Anterior lobe of pituitary gland Secondary plexus of hypophyseal portal system

Posterior lobe of pituitary gland Anterior pituitary hormones

Inferior hypophyseal artery

Chapter 47  •  Organization of Endocrine Control

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Figure 47-3  Hypothalamic-pituitary axis. The pituitary (or hypophysis) is actually two glands—an anterior pituitary and a posterior pituitary (or neurohypophysis). Although in both cases the hypothalamus controls the secretion of hormones by the pituitary, the mechanisms are very different. Anterior pituitary: Small-bodied neurons in the hypothalamus secrete releasing and inhibitory factors into a rich, funnel-shaped plexus of capillaries that penetrates the median eminence and surrounds the infundibular recess. The cell bodies of these neurons are in several nuclei that surround the third ventricle. These include the arcuate nucleus, the paraventricular and ventromedial nuclei, and the medial preoptic and periventricular regions. The capillaries (primary plexus), which are outside of the blood-brain barrier, coalesce into long portal veins that carry the releasing and inhibitory factors down the pituitary stalk to the anterior pituitary. Other neurons secrete their releasing factors into a capillary plexus that is much further down the pituitary stalk; short portal veins carry these releasing factors to the anterior pituitary. There, the portal veins break up into the secondary capillary plexus of the anterior pituitary and deliver the releasing and inhibitory factors to the “troph” cells that actually secrete the anterior pituitary hormones (GH, TSH, ACTH, LH, FSH, and PRL) that enter the systemic bloodstream and distribute throughout the body. Posterior pituitary: Large neurons in the paraventricular and supraoptic nuclei of the hypothalamus actually synthesize the hormones AVP and oxytocin (OT). These hormones travel down the axons of the hypothalamic neurons to the posterior pituitary, where the nerve terminals release the hormones, like neurotransmitters, into a rich plexus of vessels.

AVP (or antidiuretic hormone, ADH) is a neuropeptide hormone that acts on the collecting duct of the kidney to increase water reabsorption (see pp. 817–820). Oxytocin (OT) is the other neuropeptide secreted by the posterior pituitary. However, its principal biological action relates to stimulation of smooth-muscle contraction by the uterus during parturition (see pp. 1145–1146) and by the mammary gland during suckling (see p. 1150). These two posterior pituitary hormones appear to have a common ancestor—vasotocin—in amphibians and other submammalian species. The two peptide hormones secreted by the posterior pituitary are each made by hypothalamic neurons as a precursor molecule that is transported along the axons of the hypothalamic neurons to the posterior pituitary. For AVP, this precursor protein is proneurophysin II (see p. 845 and Fig. 40-8), whereas for oxytocin it is proneurophysin I. In each case, cleavage of the precursor occurs during transport along the axons from the hypothalamus to the posterior pituitary. At the time that the active neurohormone (e.g., AVP) is secreted, its residual neurophysin is co-secreted stoichiometrically. Defects in the processing of the neurophysin precursor can lead to impaired secretion of active hormone. In the case of AVP, the result is partial or complete diabetes insipidus.

PEPTIDE HORMONES Specialized endocrine cells synthesize, store, and secrete peptide hormones Organisms as primitive as fungi secrete proteins or peptides in an effort to respond to and affect their environment. In more complex organisms, peptide hormones play important developmental and other regulatory roles. Transcription of peptide hormones is regulated by both cis- and trans-acting elements (see p. 78). When transcription is active, the mRNA is processed in the nucleus and the capped message moves to the cytosol, where it associates with ribosomes on the rough endoplasmic reticulum. These peptides are destined for secretion because an amino-acid signal sequence (see p. 28) present near the N terminus targets the protein to the endoplasmic reticulum while the protein is still associated with the ribosome.

With minor modification, the secretory pathway illustrated in Figure 2-18 can describe the synthesis, processing, storage, and secretion of peptides by a wide variety of endocrine tissues. Once the protein is in the lumen of the endoplasmic reticulum, processing (e.g., glycosylation or further proteolytic cleavage) yields the mature, biologically active hormone. This processing occurs in a very dynamic setting. The protein is first transferred to the cis-Golgi domain, then through to the trans-Golgi domain, and finally to the membrane-bound secretory vesicle or granule in which the mature hormone is stored before secretion. This path­ way is referred to as the regulated pathway of hormone synthesis because external stimuli can trigger the cell to release hormone that is stored in the secretory granule as well as to increase synthesis of additional hormone. For example, binding of GHRH to somatotrophs causes them to release GH. A second pathway of hormone synthesis is the constitutive pathway. Here, secretion occurs more directly from the endoplasmic reticulum or vesicles formed in the cis Golgi. Secretion of hormone, both mature and partially processed, by the constitutive pathway is less responsive to secretory stimuli than is secretion by the regulated pathway. In both the regulated and constitutive pathways, fusion of the vesicular membrane with the plasma membrane— exocytosis of the vesicular contents—is the final common pathway for hormone secretion. In general, the regulated pathway is capable of secreting much larger amounts of hormone—on demand—than is the constitutive pathway. However, even when stimulated to secrete its peptide hormone, the cell typically secretes only a very small amount of the total hormone present in the secretory granules. To maintain this secretory reserve, many endocrine cells increase the synthesis of peptide hormones in response to the same stimuli that trigger secretion.

Peptide hormones bind to cell-surface receptors and activate a variety of signal-transduction systems Once secreted, most peptide hormones exist free in the circulation. As noted above, this lack of binding proteins contrasts with the situation for steroid and thyroid hormones, which circulate bound to plasma proteins. IGF-1 and IGF-2

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TABLE 47-3  Peptide Hormones and Their Signal-Transduction Pathways AGONISTS

RECEPTOR

LINKED ENZYME

SECOND MESSENGER

PTH

Coupled to Gαs

Adenylyl cyclase

cAMP

ANG II

Coupled to Gαi

Adenylyl cyclase (inhibited)

cAMP

AVP, ANG II, TRH

Coupled to Gαq

PLC

IP3 and DAG

ANG II

Coupled to Gi/Go

PLA2

Arachidonic acid metabolites

ANP

Guanylyl cyclase

Guanylyl cyclase

cGMP

Insulin, IGF-1, IGF-2, EGF, PDGF

Tyrosine kinase

Tyrosine kinase

Phosphoproteins

GH, erythropoietin, LIF

Associated with tyrosine kinase

JAK/STAT family of tyrosine kinases

Phosphoproteins

ANG II, angiotensin II; ANP, atrial natriuretic peptide; EGF, epidermal growth factor; LIF, leukemia inhibitory factor; STAT, signal transducer and activator of transcription.

are an exception to this rule: at least six plasma proteins bind these peptide growth factors. While traversing the circulation, peptide hormones encounter receptors on the surface of target cells. These receptors are intrinsic membrane proteins that bind the hormone with very high affinity (typically, KD ranges from 10−8 to 10−12 M). Examples of several types of peptide hormone receptors are shown in Figure 47-4. Each of these receptors has already been introduced in Chapter 3. The primary sequence of most peptide hormone receptors is known from molecular cloning, mutant receptors have been synthesized, and the properties of native and mutant receptors have been compared to assess primary structural requirements for receptor function. Despite this elegant work, too little information is available on the threedimensional structure of these membrane proteins for us to know just how the message that a hormone has bound to the receptor is transmitted to the internal surface of the cell membrane. However, regardless of the details, occupancy of the receptor can activate many different intracellular signaltransduction systems (Table 47-3) that transfer the signal of cell activation from the internal surface of the membrane to intracellular targets. The receptor provides the link between a specific extracellular hormone and the activation of a specific signal-transduction system. We discussed each of these signal-transduction systems in Chapter 3. Here, we briefly review the various signal-transduction systems through which peptide hormones act. G Proteins Coupled to Adenylyl Cyclase  cAMP, the pro­ totypical second messenger, was discovered during an investigation of the action of glucagon on glycogenolysis in the liver. In addition to playing a role in hormone action, cAMP is involved in such diverse processes as lymphocyte activation, mast cell degranulation, and even slime mold aggregation. As summarized in Figure 47-4A, binding of the appropriate hormone (e.g., PTH) to its receptor initiates a cascade of events (see pp. 56–57): (1) activation of a heterotrimeric G protein (αs or αi); (2) activation (by αs) or inhibition (by αi) of a membrane-bound adenylyl cyclase; (3) formation of intracellular cAMP from ATP, catalyzed by adenylyl cyclase; (4) binding of cAMP to the enzyme protein kinase A (PKA); (5) separation of the two catalytic subunits of PKA from the two regulatory subunits; (6) phosphorylation of serine and

BOX 47-2  Pseudohypoparathyroidism

I

nasmuch as G proteins are part of the signaling system involved in large numbers of hormone responses, molecular alterations in G proteins could be expected to affect a num­ ber of signaling systems. In the disorder pseudohypoparathyroidism, the key defect is an abnormality in a stimulatory α subunit (αs) of a heterotrimeric G protein. The result is an impairment in the ability of PTH to regulate body calcium and phosphorus homeostasis (see pp. 1058–1063). Patients with this disorder have a low serum calcium level and high serum phosphate level, just like patients whose parathyroid glands have been surgically removed. However, patients with pseudohypoparathyroidism have increased circulating concentrations of PTH; the hormone simply cannot act normally on its target tissue, hence the term pseudohypoparathyroidism. These individuals also have an increased risk of hypothyroidism, as well as of gonadal dysfunction. These additional endocrine deficiencies arise from the same defect in signaling.

threonine residues on a variety of cellular enzymes and other proteins by the free catalytic subunits of PKA that are no longer restrained; and (7) modification of cellular function by these phosphorylations. The activation is terminated in two ways. First, phosphodiesterases in the cell degrade cAMP. Second, serine/threonine-specific phosphoprotein phosphatases can dephosphorylate enzymes and proteins that had previously been phosphorylated by PKA (Box 47-2). G Proteins Coupled to Phospholipase C  As summarized in Figure 47-4B, binding of the appropriate peptide hormone (e.g., AVP) to its receptor initiates the following cascade of events (see pp. 58–61): (1) activation of Gαq; (2) activation of a membrane-bound phospholipase C (PLC); and (3) cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) by this PLC, with the generation of two signaling molecules, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The IP3 fork of the pathway includes (4a) binding of IP3 to a receptor on the cytosolic surface of the endoplasmic reticulum; (5a) release of Ca2+ from internal stores, which causes [Ca2+]i to rise by several-fold; and (6a) activation of Ca2+-dependent kinases (e.g., Ca2+-calmodulin–dependent protein kinases, protein kinase C [PKC]) by the increases in [Ca2+]i.

Chapter 47  •  Organization of Endocrine Control

N

A

B

PARATHYROID HORMONE (PTH)

C

ARGININE VASOPRESSIN (AVP)

THYROTROPIN-RELEASING HORMONE (TRH)

Extracellular space C

γ

α

β

γ

AC

G-protein complex

β

α

γ

PLC

cAMP

β

+ IP3

DAG Arachidonic acid metabolites

Ca2+ PKC

Cytosol ATRIAL NATRIURETIC PEPTIDE (ANP)

E

F

INSULIN

GROWTH HORMONE (GH)

Extracellular space N

N

N

N

α

α S S

S

S

C

S

JAK

Guanylyl cyclase domains Cytosol

C

C

cGMP

β

S

β

C

JAK

C

Tyrosine kinase domains

C

Tyrosine kinase domains Protein phosphorylation

PLA2

Phospholipase A2

PKA

D

α

Protein phosphorylation

Figure 47-4  Receptors and downstream effectors for peptide hormones. AC, adenylyl cyclase.

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SECTION VIII  •  The Endocrine System

The DAG fork of the pathway includes (4b) allosteric activation of PKC by DAG (the activity of this enzyme is also stimulated by the increased [Ca2+]i); and (5b) phosphorylation of a variety of proteins by PKC, which is activated in the plane of the cell membrane. An example of a hormone whose actions are in part mediated by DAG is TSH. G Proteins Coupled to Phospholipase A2  As summarized in Figure 47-4C, some peptide hormones (e.g., TRH) activate phospholipase A2 (PLA2) through the following cascade (see pp. 58–61): (1) activation of Gαq or Gα11, (2) stimulation of membrane-bound PLA2 by the activated Gα, (3) cleavage of membrane phospholipids by PLA2 to produce lysophospholipid and arachidonic acid, and (4) conversion—by several enzymes—of arachidonic acid into a variety of biologically active eicosanoids (e.g., prostaglandins, prostacyclins, thromboxanes, and leukotrienes). Guanylyl Cyclase  Other peptide hormones (e.g., atrial natriuretic peptide) bind to a receptor (see Fig. 47-4D) that is itself a guanylyl cyclase that converts cytoplasmic GTP to cGMP (see pp. 66–67). In turn, cGMP can activate cGMPdependent kinases, phosphatases, or ion channels. Receptor Tyrosine Kinases  For some peptide hormones, notably insulin and IGF-1 and IGF-2, the hormone receptor (see Fig. 47-4E) itself possesses tyrosine kinase activity (see pp. 68–70). This is also a property of other growth factors, including PDGF and epidermal growth factor. Occupancy of the receptor by the appropriate hormone increases kinase activity. For the insulin and IGF-1 receptor, as well as for others, this kinase autophosphorylates tyrosines within the hormone receptor, as well as substrates within the cytosol, thus initiating a cascade of phosphorylation reactions. Tyrosine Kinase–Associated Receptors  Some peptide hormones (e.g., GH) bind to a receptor that, when occupied, activates a cytoplasmic tyrosine kinase (see Fig. 47-4F), such as a member of the JAK (Janus kinase, or just another kinase) family of kinases (see pp. 70–71). As for the receptor tyrosine kinases, activation of these receptor-associated kinases initiates a cascade of phosphorylation reactions.

AMINE HORMONES Amine hormones are made from tyrosine and tryptophan Four major amine hormones are recognized. The adrenal medulla makes the catecholamine hormones epinephrine and norepinephrine from the amino acid tyrosine (see Fig. 13-8C). These hormones are the principal active amine hormones made by the endocrine system. In addition to acting as a hormone, norepinephrine also serves as a neurotransmitter in the CNS (see p. 312) and in postganglionic sympathetic neurons (see pp. 342–343). Dopamine, which is also synthesized from tyrosine, acts as a neurotransmitter in the CNS (see p. 313); it is synthesized in other tissues, but its functional role outside the nervous system is not well clarified. Finally, the hormone serotonin is made from tryptophan (see Fig. 13-8B) by endocrine cells that are located

within the gut mucosa. Serotonin appears to act locally to re­ gulate both motor and secretory function in the gut, and also acts as a neurotransmitter in the CNS (see pp. 312–313). The human adrenal medulla secretes principally epinephrine (see pp. 1030–1033). The final products are stored in vesicles called chromaffin granules. Secretion of catecholamines by the adrenal medulla appears to be mediated entirely by stimulation of the sympathetic division of the autonomic nervous system (see p. 343). Unlike the situation for many peptide hormones, in which the circulating concentration of the hormone (e.g., TSH) negatively feeds back on secretion of the releasing hormone (e.g., TRH), the amine hormones do not have such a hierarchic feedback system. Rather, the feedback of amine hormones is indirect. The higher control center does not sense circulating levels of the amine hormones (e.g., epinephrine) but rather a physiological end effect of that amine hormone (e.g., blood pressure; see pp. 534–536). The sensor of the end effect may be a peripheral receptor (e.g., stretch receptor) that communicates to the higher center (e.g., the CNS), and the efferent limb is the sympathetic outflow that determines release of the amine. Serotonin (5-hydroxytryptamine, or 5-HT), in addition to being an important neurotransmitter in the CNS (see pp. 312–313 and Fig. 13-7B), is a hormone made by neuroendocrine cells, principally located within the lining of the small intestine and larger bronchi. Unlike the other hormones that we discuss in this chapter, serotonin is not made by a specific gland. Little is known about feedback regulation or even regulation of secretion of this hormone. Serotonin arouses considerable clinical interest because of the dramatic clinical presentation of patients with unusual tumors—called carcinoid tumors—of serotonin-secreting cells. Individuals with these tumors frequently present with carcinoid syndrome, characterized by episodes of spontaneous intense flushing in a typical pattern involving the head and neck and associated with diarrhea, bronchospasm, and occasionally right-sided valvular heart disease. The primary tumors involved can occur within the intestinal tract, in the bronchial tree, or more rarely at other sites.

Amine hormones act via surface receptors Once secreted, circulating epinephrine is free to associate with specific adrenergic receptors, or adrenoceptors, located on the surface membranes of target cells. Numerous types of adrenoceptors exist and are generically grouped as α or β, each of which has several subtypes (see Table 14-2). Epinephrine has a greater affinity for β-adrenergic receptors than for α-adrenergic receptors, whereas norepinephrine acts predominantly through α-adrenergic receptors. All adrenoceptors that have been isolated from a variety of tissues and species are classic G protein–coupled receptors (GPCRs). β-adrenergic stimulation occurs through the adenylyl cyclase system. The α2 receptor also usually acts through adenylyl cyclase. However, α1-adrenergic stimulation is linked to Gαq, which activates a membrane-associated PLC that liberates IP3 and DAG. IP3 can release Ca2+ from intracellular stores, and DAG directly enhances the activity of PKC. Combined, these actions enhance the cellular activity of Ca2+dependent kinases, which produce a metabolic response that is characteristic of the specific cell.

Chapter 47  •  Organization of Endocrine Control

985

Extracellular space Stimulatory effects Epinephrine

Inhibitory effects

Dopamine

Norepinephrine

Dopamine

Norepinephrine

Adenylyl cyclase

β2

β1

D1

α2

AC

D2

γ

α1

αq

PLC

β γ

β

αs

cAMP

αi

β

γ

+

PKC

DAG

Cytosol

Ca2+ IP3

Figure 47-5  Catecholamine receptors. The β1, β2, and D1 receptors all interact with Gαs, which activates

adenylyl cyclase (AC) and raises levels of cAMP. The α2 and D2 receptors interact with Gαi, which inhibits AC. Additionally, the α1 receptor interacts with Gαq, which activates PLC, which in turn converts phosphoinositides in the cell membrane to IP3 and DAG.

As indicated in Figure 47-5, the intracellular action of a specific catecholamine is determined by the complement of receptors present on the surface of a specific cell. For example, when epinephrine binds to the β1-adrenergic receptor, it activates a Gαs protein, which stimulates adenylyl cyclase, promotes increases in [cAMP]i, and thus enhances the activity of PKA (see Table 14-2). In contrast, when the same hormone binds to a cell displaying principally α2 receptors, it activates a Gαi protein, which inhibits adenylyl cyclase, diminishes [cAMP]i, and therefore reduces PKA activity. Thus, the response of a specific cell to adrenergic stimulation (whether via circulating epinephrine or via norepinephrine released locally by sympathetic neurons) depends on the receptor repertoire displayed by the cell. As a result, the response to adrenergic agonists varies among tissues; for example, glycogenolysis in the liver or muscle (predominantly a β effect), contraction (an α1 effect) or relaxation (a β2 effect) in vascular smooth muscle, or a change in the inotropic or chronotropic state of the heart (a β1 effect). Dopamine also can interact with several GPCRs. The D1 receptor is coupled to Gαs and the D2 receptor is linked to Gαi.

STEROID AND THYROID HORMONES Cholesterol is the precursor for the steroid hormones: cortisol, aldosterone, estradiol, progesterone, and testosterone Members of the family of hormones called steroids share a common biochemical parentage: all are synthesized from cholesterol. Only two tissues in the body possess the enzymatic apparatus to convert cholesterol to active hormones. The adrenal cortex makes cortisol (the main gluco­ corticoid hormone), aldosterone (the principal mineralo­ corticoid in humans), and androgens. The gonads make either estrogen and progesterone (ovary) or testosterone (testis). In each case, production of steroid hormones is regulated by trophic hormones released from the pituitary.

For aldosterone, the renin-angiotensin system also plays an important regulatory role. The pathways involved in steroid synthesis are summarized in Figure 47-6. Cells that produce steroid hormones can use, as a starting material for hormone synthesis, the cholesterol that is circulating in the blood in association with low-density lipoprotein (LDL; see p. 968). Alternatively, these cells can synthesize cholesterol de novo from acetate (see Fig. 46-16). In humans, LDL cholesterol appears to furnish ~80% of the cholesterol used for steroid synthesis (see Fig. 47-6). An LDL particle contains both free cholesterol and cholesteryl esters, in addition to phospholipids and protein. The cell takes up this LDL particle via the LDL receptor and receptor-mediated endocytosis (see p. 42) into clathrin-coated vesicles. Lysosomal hydrolases then act on the cholesteryl esters to release free cholesterol. The cholesterol nucleus, whether taken up or synthesized de novo, subsequently undergoes a series of reactions that culminate in the formation of pregnenolone, the common precursor of all steroid hormones. Via divergent pathways, pregnenolone is then further metabolized to the major steroid hormones: the mineralocorticoid aldosterone (see Fig. 50-2), the glucocorticoid cortisol (see Fig. 50-2), the androgen testosterone (see Fig. 54-6), and the estrogen estradiol (see Fig. 55-8). Unlike the peptide and amine hormones considered above, steroid hormones are not stored in secretory vesicles before their secretion (Table 47-4). For these hormones, synthesis and secretion are very closely linked temporally. Steroid-secreting cells are capable of increasing the secretion of steroid hormones many-fold within several hours. The lack of a preformed storage pool of steroid hormones does not appear to limit the effectiveness of these cells as an endocrine regulatory system. Furthermore, steroid hormones, unlike peptide and amine hormones, mediate nearly all their actions on target tissues by regulating gene transcription. As a result, the response of target tissues to steroids typically occurs over hours to days. Like cholesterol itself, steroid hormones are poorly soluble in water. On their release into the circulation, some steroid

986

SECTION VIII  •  The Endocrine System

Remnant chylomicrons from diet

Dietary cholesterol (chylomicrons)

Uncoated vesicle

Acetyl CoA

Lysosome

Acetyl CoA

20%

Plasma membrane

LIVER

Clathrin LDL VLDL

Coated vesicle

Cholesterol

LDL receptor Amino acids

Blood

80%

Coated pit

Cholesterol Fatty acids

LDL PARTICLE:

Pregnenolone

Aldosterone Cortisol

Phospholipids and cholesterol

Testosterone Estradiol

Cholesteryl esters and triglycerides Apo B-100 protein Figure 47-6  Uptake of cholesterol and synthesis of steroid hormones from cholesterol. The cholesterol needed as the starting material in the synthesis of steroid hormones comes from two sources. Approximately 80% is taken up as LDL particles via receptor-mediated endocytosis. The cell synthesizes the remaining cholesterol de novo from acetyl coenzyme A (Acetyl CoA). Apo B-100, apolipoprotein B-100; VLDL, very-lowdensity lipoprotein.

TABLE 47-4  Differences Between Steroid and Peptide/Amine Hormones PROPERTY

STEROID HORMONES

PEPTIDE/AMINE HORMONES

Storage pools

None

Secretory vesicles

Interaction with cell membrane

Diffusion through cell membrane

Binding to receptor on cell membrane

Receptor

In cytoplasm or nucleus

On cell membrane

Action

Regulation of gene transcription (primarily)

Signal-transduction cascade(s) that affect a variety of cell processes

Response time

Hours to days (primarily)

Seconds to minutes

hormones associate with specific binding proteins (e.g., cortisol-binding globulin) that transport the steroid hormones through the circulatory system to their target tissues. The presence of these binding proteins, whose concentration in the circulation can change in response to a variety of physiological conditions, can complicate efforts to measure the amount of active steroid hormone in the circulation.

Steroid hormones bind to intracellular receptors that regulate gene transcription Steroid hormones appear to enter their target cell by simple diffusion across the plasma membrane (Fig. 47-7). Once within the cell, steroid hormones are bound with high

affinity (KD in the range of 1 nM) to receptor proteins located in the cytosol or the nucleus. As detailed in Chapter 4, binding of steroid hormone to its receptor results in a change in the receptor conformation so that the “active” receptorhormone complex now binds with high affinity to specific DNA sequences called hormone response elements (see p. 90) or steroid response elements (SREs), also called sterol regulatory elements. These sequences are within the 5′ region of target genes whose transcription is regulated by the specific steroid hormone–receptor complex. Termination of gene regulation by the steroid hormone–receptor complex is not as well understood as initiation of the signal. The receptor protein may be modified in a manner that permits dissociation of the hormone and DNA. The receptor itself could then be recycled and the steroid molecule metabolized or otherwise cleared from the cell. Steroid receptors are monomeric phosphoproteins with a molecular weight that is between 80 and 100 kDa. A remarkable similarity is seen among receptors for the glucocorticoids, sex steroids, retinoic acid, the steroid-like vitamin 1,25-dihydroxyvitamin D, and thyroid hormone. The genes encoding the receptors for these diverse hormones are considered part of a gene superfamily (see pp. 71–72). Each of these receptors has a similar modular construction with six domains (A through F). The homology among receptors is especially striking for the C domain, particularly the C1 subdomain, which is the part of the receptor molecule that is responsible for binding to DNA (see Fig. 3-14). Steroid hormone receptors dimerize on binding to their target sites on DNA. Dimerization appears essential for the regulation of gene transcription. Within the C1 DNAbinding domain of the steroid receptor monomer are two

Chapter 47  •  Organization of Endocrine Control

Extracellular space

Steroid

Cytosol

987

Steroid Nucleus

…or in nucleus.

Steroid binds to the receptor in cytosol…

Steroid receptor Steroid diffuses into the cell.

DNA

Nuclear envelope Translation

Transcription Steroid receptor hsp

mRNA

Protein

Figure 47-7  Action of steroid hormones. The activated steroid hormone receptor binds to specific stretches of DNA called steroid response elements (SREs), which stimulates the transcription of appropriate genes. hsp, heat shock protein.

zinc fingers that are involved in binding of the receptor to DNA (see p. 82). Even receptors with very different biological actions have a striking sequence similarity in this domain of the receptor. Because the specificity with which genes are regulated by a specific steroid receptor arises from the specificity of the DNA-binding domain, mutations in this region can greatly alter hormone function. For example, substitution of two amino acids in the glucocorticoid receptor causes the mutated glucocorticoid receptor to bind to DNA to which the estrogen receptor normally binds. In such a system, a glucocorticoid could have an estrogen-like effect. The activated steroid receptor, binding as a dimer to SREs in the 5′ region of a gene, regulates the rate of transcription of that gene. Each response element is identifiable as a consensus sequence of nucleotides, or a region of regulatory DNA in which the nucleotide sequences are preserved through different cell types. The effect of gene regulation by activated steroid receptors binding to an SRE is dramatically illustrated by the chick ovalbumin gene. Chicks that are not exposed to estrogen have approximately four copies of the ovalbumin mRNA per cell in the oviduct. A 7-day course of estrogen treatment increases the number of copies of message 10,000-fold! This increase in message is principally the result of an increased rate of gene transcription. However, steroid hormones can also stabilize specific mRNA molecules and increase their half-life.  N47-2 The 5′ flanking region of the gene typically has one or more SREs upstream of the TATA box, a nucleotide sequence rich in adenine and thymine that is located near the starting point for transcription (see p. 78). The activated steroid hormone receptors recognize these SREs from their specific consensus sequences. For example, one particular consensus sequence designates a site as a glucocorticoid response element if the SRE is in a cell with a glucocorticoid receptor. This same consensus sequence in a cell of the endometrium would be recognized by the activated progesterone receptor or, in the renal distal tubule, by the activated mineralocorticoid receptor. The specificity of the response thus depends on the cell’s expression of particular steroid receptors, not simply the consensus sequence. For example, the renal distal tubule cell expresses relatively more mineralocorticoid

receptors than it does progesterone receptors when compared with the endometrium. As a result, changes in plasma aldosterone regulate Na+ reabsorption in the kidney with greater sensitivity than does circulating progesterone. However, very high levels of progesterone can, like aldosterone, promote salt reabsorption. From the foregoing it should be apparent that the speci­ ficity of response of a tissue to steroid hormones depends on the abundance of specific steroid receptors expressed within a cell. Because all somatic cells have the full complement of DNA with genes possessing SREs, whether a cell responds to circulating estrogen (e.g., breast), androgen (e.g., prostate), or mineralocorticoid (e.g., renal collecting duct) depends on the receptors present in the cell. This specificity raises the obvious, but as yet unanswered, question of what regulates the expression of specific steroid receptors by specific tissues. Within a given tissue, several factors control the concentration of steroid hormone receptors. In the cytosol of all steroid-responsive tissues, steroid receptor levels usually drop dramatically immediately after exposure of the tissue to the agonist hormone. This decrease in receptor level is the result of net movement of the agonist-receptor complex to the nucleus. Eventually, the cytosolic receptors are repopulated. Depending on the tissue, this repopulation may involve new synthesis of steroid hormone receptors or simply recycling of receptors from the nucleus after dissociation of the agonist from the receptor. In addition, some steroids reduce the synthesis of their own receptor in target tissues. For example, progesterone reduces the synthesis of progesterone receptor by the uterus, thus leading to an overall net reduction or downregulation of progesterone receptor concentration in a target tissue. An interesting observation in this regard is that the genes for steroid receptor proteins do not appear to have SREs in their 5′ flanking region. Thus, this regulation of receptor number probably involves trans-acting transcriptional factors other than the steroid hormones themselves. Other factors that affect the concentration of steroid receptors in target tissues include the state of differentiation of the tissue, the presence of other hormones that affect

Chapter 47  •  Organization of Endocrine Control

N47-2  Stabilization of mRNA by Estrogen Contributed by Gene Barrett For example, in frogs, estrogen increases the half-life of the mRNA for vitellogen (which is formed by Xenopus liver) from 2.7 m. It is important that, in both cases, the abnormality of GH secretion was present from early life. Children with GH deficiency are of normal size at birth and only subsequently fall behind their peers in stature. A deficiency of GH beginning in adult life does not result in any major clinical illness. However, it is now appreciated that replacement of GH (clinically available as a recombinant protein) in adults with GH deficiency leads to increased lean body mass, decreased body fat, and perhaps an increased sense of vigor or well-being. An excess of GH after puberty results in the clinical syndrome of acromegaly (from the Greek akron [top] + megas [large]). This condition is characterized by the growth of bone and many other somatic tissues, including skin, muscle, heart, liver, and the gastrointestinal (GI) tract. The lengthening of long bones is not part of the syndrome because the epiphyseal growth plates close at the end of puberty. Thus, acromegaly causes a progressive thickening of bones and soft tissues of the head, hands, feet, and other parts of the body. If untreated, these somatic changes cause significant morbidity and shorten life as a result of joint deformity, hypertension, pulmonary insufficiency, and heart failure. GH is made by somatotrophs throughout the anterior pituitary (see pp. 978–979). Like other peptide hormones, GH is synthesized as a larger “prehormone” (Fig. 48-1). During processing through the endoplasmic reticulum and Golgi system, several small peptides are removed. GH exists in at least three molecular forms. The predominant form is

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

990.e1

N48-1  General Tom Thumb Contributed by Emile Boulpaep and Walter Boron Charles S. Stratton (1838–1883), whose stage name was General Tom Thumb, was a famous performer in the P.T. Barnum circus. Stratton’s maternal and paternal grandmothers were twin sisters of short stature. At birth, Stratton was somewhat larger than average (4.3 kg, or 9 pounds, 8 ounces) and he continued to grow normally until about 6 months of age, when he was 64 cm (25 inches) tall and weighed 6.8 kg (15 pounds). After that, he virtually ceased to grow for several years, although he was otherwise completely normal. He had siblings of normal size. At the age of 9, Stratton began to grow very slowly, reaching the height of 74 cm (2 feet, 5 inches) at age 13 and 82.6 cm (2 feet, 8.5 inches) at age 18. At age 25, Stratton married Mercy Lavinia Bumpus (stage name, Lavinia Warren), who was also a short person. They had no children, and the cause of Stratton’s short stature is not known.

The American circus impresario P.T. Barnum heard of Stratton and recruited him for his circus when Stratton was age 5. Barnum coached Stratton, who became a song-and-dance man and comedian. Tours of both the United States and Europe were great successes, and Stratton became an international celebrity and, under the guidance of Barnum, a wealthy man.

REFERENCES Wikipedia. s.v. General Tom Thumb. Last modified May 13, 2015. http://en.wikipedia.org/wiki/General_Tom_Thumb. Accessed June 5, 2015. Wikipedia. s.v. Lavinia Warren. Last modified May 6, 2015. http://en.wikipedia.org/wiki/Lavinia_Warren. Accessed June 5, 2015.

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

Pre-mRNA 3´

5´

Intron

Exon

pre–22.5-kDa growth hormone mRNA

pre–20-kDa growth hormone mRNA

Rough ER

pre-prohormone

Golgi prohormone

22-kDa growth hormone (191 amino acids).

prohormone

Secretory granules

may exert fewer of the acute metabolic actions of GH. Once synthesized, GH is stored in secretory granules in the cytosol of the somatotrophs until secreted.

GH is in a family of hormones with overlapping activity

Nucleus

pre-prohormone

991

20-kDa growth hormone (176 amino acids).

Secreted hormones

Figure 48-1  Synthesis of GH. Somatotrophic cells in the anterior pituitary are responsible for the synthesis of GH. The cell transcribes five exons to form GH mRNA for either the 22-kDa protein (191 amino acids) or the 20-kDa protein (176 amino acids). Alternative splicing in the third exon, which removes the RNA-encoding amino acids 32 to 46, is responsible for the two isoforms found in the pituitary. Both mRNAs have a signal sequence that causes them to be translated in the rough endoplasmic reticulum (ER) and enter the secretory pathway. Subsequent processing converts the two pre-pro-GHs first to the pro-GHs and then to the mature GHs. The cleavage of the pro sequence and disulfide-bond formation occur during transit through the Golgi bodies. The somatotroph stores mature GH in granules until GHRH stimulates the somatotroph to secrete the hormones. The 22-kDa version is the dominant form of GH.

a 22-kDa polypeptide with two intramolecular sulfhydryl bonds. Alternative splicing generates a 20-kDa form of GH. Other GH forms include a 45-kDa protein, which is a dimer of the 22-kDa form, as well as larger forms that are multimers of monomeric GH. There is little information to suggest that the different principal forms of GH (i.e., the 20- and 22-kDa versions) vary in their activity, but the 20-kDa form

GH appears to be a single-copy gene, but four other hormones have significant homology to GH. Most striking are three hormones made by the placenta: placental-variant GH (pvGH) and human chorionic somatomammotropins 1 and 2 (hCS1 and hCS2; Table 48-1). Human genes for these hormones are located in the GH gene cluster on the long arm of chromosome 17. The multiple genes in this cluster have an identical intron structure and encode proteins of similar size with substantial amino-acid sequence homology. pvGH is a 191–amino-acid peptide that is 93% identical to the 22-kDa form of GH. With virtually the same affinity as GH for the hepatic GH receptor, pvGH mimics some of the biological actions of GH and may be an important modulator of systemic IGF-1 production during pregnancy. (As discussed below, a major action of GH is to stimulate secretion of IGF-1.) The hCSs are also called human placental lactogens (hPLs). The affinity of the two forms of hCS for the GH receptor is 100- to 1000-fold less than that of either GH or pvGH. As a result, the hCSs are less effective in promoting production of IGF-1 or IGF-2. The somatomammotropins are primarily lactogenic, priming the breast for lactation after birth (see Table 56-6). The pituitary hormone prolactin (PRL; see Table 48-1) is the fourth hormone with homology to GH. The principal physiological role of PRL involves promotion of milk production in lactating women (see pp. 1148–1150). PRL is made by lactotrophs in the anterior pituitary. Its homology to GH suggests that the two hormones, despite their divergent actions, arose from some common precursor by a geneduplication event. The sequence homology between these proteins is underscored by the observation that GH and PRL have similar affinities for the PRL receptor. The converse is not true—that is, PRL has no significant affinity for the GH receptor and thus has no growth-promoting activity. As discussed below, the PRL and GH receptors are coupled to an intracellular signaling system that involves stimulation of the JAK family of tyrosine kinases (see p. 70) as an early postreceptor event. Men, like women, make PRL throughout their lives. However, no physiological role for PRL in males has been defined. Both men and women with disorders involving hypersecretion of GH or PRL can develop galactorrhea (breast milk secretion). Although GH and PRL are normally secreted by distinct cell populations in the anterior pituitary, some benign GH-producing pituitary adenomas (i.e., tumors) secrete PRL along with GH.

Somatotrophs secrete GH in pulses Whereas growth occurs slowly over months and years, the secretion of GH is highly episodic, varying on a minute-tominute basis. Most physiologically normal children experience episodes or bursts of GH secretion throughout the day, most prominently within the first several hours of sleep.

992

SECTION VIII  •  The Endocrine System

TABLE 48-1  Homology of GH to Chorionic Hormones and Prolactin HORMONE

NUMBER OF AMINO ACIDS

HOMOLOGY (%)

CHROMOSOME

hGH (human growth hormone)

191

100

17

pvGH (placental-variant GH)

191

93

17

hCS1 (human chorionic somatomammotropin 1)

191

84

17

hCS2 (human chorionic somatomammotropin 2)

191

84

17

hPRL (human prolactin)

199

16

6

Sleep 20

Growth hormone (µg/L)

15

10

5

0 8 AM

8 PM

Midnight

4 AM

Figure 48-2  Bursts in plasma levels of GH, sampled in the blood plasma of a 23-year-old woman every 5 minutes over a 24-hour period. Each peak in the plasma GH concentration reflects bursts of hundreds of GH-secretory pulses by the somatotrophs of the anterior pituitary. These bursts are most common during the first few hours of sleep. The integrated amount of GH secreted each day is higher during pubertal growth than in younger children or in adults. (Data from Hartman ML, Veldhuis JD, Vance ML, et al: Somatotropin pulse frequency and basal concentrations are increased in acromegaly and are reduced by successful therapy. J Clin Endocrinol Metab 70:1375, 1990.)

Underlying each peak in plasma levels of GH, illustrated for an adult in Figure 48-2, are bursts of many hundreds of pulses of GH secretion by the somatotrophs in the anterior pituitary. With the induction of slow-wave sleep, several volleys of GH pulses may occur; it is estimated that >70% of total daily GH secretion occurs during these periods. This pulsatile secretion underlines the prominent role of the CNS in the regulation of GH secretion and growth. The circulating GH concentrations may be up to 100-fold higher during the bursts of GH secretion (i.e., the peaks in Fig. 48-2) than during intervening periods. The pattern of bursts depends on sleep-wake patterns, not on light-dark patterns. Exercise, stress, high-protein meals, and fasting also cause a rise in the mean GH level in humans. In circumstances in which GH secretion is stimulated (e.g., fasting or consumption of a high-protein diet), the increased GH output results from an increase in the frequency—rather than the amplitude—of pulses of GH secretion by the somatotrophs.

GH secretion is under hierarchical control by GH–releasing hormone and somatostatin The coordination of GH secretion by the somatotrophs during a secretory pulse presumably occurs in response to both positive and negative hypothalamic control signals.

GH-Releasing Hormone  Small-diameter neurons in the arcuate nucleus of the hypothalamus secrete growth hormone–releasing hormone (GHRH), a 43–amino-acid peptide that reaches the somatotrophs in the anterior pituitary via the hypophyseal portal blood (Fig. 48-3). As the name implies, this neuropeptide promotes GH secretion by the somatotrophs. GHRH is made principally in the hypothalamus, but it can also be found in neuroectodermal tissue outside the CNS; it was first isolated and purified from a pancreatic islet cell tumor of a patient with acromegaly. GHRH Receptor  GHRH binds to a G protein–coupled receptor (GPCR) on the somatotrophs and activates Gαs, which in turn stimulates adenylyl cyclase (see pp. 56–57). The subsequent rise in [cAMP]i causes increased gene transcription and synthesis of GH. In addition, the rise in [cAMP]i opens Ca2+ channels in the plasma membrane and causes [Ca2+]i to rise. This increase in [Ca2+]i stimulates the release of preformed GH. Ghrelin  A relatively newly discovered hormone, ghrelin consists of 28 amino acids. One of the serine residues is linked to an octanol group, and only this acylated form of the peptide is biologically active.  N48-2  Distinct endocrine cells within the mucosal layer of the stomach release

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

N48-2  Ghrelin Contributed by Emile Boulpaep and Walter Boron Circulating forms of ghrelin include both the acyl and deacylated species, and this has complicated efforts to define the physiological responses of ghrelin to dietary manipulation as well as the responses to exogenously administered hormone. Ghrelin has 28 amino acids and is also known as ghrelin-28. The sequence of human ghrelin (amino acids 24 to 51 of the full, immature peptide), using the single-letter code, is as follows: GSSFLSP EHQRVQQRKE SKKPPAKLQP R. Ghrelin-27 has only 27 amino acids, lacking the C-terminal arginine of ghrelin-28.

REFERENCE UniProt Knowledgebase [results for ghrelin]. http://www .uniprot.org/uniprot/?query=ghrelin&sort=score. Accessed September 2014.

992.e1

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

2 Cells in the periventricular region release somatostatin, a 14–amino acid peptide that is a potent inhibitor of growth hormone (GH) secretion, into the long portal veins.

993

1 Small-bodied neurons in the arcuate nucleus secrete growth hormone–releasing hormone (GHRH), a 43–amino acid peptide that reaches the somatotrophs via long portal veins. Arcuate nucleus

Periventricular region

Hypothalamus Released GHRH

Released somatostatin

GHRH

Anterior lobe of pituitary

GHRH receptor

Somatotroph γ

β

α

SS receptor

AC

SS

GH

γ

cAMP

β

Somatotrophs

α

(Stimulating) G-protein

(Inhibitory) G-protein

2+

Ca

PKA Ca2+

3 GHRH causes somatotrophs to synthesize and release GH. 4 Somatostatin inhibits the release of GH by somatotrophs. Figure 48-3  Synthesis and release of GHRH and SS, and the control of GH release. GHRH raises [cAMP]i and [Ca2+]i in the somatotrophs and thereby stimulates release of GH stored in secretory granules. SS inhibits adenylyl cyclase (AC), lowers [Ca2+]i, and thereby inhibits release of GH. PKA, protein kinase A.

ghrelin in response to fasting. Endocrine cells throughout the GI tract also make ghrelin, although the highest ghrelin concentrations are in the fundus of the stomach. The arcuate nucleus of the hypothalamus also makes small amounts of ghrelin. Infusion of ghrelin either into the bloodstream or into the cerebral ventricles markedly increases growth hormone secretion. Indeed, ghrelin appears to be involved in the postmeal stimulation of growth hormone secretion. It has been more difficult to define the extent to which ghrelin—versus GHRH and somatostatin (SS)—contributes to the changes in normal growth hormone secretion in response to fasting, amino-acid feeding, and carbohydrate feeding. Ghrelin also is orexigenic (i.e., it stimulates appetite; see p. 1003), thereby contributing to body mass regulation as well as linear growth.

Ghrelin Receptor  The hormone ghrelin binds to a GPCR designated GH secretagogue receptor 1a (GHSR1a). This receptor was first identified because it binds synthetic peptide ligands that stimulate GH secretion. In this regard, GHSR1a is like the GHRH receptor (GHRHR); however, GHSR1a does not bind GHRH. Somatostatin  The hypothalamus also synthesizes SS, a 14–amino-acid neuropeptide. SS is made in the periventricular region of the hypothalamus and is secreted into the hypophyseal portal blood supply. It is a potent inhibitor of GH secretion. SS is also made elsewhere in the brain and in selected tissues outside the CNS, such as the pancreatic islet δ cells (see p. 1053) and D cells in the GI tract (see pp. 868– 870 and Table 41-1). Within the CNS, the 14–amino-acid

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SECTION VIII  •  The Endocrine System

form of SS (SS-14) dominates. The GI tract predominantly expresses a 28–amino-acid splice variant; the C-terminal 14 amino acids of SS-28 are identical to those of SS-14. It appears that the primary regulation of GH secretion is stimulatory, because sectioning the pituitary stalk, and thereby interrupting the portal blood flow from the hypothalamus to the pituitary, leads to a decline in GH secretion. Conversely, sectioning of the stalk leads to a rise in PRL levels, presumably because dopamine made in the hypothalamus normally inhibits PRL secretion in the anterior pituitary (see pp. 1149–1150). It also appears that the pulses of GH secretion are entrained by the pulsatile secretion of GHRH (as opposed to the periodic loss of SS inhibition). SS Receptor  SS binds to a GPCR called SSTR found on somatotrophs and activates Gαi, which inhibits adenylyl cyclase. As a result, [Ca2+]i decreases, which diminishes the responsiveness of the somatotroph to GHRH. When somatotrophs are exposed to both GHRH and SS, the inhibitory action of SS prevails.

Both GH and IGF-1 negatively feed back on GH secretion by somatotrophs In addition to being controlled by GHRH, ghrelin, and SS, somatotroph secretion of GH is under negative-feedback control via IGF-1. As discussed below, GH triggers the secretion of IGF-1 from GH target tissues throughout the body (Fig. 48-4, No. 1). Indeed, IGF-1 mediates many of the growth-promoting actions of GH. IGF-1 synthesized in tissues such as muscle, cartilage, and bone may act in a paracrine or autocrine fashion to promote local tissue growth. In contrast, circulating IGF-1, largely derived from hepatic secretion, exerts endocrine effects. Circulating IGF-1 suppresses GH secretion through both direct and indirect mechanisms. First, circulating IGF-1 exerts a direct action on the pituitary to suppress GH secretion by the somatotrophs (see Fig. 48-4, No. 2), probably inhibiting GH secretion by a mechanism different from that of SS. In its peripheral target cells, IGF-1 acts through a receptor tyrosine kinase (see pp. 68–70) and not by either the Ca2+ or cAMP messenger systems. IGF-1 presumably acts by this same mechanism to inhibit GH secretion in somatotrophs. Second, circulating IGF-1 inhibits GH secretion via two indirect feedback pathways, both targeting the hypothalamus. IGF-1 suppresses GHRH release (see Fig. 48-4, No. 3) and also increases SS secretion (see Fig. 48-4, No. 4). Yet another feedback system, independent of IGF-1, reduces GH secretion. Namely, GH itself appears to inhibit GH secretion in a short-loop feedback system (see Fig. 48-4, No. 5).

GH has short-term anti-insulin metabolic effects as well as long-term growth-promoting effects mediated by IGF-1 Once secreted, most GH circulates free in the plasma. However, a significant fraction (~40% for the 22-kDa GH) is complexed to a GH-binding protein formed by proteolytic cleavage of the extracellular domain of GH receptors in

GH target tissues. This protein fragment binds to GH with high affinity, thereby increasing the half-life of GH and competing with GH target tissues for GH. In the circulation, GH has a half-life of ~25 minutes. GH Receptor  GH binds to a receptor (GHR) on the surface of multiple target tissues. The monomeric GHR is a 620–amino-acid protein with a single membrane-spanning segment. The molecular weight of GHR (~130 kDa) greatly exceeds that predicted from its amino-acid composition (~70 kDa) as a result of extensive glycosylation. Like other members of the type I cytokine receptor family, GHR is a tyrosine kinase–associated receptor (see pp. 70–71). When one GH molecule simultaneously binds to sites on two GHR monomers and acts as a bridge, the monomers dimerize (see Fig. 3-12D). Receptor occupancy increases the activity of a tyrosine kinase (JAK2 family) that is associated with, but is not an integral part of, the GH receptor. This tyrosine kinase triggers a series of protein phosphorylations that modulate target cell activity. Short-Term Effects of GH  GH has certain short-term (minutes to hours) actions on muscle, adipose tissue, and liver that may not necessarily be related to the more longterm growth-promoting actions of GH. These acute metabolic effects (Table 48-2) include stimulation of lipolysis in adipose tissue, inhibition of glucose uptake by muscle, and stimulation of gluconeogenesis by hepatocytes. These actions oppose the normal effects of insulin (see pp. 1035–1050) on these same tissues and have been termed the anti-insulin or diabetogenic actions of GH. Chronic oversecretion of GH, such as occurs in patients with GH-producing tumors in acromegaly, is accompanied by insulin resistance and often by glucose intolerance or frank diabetes. Long-Term Effects of GH via IGF-1  Distinct from these acute actions of GH is its action to promote tissue growth by stimulating target tissues to produce IGFs. In 1957, Salmon and Daughaday reported that GH itself does not have growth-promoting action on epiphyseal cartilage (the site where longitudinal bone growth occurs). In those experiments, the addition of serum from normal animals, but not from hypophysectomized (GH-deficient) animals, stimulated cartilage growth in vitro (assayed as incorporation of radiolabeled sulfate into cartilage). The addition of GH to GH-deficient serum did not restore the growth-promoting activity seen with normal serum. However, when the GHdeficient animals were treated in vivo with GH, their plasma promoted cartilage growth in vitro. This finding led to the hypothesis that, in animals, GH provokes the secretion of another circulating factor that mediates the action of GH.

TABLE 48-2  Diabetogenic Effects of GH TARGET

EFFECT

Muscle

↓ Glucose uptake

Fat

↑ Lipolysis

Liver

↑ Gluconeogenesis

Muscle, fat, and liver

Insulin resistance

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

4 IGF-1 indirectly inhibits GH release by increasing secretion of somatostatin from nuclei in the periventricular region. Periventricular region

Hypothalamus

3 IGF-1 indirectly inhibits GH release by suppressing GHRH release from the arcuate nucleus in the hypothalamus.

Arcuate nucleus

Released somatostatin

Released GHRH

5 GH inhibits its own secretion via “short-loop” feedback on somatotrophs.

IGF-1

IGF-1

Somatotrophs Anterior lobe of pituitary 1 GH stimulates secretion of IGF-1 from peripheral target tissue.

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GH

Target tissue

IGF-1

2 IGF-1 then directly inhibits GH release by suppressing the somatotrophs.

Figure 48-4  GH and IGF-1 (also called somatomedin C) negative-feedback loops. Both GH and IGF-1 feed back—either directly or indirectly—on the somatotrophs in the anterior pituitary to decrease GH secretion. GH itself inhibits GH secretion (“short loop”). IGF-1, whose release is stimulated by GH, inhibits GH release by three routes, one of which is direct and two of which are indirect. The direct action is for IGF-1 to inhibit the somatotroph. The first indirect pathway is for IGF-1 to suppress GHRH release in the hypothalamus. The second is for IGF-1 to increase secretion of SS, which in turn inhibits the somatotroph.

Initially called sulfation factor because of how it was assayed, this intermediate was subsequently termed somatomedin because it mediates the somatic effects of GH. We now know that somatomedin is in fact two peptides resembling proinsulin and thus termed insulin-like growth factors 1 and 2 (Fig. 48-5). Indeed, the IGFs exert insulin-like actions

in isolated adipocytes and can produce hypoglycemia in animals and humans. IGF-1 and IGF-2 are made in various tissues, including the liver, kidney, muscle, cartilage, and bone. As noted above, the liver produces most of the circulating IGF-1, which more closely relates to GH secretion than does IGF-2.

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SECTION VIII  •  The Endocrine System

Human insulin Of 29 amino acids in IGF-1, 13 are homologous with insulin B domain. IGF-1

B 30 AA

C 31 AA

13/29 homology 29 AA

11/21 homology 12 AA

22/29 homology IGF-2

29 AA

A 21 AA

21 AA 18/21 homology

12 AA

21 AA

D 6 AA 4/6 homology D 6 AA

Figure 48-5  Structure of the IGFs. Insulin, IGF-1, and IGF-2 share three domains (A, B, and C), which have a high degree of amino-acid (AA) sequence homology. The C region is cleaved from insulin (as the C peptide) during processing, but is not cleaved from either IGF-1 or IGF-2. In addition, IGF-1 and IGF-2 also have a short D domain.

GROWTH-PROMOTING HORMONES IGF-1 is the principal mediator of the growth-promoting action of GH The synthesis of IGF-1 and, to a lesser extent, IGF-2 depends on circulating GH. As described above, the periodic nature of GH secretion results in a wide range of plasma GH concentrations. In contrast, plasma [IGF-1] does not vary by more than ~2-fold over a 24-hour period. The plasma [IGF-1] in effect integrates the pulsatile, highly fluctuating GH concentration. The reason for the relatively steady plasma levels of IGF-1 is that like GH—but unlike most peptide hormones—IGF-1 circulates bound to several IGF-1–binding proteins. These binding proteins are made principally in the liver, but they are also manufactured by other tissues. More than 90% of IGF-1 measured in the serum is bound to these proteins. At least six distinct IGF-binding proteins have been identified. In addition to providing a buffer pool in plasma of bound IGF, these proteins may aid the transfer of IGF to the tissue receptors, thereby facilitating the action of these hormones. The local free fraction of IGF-1 is probably the more biologically active component that binds to the receptor and stimulates tissue growth. Like other peptide hormones, IGF-1 and IGF-2 are synthesized via the secretory pathway (see pp. 34–35) and are secreted into the extracellular space, where they may act locally in a paracrine fashion. In the extracellular space, the IGFs encounter binding proteins that may promote local retention of the secreted hormone by increasing the overall molecular size of the complex. This action inhibits the entry of the IGFs into the vascular system. Thus, local concentrations of the IGFs are likely to be much higher than plasma concentrations. Whether made locally or reaching tissues through the circulation, IGF-1 acts via a specific receptor tyrosine kinase (see pp. 68–70), a heterotetramer that is structurally related to the insulin receptor (Fig. 48-6). Like the insulin receptor (see pp. 1041–1042), the IGF-1 receptor (IGF1R) has two completely extracellular α chains and two transmembrane β chains. Also like in the insulin receptor, the β chains have intrinsic tyrosine kinase activity. Binding of IGF-1 to its receptor enhances receptor autophosphorylation as well as

phosphorylation of downstream effectors. The structural homology between the insulin and IGF-1 receptors is sufficiently high that insulin can bind to the IGF-1 receptor, although with an affinity that is about two orders of magnitude less than that for IGF-1. The same is true for the binding of IGF-1 to insulin receptors. In fact, the homology between the insulin and IGF-1 receptors is so strong that hybrid receptors containing one α-β chain of the insulin receptor and one α-β chain of the IGF-1 receptor are present in many tissues. These hybrid receptors bind both insulin and IGF-1, but their affinity for IGF-1 is greater. Given the structural similarity between insulin and IGFs and between the insulin receptor and the IGF-1 receptor, it is not surprising that IGFs can exert insulin-like actions in vivo. This effect has been particularly well studied for IGF-1, which, like insulin, induces hypoglycemia when injected into animals. This action is largely the result of increased uptake of glucose into muscle tissue. IGF-1 is less effective in mimicking insulin’s action on adipose and liver tissue; in humans, these tissues have few IGF-1 receptors. In muscle, IGF-1 promotes the uptake of radiolabeled amino acids and stimulates protein synthesis at concentrations that do not stimulate glucose uptake. Thus, IGF-1 promotes growth at lower circulating concentrations than those required to produce hypoglycemia (Box 48-1).

IGF-2 acts similarly to IGF-1 but is less dependent on GH The physiology of IGF-2 differs from that of IGF-1 both in terms of control of secretion and receptor biology. Regarding control of secretion, IGF-2 levels depend less on circulating GH than do IGF-1 levels. In GH deficiency—as seen in pituitary dwarfism—circulating levels of IGF-1, but not IGF-2, are decreased. In states of excessive GH secretion, plasma IGF-1 level is reliably elevated, whereas plasma IGF-2 level is not. Regarding receptor biology, although IGF-2 binds to the IGF-1 receptor, it preferentially binds to the so-called IGF-2 receptor (IGF2R). This IGF-2 receptor is a single-chain polypeptide that is structurally very distinct from the IGF-1 receptor and is not a receptor tyrosine kinase (see Fig. 48-6). After IGF2R binds IGF-2, the internalization of the complex

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

Extracellular space

997

IGF-1 RECEPTOR N

IGF-2 MANNOSE-6PHOSPHATE RECEPTOR

N

INSULIN RECEPTOR α N

N

N

S S

S S

S S

α S S

S S

S S

β

Receptor domain

β

C

C

C

C

Tyrosine kinase domains

C

Tyrosine kinase domains

Cytosol Figure 48-6  Comparison of insulin, IGF-1, and IGF-2 receptors. Both the insulin and IGF-1 receptors are

heterotetramers joined by disulfide bonds. For both, the cytoplasmic portions of the β subunits have tyrosine kinase domains as well as autophosphorylation sites. The IGF-2 receptor (also called the mannose-6-phosphate [M6P] receptor) is a single polypeptide chain with no kinase domain.

BOX 48-1  Plasma Level of IGF-1 as a Measure of GH Secretion

T

he plasma concentration of IGF-1 is a valuable measure of GH secretion. The wide swings in plasma [GH] that result from the pulsatile secretion of this hormone have confounded efforts to use GH measurements to diagnose disorders of GH deficiency or excess. However, an increased circulating concentration of IGF-1 is one of the most useful clinical measures of the excess GH secretion that occurs in acromegaly (i.e., GH excess in adults) and gigantism (i.e., GH excess in children). Measurement of plasma [IGF-1] has also helped to explain the genesis of a particular type of dwarfism known as Laron dwarfism. These patients were initially identified as persons with growth failure mimicking that of typical pituitary dwarfism; however, plasma [GH] is normal or elevated, and treatment

clears IGF-2 from the blood plasma. In an unrelated function, IGF2R in the trans Golgi binds—at a site different from that for IGF-2 binding—newly synthesized lysosomal hydrolases tagged with mannose-6-phosphate (M6P) for trafficking to the lysosomes.

with GH is ineffective in reversing the growth failure. It was subsequently demonstrated that these individuals have mutations of their GH receptors that make the receptors nonfunctional. Thus, the mutant GH receptors cannot trigger the production of IGFs. With the availability of recombinant IGF-1, effective treatment of these children may be possible with restoration of growth. Despite the structural similarity of their receptors, IGF-1 and insulin exert different actions on tissues. IGF-1 has a more marked effect on growth, and insulin has a more significant effect on glucose and lipid metabolism. However, the differences in the postreceptor signaling pathways triggered by the two hormones have not been fully defined.

Despite these differences, IGF-2 does share with IGF-1 (and also with insulin) the ability to promote tissue growth and to cause acute hypoglycemia. These properties appear to result from IGF-2’s structural similarity to proinsulin and its ability to bind to the IGF-1 receptor.

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SECTION VIII  •  The Endocrine System

20

1000

Plasma [IGF-1] (µg/L)

16

The pubertal peak rate of growth corresponds to the peak serum concentrations of IGF-1.

800

600

12

400

8

IGF-1 in serum

Rate of height increase (cm/yr)

4

200 Rate of height increase 0

0

10 12

20 Age

30

40

0

Figure 48-7  Serum IGF-1 levels and height velocity as a function of age. The red curve shows the mean plasma concentrations of IGF-1 as a function of age in human females. The curve for males is similar, but the peak is shifted to an older age by 3 to 4 years. The brown curve indicates for females the mean height velocity—the rate at which height increases (cm/yr). The pubertal peak rate of growth corresponds to the peak serum concentrations of IGF-1. (Data from Reiter EO, Rosenfeld RG: Normal and aberrant growth. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR [eds]: Williams Textbook of Endocrinology, 9th ed. Philadelphia, WB Saunders, 1998, pp 1427–1507.)

Growth rate parallels plasma levels of IGF-1 except early and late in life Illustrated in Figure 48-7 is the mean concentration of total IGF-1 (both free and bound) found in human serum as a function of age. Also shown is the normal rate of height increase (cm/yr). During puberty, the greatest growth rates are observed at times when plasma [IGF-1] is highest. A similar comparison can be made using GH, provided care is taken to obtain multiple measurements at each age and thereby account for the pulsatile secretion and marked diurnal changes that occur in plasma [GH]. Whereas, during puberty, growth rate parallels plasma [IGF-1], the two diverge at both younger and older ages. A first period of life for this divergence is very early childhood (see Fig. 48-7), which is characterized by a very rapid longitudinal growth rate, but quite low IGF-1 levels. If this time frame is extended back to intrauterine life, then the discordance is even greater. Indeed, children with complete GH deficiency have very low plasma [IGF-1] levels but are of normal length and weight at birth. This observation suggests that during intrauterine life, factors other than GH and IGF-1 are important regulators of growth. One of these additional factors may be insulin, as discussed below. Another explanation for the divergence between growth rate and IGF-1 levels may be that IGF-2 is an important mediator of intrauterine growth. Plasma [IGF-2] is greater during fetal life than later and peaks just before birth. Plasma [IGF-2] plummets soon after birth, but then it gradually doubles between birth and age 1 year, and remains at this level until at least the age of 80 years. Thus, IGF-2 levels are at adult levels during the first several years of life, when IGF-1 levels are low but growth is rapid. However, several other

hormones may also contribute to somatic growth during the first several years of life. By age 3 or 4 years, GH and IGF-1 begin to play major roles in the regulation of growth. The concentrations of these hormones rise throughout childhood and peak during the time of the pubertal growth spurt. The rate of long-bone growth in the pubertal growth spurt is exceeded only during intrauterine life and early childhood. The frequency of pituitary GH secretory pulses increases markedly at puberty. The factors responsible for this acceleration are not clear. However, the accompanying sexual maturation likely plays some role, because both estradiol and testosterone appear to promote GH secretion. During adulthood, longitudinal growth essentially ceases, yet secretion of GH and of IGF-1 continues to be highly regulated, although the circulating concentrations of both hormones decline during aging. For many years, the continued secretion of these hormones in adults was considered to be largely vestigial. This belief was reinforced by the observation that cessation of GH secretion and the consequent decline of IGF-1 after trauma, a pituitary tumor, or surgical removal of the pituitary did not result in any clear clinical syndrome. However, in GH-deficient adults replacement with recombinant human GH leads to remarkable increases in body muscle mass, decreases in fat mass, and improved nitrogen balance (a measure of protein nutrition). These findings support the conclusion that—even after linear growth ceases after puberty—GH and IGF-1 remain important regulators of body composition and appear to promote anabolic actions in muscle. Indeed, some investigators have suggested that supplementing physiologically normal adults with GH or IGF-1 may reverse some of the effects of aging,

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

including loss of muscle mass, negative nitrogen balance, and osteoporosis. Nutritional factors also modulate both GH secretion and IGF-1 production. In both children and adults, GH secretion is triggered by high dietary protein intake. Teleologically, this is intriguing in that it may provide linkage between the availability of amino acids to serve as substrates for body protein synthesis (growth) and the endocrine stimulus of cells to grow. This relationship is not simple, however, because the rise in GH levels in the setting of protein intake is not sufficient to stimulate IGF-1 production fully. This principle is well illustrated by fasting, which is associated with a decline in IGF-1 even with increased GH. During fasting, insulin levels are low. Thus, increased insulin appears to be required, at least in some tissues, for GH to stimulate IGF-1 effectively.

Thyroid hormones, steroids, and insulin also promote growth Although the discussion to this point emphasizes the action of GH and the GH-induced growth factors as modulators of somatic growth, we could regard them as necessary but not sufficient agents for normal growth. Certain other hormones, as well as receptive growth-responsive cartilage, are required. Because growth is a difficult phenomenon to study, especially in humans (few scientists want to follow an experiment over 10 to 20 years), much of our understanding of endocrine regulation of normal growth derives from observations of abnormal growth as it occurs in clinical syndromes of endocrine excess or deficiency. Several of the more important of these endocrine influences are illustrated here. The exact mechanism by which growth is regulated by these agents is not always well understood. Thyroid Hormones  Next to GH, perhaps the most prominent among the growth-promoting hormones are the thyroid hormones thyroxine and triiodothyronine, which we discuss in Chapter 49. In many nonhuman species, thyroid hor­ mone plays a major role in tissue growth and remodeling. For example, resorption of the tadpole tail during morphogenesis requires thyroid hormone. In humans, severe deficiency of thyroid hormones early in life causes dwarfism and mental retardation (cretinism; see pp. 1013–1014). In children with normal thyroid function at birth, development of hypothyroidism at any time before epiphyseal fusion leads to growth retardation or arrest. Much of the loss in height that occurs can be recovered through thyroid hormone treatment, a phenomenon called catch-up growth. However, because the diagnosis of hypothyroidism may elude detection for many months or years, delays in initiating treatment can lead to some loss of potential growth. A child’s growth curve can provide a particularly sensitive early indicator of hypothyroidism. Sex Steroids  As with thyroid hormones, the importance of sex steroids for growth is most readily understood by considering the effects of deficiency or excess of these hormones. Androgen or estrogen excess occurring before the pubertal growth spurt accelerates bone growth. However, the sex steroids also accelerate the rate at which the skeleton

999

matures and thus shorten the time available for growth before epiphyseal closure occurs. Most of the time, the dominant effect of sex steroids is to narrow the growth window, thereby diminishing ultimate longitudinal bone growth. This effect is well illustrated in settings in which children are exposed to excessive sex steroid at an early age. The sex steroids can come from endogenous sources (e.g., early maturation of the hypothalamic-pituitary-gonadal axis that produces premature puberty, or tumors that secrete estrogen or androgen) or from exogenous sources (e.g., children who take sex steroids prescribed for others). Again, the growth curve is useful in that it typically shows an increase in growth rate, followed by an early leveling off of growth associated with the development of secondary sexual characteristics. Glucocorticoids  An excess of adrenal glucocorticoids inhibits growth. Growth ceases in children who produce too much cortisol, as a result of either adrenal or pituitary tumors (which secrete adrenocorticotropic hormone [ACTH] and cause secondary increases in plasma cortisol levels). The use of synthetic glucocorticoids in treating various serious illnesses (e.g., asthma, organ transplantation, various chronic autoimmune processes) also can arrest growth. Restoration of normal growth does not occur until the glucocorticoid levels return toward normal. Neither GH nor IGF-1 concentrations drop significantly during glucocorticoid treatment. The failure of GH administration to correct the growth retardation that occurs in glucocorticoid-treated children further confirms that GH deficiency cannot account for the growth failure associated with glucocorticoid excess. Because linear growth is related to cartilage and bone synthesis at the growth plates, glucocorticoids presumably are acting at least in part at these sites to impair growth. However, the specific biochemical locus at which glucocorticoids act remains unclear. In adults, as in children, glucocorticoid excess impairs tissue anabolism and thus may manifest as wasting in some tissues (e.g., bone, muscle, subcutaneous connective tissue), rather than growth failure. This tissue wasting results in some of the major clinical morbidity associated with glucocorticoid excess (i.e., osteoporosis, muscle weakness, and bruising). In glucocorticoid deficiency, growth is not substantially affected. However, other deleterious effects of cortisol deficiency (e.g., hypoglycemia, hypotension, weakness; see p. 1019) dominate. Insulin  Insulin is also an important growth factor, particularly in utero. For example, women with diabetes frequently have high blood levels of glucose during pregnancy and deliver babies of high birth weight (fetal macrosomia). The developing fetus exposed to glucose concentrations that are higher than normal secretes additional insulin. Hyperinsulinemia results in increased fetal growth. Fetal macrosomia can create significant obstetric difficulties at the time of delivery. Conversely, infants born with pancreatic agenesis or with one of several forms of severe insulin resistance are very small at birth. One form of this condition, leprechaunism, is the result of a defect in the insulin receptor (see Box 51-4). Thus, it appears that insulin, acting through its own receptor, is an intrauterine growth factor.

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SECTION VIII  •  The Endocrine System

BOX 48-2  Anabolic-Androgenic Steroids

W

e are all unfortunately familiar with the potential for abuse of anabolic-androgenic steroids by bodybuilders and competitive athletes. Illicit use of these agents appears to be widespread in sports, where strength is closely linked to overall performance. Not only naturally occurring androgens (e.g., testosterone, dihydrotestosterone, androstenedione, and dehydroepiandrosterone), but also many synthetic androgenic steroids—as well as GH—serve as performance enhancers. In addition to the sought-after “beneficial” effects of increasing muscle mass and strength, each of these agents carries with it a plethora of adverse side effects. Some—such as oily skin, acne, and hair growth—are principally cosmetic. Others—including liver function abnormalities, mood changes with aggressive behavior, and hepatocellular carcinoma—are much more serious. Illicit use of these agents by younger athletes, especially teenagers, is also problematic with regard to alterations in growth and sexual maturation.

Severe insulin deficiency produces a marked catabolic effect associated with wasting of lean body mass in both children and adults. The acute adverse effects of such deficiency (dehydration and acidosis) dominate the clinical picture. Mild insulin deficiency, as seen in patients with chronically undertreated diabetes, diminishes growth in affected children. Improved diabetes management may allow restoration of normal growth rates and possibly even some transient accelerated or catch-up growth. Thus, with good care, children with diabetes can expect to achieve normal adult height (Box 48-2).

The musculoskeletal system responds to growth stimuli of the GHRH–GH–IGF-1 axis Longitudinal growth involves lengthening of the somatic tissues (including bone, muscle, tendons, and skin) through a combination of tissue hyperplasia and hypertrophy. Each of these tissues remodels its structure throughout life. For bone, longitudinal growth occurs by the hyperplasia of chondrocytes at the growth plates of the long bones, followed by endochondral ossification. The calcified cartilage is remodeled as it moves toward the metaphyses of the bone, where it is eventually replaced by cortical bone (see pp. 1056–1057) and trabecular bone (see p. 1057). This process continues until epiphyseal closure occurs toward the completion of puberty. The process of cartilage formation and longitudinal bone growth begins as the cellular elements capable of forming cartilage divide along the growth plate and then migrate toward the more mature bone. These cells synthesize the extracellular matrix of cartilage, which includes type II collagen, hyaluronic acid, and mucopolysaccharides. These cells appear to respond directly to GH by proliferating and increasing production of the extracellular matrix. This response involves local generation of IGF-1 within the cartilage as an early event in the growth process. As the cells more closely approach the already formed cortical and trabecular bone, ossification of the extracellular matrix begins, and eventually the cellular elements become isolated by the

TABLE 48-3  Other Growth Factors Affecting Growth Nerve growth factor (NGF) Fibroblast growth factor (FGF) Angiogenesis factor Vascular endothelial growth factor (VEGF) Epidermal growth factor (EGF) Hepatocyte growth factor (HGF)

calcifying cartilage. However, this calcified cartilage is not structurally the same as normal bone, and soon after formation it begins to be remodeled by an ingrowth of cellular elements (osteoclasts and osteoblasts) from adjacent bone. Eventually, it is replaced by normal bone, and becomes part of the metaphysis of the long bone. In most children, growth ceases within several years after completion of puberty, when the chondrocytes at the growth plates of the long bones cease dividing and calcify the previously cartilaginous surrounding matrix. After puberty, radial growth occurs as bones increase their diameter through a process of endosteal bone resorption and periosteal bone deposition. This process is not strictly compartmentalized; that is, resorption and deposition of bone occur at both the periosteal and endosteal surfaces. However, during periods of growth, the rate of periosteal deposition exceeds the rate of endosteal resorption, and the bone shafts grow in width and thickness. As may be expected, numerous disorders disrupt the complex process of endochondral bone growth on a genetic or congenital basis (e.g., defects in collagen or mucopoly­ saccharide synthesis) and lead to genetic forms of dwarfism. In these settings, the GHRH–GH–IGF-1 axis is entirely intact and appears to regulate normally. No apparent compensation occurs for the short stature by increased GH secretion, a finding suggesting that the axis is not sensitive to the growth process per se, but simply to the intermediate chemical mediator IGF-1. GH and IGF-1 clearly play important roles in mediating longitudinal bone growth and also modulate growth of other tissues. Thus, proportional growth of muscle occurs as bones elongate, and the visceral organs enlarge as the torso increases in size. The mechanisms by which GH and IGF-1 coordinate this process and the way in which other hormones or growth factors may be involved continue to be investigated. It is clear that, whereas GH and, more recently, IGF-1 have been considered the major hormones responsible for somatic growth, other tissue growth factors play an important, albeit incompletely defined, role. Table 48-3 lists some of these growth factors. In general, the tissue growth factors have more tissue-specific actions on organogenesis and their growth-promoting activity than the IGFs, and they appear to act largely in a paracrine or autocrine fashion.

REGULATION OF BODY MASS The multiple hormonal factors that influence longitudinal growth—discussed in the previous two subchapters—are themselves responsive to the nutritional intake of a growing

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

1001

individual. For example, amino acids and carbohydrates promote insulin secretion, and amino acids stimulate GH secretion (see pp. 992–994). In addition, the availability of an adequate balanced nutrient supply likely exerts both direct and indirect influences to promote tissue growth. Independent of any hormonal factors, glucose, fatty acids, and amino acids can each influence the transcription of specific genes. Similarly, amino acids can directly activate the signaling pathways involved in regulating messenger RNA (mRNA) translation. Beyond the effects of macronutrients, the effects of micronutrients can be similarly important in regulating cell growth and, by extension, growth of the organism. An example is iodine, a deficiency of which can produce dwarfism (see p. 1009). In a more global fashion, the effect of nutrient limitation on height can be appreciated by considering the differences in mean height between men in North Korea (165 cm) and South Korea (171 cm). As mentioned on page 990, nutritional deprivation early in life can markedly limit longitudinal growth. Perhaps equally fascinating, and only recently appreciated, is that nutritional deprivation early in life also appears to predispose affected individuals to obesity when they reach middle age. This phenomenon was first noted in epidemiological studies in several European countries that revealed a positive correlation between middleaged obesity and being born during periods of deprivation during and immediately following the Second World War. Such findings suggest that some level of genetic programming occurs early in life that both diminishes longitudinal growth and predisposes persons to body mass accretion.

sleeping) is known as the RMR (see p. 1170), which amounts to ~2100 kcal/day for a young 70-kg adult. The RMR supports maintenance of body temperature, the basal functioning of multiple body systems (e.g., heartbeat, GI motility, ventilation), and basic cellular processes (e.g., synthesizing and degrading proteins, maintaining ion gradients, metabolizing nutrients). 2. Activity-related energy expenditure. As we wake up in the morning and begin to move about, we expend more energy than resting metabolism. Exercise or physical work can have a major impact on total daily energy expenditure and varies widely across individuals, and within an individual on a day-to-day basis. We also expend energy in activities not classically regarded as exercise or heavy work, such as tapping the foot while sitting in a chair, looking about the room during a physiology lecture, typing at a keyboard—activities dubbed non–exercise-associated thermogenesis or NEAT. Such energy expenditures can vary 3- to 10-fold across individuals and can account for 500 kcal or more of daily energy expenditure. NEAT differences, over time, could contribute considerably to differences in weight gain by individuals with identical caloric intake. 3. Diet-induced thermogenesis. Eating requires an additional component of energy expenditure for digesting, absorbing, and storing food. Typically, diet-induced thermogenesis accounts for 10% of daily energy expenditure. Proteins have a higher thermic effect than either carbohydrates or fats (i.e., the metabolism and storage of proteins requires more energy).

The balance between energy intake and expenditure determines body mass

Each of these three components of energy expenditure can vary considerably from day to day and is subject to regulation. For example, thyroid hormone is a major regulator of thermogenesis (see p. 1013). Overproduction of thyroid hormone increases both RMR and NEAT, whereas thyroid hormone deficiency has the opposite effect.  N48-3

At any age or stage of life the factors that govern body mass accretion relate specifically to the energy balance between intake and expenditure. If energy intake exceeds expenditure over time—positive energy balance (see p. 1173)—body mass will increase, assuming the diet is not deficient in essential macronutrients or micronutrients. Small positive deviations from a perfect energy balance, over time, contribute to the major increase in body weight—the “obesity epidemic”—that affects many middle-aged adults, and increasingly adolescents, in developed societies. For example, if energy intake in the form of feeding exceeds energy expenditure by only 20 kcal (1 tsp of sugar) daily, over 1 year a person would gain ~1 kg of fat, and over 2 decades, ~20 kg. Indeed, it is remarkable that many adults maintain a consistent body weight for decades essentially in the absence of conscious effort. Thus, a finely tuned regulatory system must in some manner “monitor” one or more aspects of body mass, direct the complex process of feeding (appetite and satiety) to replete perceived deficiencies, and yet avoid excesses.

Energy expenditure comprises resting metabolic rate, activity-related energy expenditure, and diet-induced thermogenesis We can group energy expenditure into three components: 1. Resting metabolic rate (RMR). The metabolism of an individual who is doing essentially nothing (e.g.,

Hypothalamic centers control the sensations of satiety and hunger Classic studies in which investigators made lesions in, or electrically stimulated, specific brain regions identified two areas in the hypothalamus that are important for controlling eating. A satiety center is located in the ventromedial nucleus (VMN; see Fig. 47-3). Electrical stimulation of the satiety center elicits sensations of satiety, even when an animal is in the presence of food. Conversely, a lesion of the satiety center causes continuous food intake (hyperphagia) even in the absence of need. A hunger (or feeding) center is located in the lateral hypothalamic area (see Fig. 47-3). Electrical stimulation of this center elicits a voracious appetite, even after an animal has ingested adequate amounts of food. A lesion of the hunger center causes complete and lasting cessation of food intake (aphagia).

Leptin tells the brain how much fat is stored Only in the last 2 decades have we begun to understand regulatory mechanisms that maintain body mass, an advance made possible by the study of mouse models of obesity. One

Chapter 48  •  Endocrine Regulation of Growth and Body Mass 1001.e1

N48-3  Effect of Hyperthyroidism on Basal Metabolic Rate Contributed by Emile Boulpaep and Walter Boron One of the earliest tests for hyperthyroidism was to measure basal metabolic rate (BMR), as discussed in the text on page 1170. This method is not used today because BMR can be affected by other factors (e.g., body size, fever, catecholamines, fasting), so changes cannot be related specifically to the thyroid. In addition, it is cumbersome to measure BMR accurately compared with obtaining serum estimates of thyroid hormone concentrations or activity. Nevertheless, all things being equal, thyroid hormone increases the BMR. The difference between BMR and RMR is discussed on page 1170.

1002

SECTION VIII  •  The Endocrine System

A

Ob

B

Db

C

Ob

Db

A = Ob mouse + Wt mouse B = Db mouse + Wt mouse C = Ob mouse + Db mouse Figure 48-8  Parabiosis experiments. In parabiotically coupled mice, ~1% of the cardiac output of one mouse goes to the other, and vice versa, so that the animals exchange blood-borne factors. Wt, wild type.

monogenic model is the Ob/Ob strain of hyperphagic mice that develop morbid obesity; affected mice typically weigh >100% more than unaffected animals of the same strain. In parabiosis experiments in which an Ob/Ob mouse was surgically connected to a wild-type mouse (Fig. 48-8A), the Ob/ Ob mouse lost weight, which suggests that such mice lack a blood-borne factor. Another model of monogenic obesity is the (Db/Db) mouse, named Db because it secondarily develops type 2 diabetes (see Box 51-5). Like Ob/Ob mice, Db/ Db mice are hyperphagic, with adult body weights ~100% greater than those of lean littermates. However, in parabiosis experiments connecting a Db/Db and a wild-type mouse (see Fig. 48-8B), the wild-type mouse starved. Finally, in parabiosis experiment connecting an Ob/Ob to a Db/Db mouse (see Fig. 48-8C), the Ob mouse lost weight but the Db mouse remained obese. These results indicate the following: 1. The Db mouse makes an excess of the blood-borne factor that cures the Ob mouse. 2. The Db mouse lacks the receptor for this factor. 3. Absence of the receptor in the Db mouse removes the negative feedback, which leads to high levels of the bloodborne factor. In 1994, Jeffrey Friedman and his colleagues used positional cloning to identify leptin (from the Greek leptos [thin]), the blood-borne factor lacked by Ob mice. Leptin is a 17-kDa protein made almost exclusively in adipocytes. The replacement of leptin in Ob/Ob mice leads to rapid weight loss. In 1995, Tepper and collaborators cloned the leptin receptor (LEP-R). The deficiency of this receptor in Db mice makes them leptin resistant. LEP-R is a tyrosine kinase– associated receptor (see Fig. 3-12D) that signals through JAK2 and STAT (see Fig. 4-14). Among the several splice variants of LEP-R, the “long-form” is richly expressed in the arcuate nucleus of the hypothalamus and several other CNS sites. Although leptin acts on numerous tissues, it somehow crosses the blood-brain barrier (see pp. 284–287) and modulates neurons in the arcuate nucleus of the hypothalamus that

secrete pro-opiomelanocortin (see Fig. 50-4) and influence feeding behavior. These same neurons also have insulin receptors. Plasma leptin levels in humans appear to rise in proportion to the mass of adipose tissue (Box 48-3). Conversely, the absence of leptin produces extreme hyperphagia, as in Ob/Ob mice. Plasma leptin has a half-time of ~75 minutes, and acute changes in food intake or fasting do not appreciably affect leptin levels. In contrast, insulin concentrations change dramatically throughout the day in response to dietary intake. Thus, it appears that leptin in some fashion acts as an intermediate- to long-term regulator of CNS feeding behavior, whereas insulin (in addition to intestinal hormones like glucagon-like peptide 1 [GLP-1] and cholecystokinin [CCK]) is a short-term regulator of the activity of hypothalamic feeding centers. In addition to acting to control appetite, leptin promotes fuel utilization. Indeed, leptin-deficient humans paradoxically exhibit some characteristics of starvation (e.g., fuel conservation via decreased thermogenesis and basal metabolic rate).

Leptin and insulin are anorexigenic (i.e., satiety) signals for the hypothalamus At least two classes of neurons within the arcuate nucleus contain receptors for leptin and insulin. These neurons, in turn, express neuropeptides. One class of neurons produces pro-opiomelanocortin (POMC), whereas the other produces neuropeptide Y (NPY) and agouti-related protein (AgRP). POMC Neurons  Insulin and leptin each stimulate largely distinct subgroups of POMC-secreting neurons (Fig. 48-9), which produce POMC. At their synapses, POMC neurons release a POMC cleavage product, the melanocortin αmelanocyte–stimulating hormone (α-MSH; see Fig. 50-4), which in turn binds to MC3R and MC4R melanocortin receptors on second-order neurons. Stimulation of these receptors not only promotes satiety and decreases food intake—that is, α-MSH is anorexigenic (from the Greek a [no] + orexis [appetite])—but also increases energy expenditure via activation of descending sympathetic pathways. An indication of the importance of this pathway is that ~4% of individuals with severe, early-onset obesity have mutations in MC3R or MC4R. POMC neurons also synthesize another protein—cocaine- and amphetamine-regulated transcript (CART)—which, like α-MSH, is anorexigenic. NPY/AgRP Neurons  In addition to stimulating POMC neurons, both insulin and leptin can also suppress neurons in the arcuate nucleus that release NPY and AgRP at their synapses (see Fig. 48-9). NPY activates NPY receptors (which are GPCRs)—predominantly Y1R and Y5R—on secondary neurons, thereby stimulating eating behavior. AgRP binds to and inhibits MC4R melanocortin receptors on the secondary neurons in the POMC pathway, thereby inhibiting the anorexigenic effect of α-MSH. Thus, both NPY and AgRP are orexigenic. The yellow obese or agouti mouse overexpresses the agouti protein, which inhibits melanocortin receptors. Overinhibition of MC1R on melanocytes inhibits the dispersion of pigment granules (leading to yellow rather than dark fur). Overinhibition of MC3R and MC4R on

Chapter 48  •  Endocrine Regulation of Growth and Body Mass

1003

BOX 48-3  Human Obesity

O

ne approach for gauging the extent to which human body mass is appropriate for body height is to compute the body mass index (BMI): BMI =

Weight in kg (Height in m)2

BMIs fall into four major categories:  N48-4 Underweight: 600 kDa), and it accounts for approximately half of the protein content of the thyroid gland. It has relatively few tyrosyl residues (~100 per molecule of Tg), and only a few of these (99.98% of the hormone circulates tightly bound to protein. T3 is bound only slightly less: ~99.5% is protein bound. Because the free or unbound hormone in the circulation is responsible for the actions of the thyroid hormones

Chapter 49  •  The Thyroid Gland

BOX 49-1  Iodine Deficiency

I

n areas where soil is relatively iodine deficient, human iodine deficiency is common. Because seawater and seafood contain large amounts of iodide, iodine deficiency is more common in inland areas, particularly in locales that rely on locally grown foods. For example, in inland areas of South America along the Andes Mountains, in central Africa, and in highland regions of Southeast Asia, iodine deficiency is common. In the early 1900s, investigators first recognized that iodide is present in high concentrations in the thyroid and that iodine deficiency promotes goiter formation. These observations led to efforts to supplement dietary iodine. Iodine deficiency causes thyroid hormone deficiency. The pituitary responds to this deficit by increasing the synthesis of TSH (see p. 1014), which in turn increases the activity of the iodinetrapping mechanism in the follicular cell in an effort to overcome the deficiency. The increased TSH also exerts a trophic effect that increases the size of the thyroid gland. If this trophic effect persists for sufficient time, the result is an iodine-deficiency goiter. Goiter is the generic term for an enlarged thyroid. If this effort at compensation is not successful (i.e., if insufficient thyroid hormone levels persist), the person will develop signs and symptoms of goitrous hypothyroidism. When iodine deficiency occurs at critical developmental times in infancy, the effects on the CNS are particularly devastating and produce the syndrome known as cretinism (see p. 1013). Persons so affected have a characteristic facial appearance and body habitus, as well as severe mental retardation. Dietary supplementation of iodine in salt and bread has all but eliminated iodine deficiency in North America. In many nations, especially in mountainous and landlocked regions of developing countries, iodine deficiency remains a major cause of preventable illness.

on their target tissues, the large amount of bound hormone has considerably confounded our ability to use simple measurements of the total amount of either T4 or T3 in the plasma to provide a reliable index of the adequacy of thyroid hormone secretion. For example, the amount of TBG in the serum can change substantially in different physiological states. Pregnancy, oral estrogen therapy, hepatitis, and chronic heroin abuse can all elevate the amount of TBG and hence the total concentration of T4 and T3. Decreased levels of TBG, associated with diminished concentration of total T4 and T3, can accompany steroid usage and nephrotic syndrome. However, despite the marked increases or decreases in the amounts of circulating TBG, the concentrations of free T4 and T3 do not change in the aforementioned cases. Box 49-2 indicates how one can calculate levels of free T4 or T3, knowing the concentration of TBG and the concentration of total T4 or total T3. The liver makes each of the thyroid-binding proteins. TBG is a 54-kDa glycoprotein consisting of 450 amino acids. It has the highest affinity for T4 and T3 and is responsible for most of the thyroid-binding capacity in the plasma. The extensive binding of thyroid hormones to plasma proteins serves several functions. It provides a large buffer pool of thyroid hormones in the circulation, so that the active concentrations of hormone in the circulation change very little on a minute-to-minute basis. The binding to plasma proteins markedly prolongs the half-lives of both T4 and T3. T4 has a

1009

BOX 49-2  Free versus Bound Thyroxine

M

ost of the T4 and T3 in the serum is bound to proteins, the most important of which is TBG. For the binding of T4 to TBG, the reaction is K T4 + TBG ← → T4 TBG

K=

[ T4 TBG] [ T4 ][TBG]

The binding constant K is ~2 × 1010  M−1 for T4. The comparable binding constant for T3 is ~5 × 108  M−1. Approximately one third of TBG’s binding sites are occupied by T4. Therefore, we have all the information we need to compute the concentration of free T4: [ T4 ]FREE =

[ T4 TBG] K[TBG]

A reasonable value for [T4TBG] would be 100 nM, and for [TBG], 250 nM. Thus, [ T4 ]FREE =

(100 nM) ( 2 × 1010 M−1) ⋅ ( 250 nM)

= 0.20 × 10 −10 M = 20 pM

Because the bound T4 in this example is 100 nM, and the free T4 is only 20 pM, we can conclude that only ~0.02% of the total T4 in the plasma is free. Because 99.98% of the total T4 in the plasma is bound, moderate fluctuations in the rate of T4 release from the thyroid have only tiny effects on the level of free T4. To simplify, we have not included the minor contribution of albumin and TTR in this sample calculation.

half-life of 8 days, and T3, of ~24 hours; each is longer than the half-life of steroid or peptide hormones. Finally, because much of the T3 in the circulation is formed by the conversion of T4 to T3 in extrathyroidal tissues, the presence of a large pool of T4 in the plasma provides a reserve of prohormone available for synthesis of T3. This reserve may be of particular importance because T3 is responsible for most of the biological activity of thyroid hormones.

Peripheral tissues deiodinate T4 to produce T3 The thyroid synthesizes and stores much more T4 than T3, and this is reflected in the ~10 : 1 ratio of T4 to T3 secreted by the thyroid. However, certain tissues in the body have the capacity to selectively deiodinate T4, thereby producing either T3 or rT3. T3 and rT3 can each be further deiodinated to various DITs and MITs (Fig. 49-4); both DITs and MITs are biologically inactive. Both iodine atoms on the inner ring, and at least one iodine atom on the outer ring, appear essential for biological activity. Similarly, the loss of the amino group renders T4 or T3 inactive. The importance of the peripheral deiodination of T4 to T3 can be readily appreciated from the observation that persons whose thyroids have been removed have normal circulating concentrations of T3 when they receive oral T4 supplementation.

1010

SECTION VIII  •  The Endocrine System

T4

T3

T2

T1

T0

T3

Thyronine T4

rT3

5´/3´-monodeiodinase (type 1 and type 2) 5/3-monodeiodinase (type 3)

Figure 49-4  Peripheral metabolism of T4. The 5′/3′-monodeiodinases (type 1 and type 2; green arrows)

remove I from the outer benzyl ring, whereas the 5/3-monodeiodinase (type 3; orange arrows) removes I from the inner benzyl ring. Thus, the action of the 5′/3′-monodeiodinases on T4 yields T3, whereas the action of the 5/3-monodeiodinase yields rT3. Sequential deiodination yields T0 (thyronine).

Inasmuch as T3 is biologically much more active than the far more abundant T4, the regulated conversion of T4 to T3 in peripheral tissues—as well the conversion of T4 and T3 to inactive metabolites—assumes considerable importance. These conversions are under the control of three deiodinases. Two deiodinases are 5′/3′-deiodinases that remove an I from the outer ring and thereby convert T4 to T3 (see Fig. 49-4). The first of these 5′/3′-deiodinases—type 1 deiodinase—is present at high concentrations in the liver, kidneys, skeletal muscle, and thyroid. It appears to be responsible for generating most of the T3 that reaches the circulation. The second 5′/3′-deiodinase—type 2 deiodinase—is found predominantly in the pituitary, central nervous system (CNS), and placenta, and is involved in supplying those tissues with T3 by local generation from plasma-derived T4. As shown below, the type 2 enzyme in the pituitary is of particular importance because the T3 that is generated there is responsible for the feedback inhibition of the release of thyrotropin (or thyroid-stimulating hormone, TSH). A third 5/3-deiodinase—type 3 deiodinase—removes an I from the inner ring, thereby converting T4 to the inactive rT3. Because the 3′ and 5′ positions in T4 are equivalent stereochemically, removing either of these by type 1 or type 2 deiodinase yields T3. Similarly, removal of the I from either the 3 or the 5 position of the inner ring of T4 by type 3 deiodinase yields rT3. Further deiodination by any of the three enzymes ultimately yields T0 (i.e., thyronine). The relative activity of the outer-ring deiodinases changes in response to physiological and pathological stimuli. Caloric restriction or severe stress inhibits the type 1 deiodinase; this process decreases the conversion of T4 to T3—and thus reduces the levels of T3. In contrast, levels of rT3 rise by default in these situations, in part because of reduced

conversion to DITs. These decreases in T3 levels are accompanied by a decline in metabolic rate. You may think that because plasma levels of T3 fall, there would be a compensatory rise in TSH, the secretion of which is inhibited by T3. However, because type 2 deiodinase mediates the conversion of T4 to T3 within the pituitary and CNS, and because caloric restriction does not affect this enzyme, local T3 levels in the pituitary are normal. Thus, the thyrotrophs in the pituitary continue to have adequate amounts of T3, and no compen­ satory rise in TSH occurs. Teleologically, the rationale to restrain calorie expenditure in settings of decreased caloric intake is appealing.   N49-2

ACTION OF THYROID HORMONES Thyroid hormones act through nuclear receptors in target tissues Thyroid hormones act on many body tissues to exert both metabolic and developmental effects. Once T4 and T3 leave the plasma, they enter the cell either by diffusing through the lipid of the cell membrane or by carrier-mediated transport (Fig. 49-5). Most, but not all, of the actions of thyroid hormones occur as thyroid hormones bind to and activate nuclear receptors (see pp. 71–72). The multitude of thyroid hormone actions is mirrored by the ubiquitous expression of thyroid hormone receptors (TRs) throughout the body’s tissues. There are actually two TR genes—α (chromosome 17) and β (chromosome 3)—and at least two isoforms of TRβ. The expression of these receptor genes is tissue specific and varies with time of development. The liver expresses TRβ, whereas TRα predominates in the brain. During development, the amount of α expressed may vary 10-fold or

Chapter 49  •  The Thyroid Gland 1010.e1

N49-2  Effect of Calorie Restriction on Type 1 Deiodinase Contributed by Eugene Barrett As noted in the text, calorie restriction inhibits type 1 monodeiodinase, reducing the conversion of T4 to T3 and thereby lowering circulating levels of T3. This effect of caloric restriction makes sense for someone who is starving because it tends to conserve body stores of fuel. On the other hand, this effect makes it more difficult to lose weight intentionally while dieting.

Chapter 49  •  The Thyroid Gland

99.5% is bound to TBG (thyroxinbinding globulin). T3

5´/3´ monodeiodinase activity removes the 5´ iodine, converting T4 to T3.

MCT8 (SLC16A2)

Thyroid hormone receptor (TR) Retinoid X receptor (RXR)

T3 3´/5´ Monodeiodinase

T4

Gene Transcription

Thyroid response element (TRE)

mRNA

T4

99.98% is bound to TBG.

Nuclear envelope Na–K pump Gluconeogenic enzymes Respiratory enzymes

I

MCT8 (SLC16A2)

Blood plasma

1011

Cytosol

Myosin heavy chain β-adrenergic receptors Many others

Nucleus

T4 and T3 enter the cell either by diffusion or by carrier-mediated transport. Figure 49-5  Action of thyroid hormones on target cells. Free extracellular T4 and T3 enter the target cell via facilitated diffusion. Once T4 is inside the cell, a cytoplasmic 5′/3′-monodeiodinase converts much of the T4 to T3, so that the cytoplasmic levels of T4 and T3 are about equal. T3 or T4 activates thyroid hormone receptors—already bound to nuclear DNA at thyroid response elements in the promoter region of certain genes—and thereby regulates the transcription of these genes. Of the total thyroid hormone bound to receptor, ~90% is T3. The receptor that binds to the DNA is preferentially a heterodimer of the thyroid hormone receptor and retinoid X receptor. MCT8, monocarboxylate transporter 8.

more. Both receptors bind to DNA response elements, predominately as heterodimers in association with the retinoid X receptor (RXR), and alter the transcription of specific genes. Biologically, T3 is much more important than T4. This statement may be surprising inasmuch as the total concentration of T4 in the circulation is ~50-fold higher than that of total T3. Nevertheless, T3 has greater biological activity for three reasons. First, T4 is bound (only 0.01 to 0.02% is free) more tightly to plasma proteins than is T3 (0.50% is free). The net effect is that the amounts of free T4 and free T3 in the circulation are comparable. Second, because the target cell converts some T4—once it has entered the cell—to T3, it turns out that T4 and T3 are present at similar concentrations in the cytoplasm of target cells. Third, the TR in the nucleus has ~10-fold greater affinity for T3 than for T4, so that T3 is more potent on a molar basis. As a result, T3 is responsible for ~90% of the occupancy of TRs in the euthyroid state.   N49-3 When T3 or T4 binds to the TR in the nucleus, the hormone-bound receptor either activates or represses the transcription of specific genes. As discussed above, TR preferentially binds to DNA as a heterodimer of TR and RXR (see Table 3-6). TR belongs to the superfamily of nuclear receptors that may contain domains A through F (see Fig. 3-14). Three regions are especially important for TR: (1) The amino-terminal A/B region contains the first of two transactivation domains, which are responsible for transducing receptor binding into a conformational change in the DNA and thereby initiating transcription. (2) The middle or C region is responsible for DNA binding through two zinc fingers (see p. 82) as well as dimerization between receptors.

(3) the E region, toward the carboxyl terminus, is responsible for binding the ligand (T3 or T4), and also for dimerization.

Thyroid hormones can also act by nongenomic pathways In addition to binding to receptors in the nucleus, T4 and T3 bind to sites in the cytosol, microsomes, and mitochondria. This observation has raised the issue of whether thyroid hormones exert actions through mechanisms not involving transcriptional regulation. Nongenomic actions of thyroid hormones have been observed in several tissues, including heart, muscle, fat, and pituitary. Thyroid hormones can act via nongenomic pathways to enhance mitochondrial oxidative phosphorylation—or at least energy expenditure as measured by O2 consumption. Nongenomic targets of thyroid hormones include ion channels, second messengers, and protein kinases. It is less clear whether these actions occur via TRα or TRβ—similar to the nongenomic actions of estrogens, which involve the estradiol receptor (see p. 989)— or whether other high-affinity thyroid-binding proteins are involved.

Thyroid hormones increase basal metabolic rate by stimulating futile cycles of catabolism and anabolism Investigators have long observed that excess thyroid hormone raises the basal metabolic rate (BMR) as measured by either heat production (direct calorimetry) or O2 consumption (indirect calorimetry). Conversely, thyroid hormone deficiency is accompanied by a decrease in BMR. Figure 49-6 illustrates the effect of thyroid hormone levels on BMR, and Table 49-1 summarizes the effect of the thyroid hormones

Chapter 49  •  The Thyroid Gland 1011.e1

N49-3  Sick Euthyroid Syndrome Contributed by Eugene Barrett Many hospitalized patients who are extremely ill exhibit abnormal results on thyroid function tests. However, the thyroid activity of most of these patients is actually appropriate and needs no correction. Many of these patients are in an intensive care unit (ICU) setting, and it is extremely important to distinguish true thyroid disease from this so-called sick euthyroid syndrome. Sick euthyroid syndrome can take many forms, but the most common is a low or lower-than-normal total T4 level and a low T3 level. In true hypothyroidism, the diminished levels of thyroid hormones decrease feedback inhibition on the pituitary gland and lead to increased levels of TSH (see p. 1014). In sick euthyroid syndrome, the TSH level is usually normal. Although the reasons for this situation are not completely understood, at least one explanation may lie in the distinction between type 1 deiodinase, which is found in the periphery, and type 2 deiodinase, which is present in the pituitary. In sick euthyroid syndrome, the activity of type 1 or peripheral 5′/3′ deiodinase decreases, so that there is less conversion of peripheral T4 to T3, but more conversion to rT3. As a result, peripheral T3 levels fall. However, as was described in the main text with regard to starvation, type 2 deiodinase is not affected by stress nearly as much as is type 1 deiodinase; therefore, the pituitary gland continues to sense normal levels of T3, and it responds to what it perceives as normal levels of feedback inhibition from local T3 on the production and release of TSH. However, other factors must also be involved, inasmuch as this mechanism does not adequately account for the decrease in total T4. Patients with sick euthyroid syndrome may appear profoundly hypothyroid, exhibiting hypothermia and a sluggish sensorium, but they are not hypothyroid, and they should not receive thyroid hormone replacement. In fact, treating sick euthyroid patients with thyroid hormone yields either no improvement or a worse outcome.

1012

SECTION VIII  •  The Endocrine System

hepatic gluconeogenic activity (see p. 1176). This effect generally does not result in increases in plasma [glucose], provided the pancreas responds by augmenting insulin secretion. Thyroid hormones also enhance the availability of the starting materials required for increased gluconeogenic activity (i.e., amino acids and glycerol), and they specifically induce the expression of several key gluconeogenic enzymes, including phosphoenolpyruvate carboxykinase, pyruvate carboxylase, and glucose 6-phosphatase.

30 20 10

Hyperthyroid

0 Percent change in basal –10 metabolic rate –20

Normal

Hypothyroid

–30 –40 –50

0

100 200 Thyroid-hormone production rate (µg/day)

300

Figure 49-6  Effect of thyroid hormone on BMR. This graph shows the dependence of BMR on the daily rate of thyroid hormone secretion (T4 and T3). We use the secretion rate because it is difficult to know whether to use free T4 or free T3. Thus, the secretion rate is a crude measure of effective thyroid hormone levels. (Data from Guyton AC, Hall JE: Textbook of Medical Physiology, 9th ed. Philadelphia, WB Saunders, 1996.)

TABLE 49-1  Physiological Effects of the Thyroid Hormones (T3 and T4) LOW LEVEL OF THYROID HORMONES (HYPOTHYROID)

HIGH LEVEL OF THYROID HORMONES (HYPERTHYROID)

Basal metabolic rate

↓

↑

Carbohydrate metabolism

↓ Gluconeogenesis ↓ Glycogenolysis Normal serum [glucose]

↑ Gluconeogenesis ↑ Glycogenolysis Normal serum [glucose]

Protein metabolism

↓ Synthesis ↓ Proteolysis

↑ Synthesis ↑ Proteolysis Muscle wasting

Lipid metabolism

↓ Lipogenesis ↓ Lipolysis ↑ Serum [cholesterol]

↑ Lipogenesis ↑ Lipolysis ↓ Serum [cholesterol]

Thermogenesis

↓

↑

Autonomic nervous system

Normal levels of serum catecholamines

↑ Expression of β adrenoceptors (increased sensitivity to catecholamines, which remain at normal levels)

PARAMETER

on several parameters. Thyroid hormones increase the BMR by stimulating both catabolic and anabolic reactions in pathways affecting fats, carbohydrates, and proteins. Carbohydrate Metabolism  Thyroid hormones raise the rate of hepatic glucose production, principally by increasing

Protein Metabolism  The amino acids required for increased hepatic gluconeogenesis stimulated by thyroid hormones come from increased proteolysis, predominantly in muscle. Thyroid hormones also increase protein synthesis. Because the increases in protein degradation usually outweigh the increases in synthesis, a net loss of muscle protein occurs. The catabolic effect is exaggerated when T3 is present in great excess, so that muscle wasting and weakness, as well as increased nitrogen loss in the urine as urea (see pp. 770–772 and 965), can be prominent features of clinical thyrotoxicosis (hyperthyroidism). Lipid Metabolism  Thyroid hormones increase the degradation of stored triacylglycerols in adipose tissue, releasing fatty acids (FAs) and glycerol. The FAs provide fuel for the liver to support the energy demand of gluconeogenesis, and the glycerol provides some of the starting material for gluconeogenesis. Thyroid hormones not only increase lipolysis but also enhance lipogenesis. Indeed, modest amounts of thyroid hormones are needed for the normal synthesis of FAs by liver. Very high levels of T3 shift the balance in favor of lipolysis, with resulting generalized fat mobilization and loss of body fat stores. By accelerating the rates of glucose production, protein synthesis and degradation, as well as lipogenesis and lipolysis, the thyroid hormones stimulate energy consumption. Therefore, to the extent that thyroid hormones stimulate both synthesis and degradation, they promote futile cycles that contribute significantly to the increased O2 consumption seen in thyrotoxicosis (hyperthyroidism). How, at the molecular level, thyroid hormones affect the BMR in states of both spontaneous and experimentally induced thyroid hormone excess or deficiency has been a difficult question to answer. The changes in metabolic rate do not appear to be determined by changes in the expression of a single gene. Several specific examples of the effects of thyroid hormones on target tissues serve to illustrate their general mechanism of action. Na-K Pump Activity  In muscle, liver, and kidney, thyroid hormone–induced increases in oxygen consumption are paralleled by increases in the activity of the Na-K pump in the plasma membrane (see pp. 115–117). This increase in transport is the result, at least in part, of an increase in the synthesis of new transporter units that are inserted into the plasma membrane. At least in some tissues, the blockade of the increases in Na-K pump activity with ouabain also blocks the increase in O2 consumption. T3 stimulates the transcription of the genes for both the α and β subunits of the Na-K pump. In addition, T3 increases translation by stabilizing the mRNA that encodes the Na-K pump. Increases in pump activity consume additional ATP, which results in increased

Chapter 49  •  The Thyroid Gland

O2 consumption and heat generation. Inasmuch as states of thyroid hormone excess are not accompanied by any noticeable derangement of plasma electrolyte levels, presumably the increase in Na-K pump activity is compensated in some manner by a leak of Na+ and K+, although such pathways have not yet been defined. Overall, the increased activity of the Na-K pump (with an accompanying cation leak) would result in a futile cycle in which energy was consumed without useful work. Thermogenesis  In rodents, thyroid hormones may affect metabolic rate and thermogenesis through another futile cycle mechanism. Brown fat in these animals expresses a mitochondrial uncoupling protein (UCP), or thermogenin, that dissociates oxidative phosphorylation from ATP generation. Thus, mitochondria consume O2 and produce heat without generating ATP. Both T3 and β-adrenergic stimulation (acting through the β3 receptor) enhance respiration in brown adipose tissue by stimulating this uncoupling mechanism. We discuss thermogenin—and the vital role it plays in helping to keep newborn humans warm—on page 1166. Thyroid hormones also increase the BMR by increasing the thermogenic effects of other processes. An example is the effect of adrenergic stimulation on thermogenesis, discussed above. In humans, plasma concentrations of catecholamines are normal in states of both excess and deficient T3 and T4. However, excess thyroid hormone raises the sensitivity of tissues to the action of adrenergic hormones. In heart, skeletal muscle, and adipose tissue, this effect is the result, at least in part, of increased expression of β-adrenergic receptors by these tissues. In patients who are acutely thyrotoxic, treatment with β-receptor antagonists is one of the first priorities. This treatment blunts the hypersympathetic state induced by the excess of thyroid hormones. Thyroid hormones may also exert postreceptor effects that enhance adrenergic tone. In the heart, thyroid hormones also regulate the expression of specific forms of myosin heavy chain. Specifically, in rodents, thyroid hormone increases the expression of the myosin α chain, thereby favoring the α/α isoform of myosin heavy chain (see Table 9-1). This isoform is associated with greater activity of both actin and Ca2+-activated ATPase, faster fiber shortening, and greater contractility.

Thyroid hormones are essential for normal growth and development In amphibians, thyroid hormone regulates the process of metamorphosis. Removing the thyroid gland from tadpoles causes development to arrest at the tadpole stage. Early administration of excess thyroid hormone can initiate premature metamorphosis. Iodothyronines are present even farther down the phylogenetic tree, at least as far as primitive chordates, although these animals lack a thyroid gland per se. However, the biological actions of iodothyronines in many species are not known. Thyroid hormones are essential for normal human development as well, as starkly illustrated by the unfortunate condition of cretinism in regions of endemic iodine deficiency. Cretinism is characterized by profound mental retardation, short stature, delay in motor development, coarse hair, and a protuberant abdomen. Correction of iodine deficiency

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has essentially eliminated endemic cretinism in developed nations. Sporadic cases continue to occur, however, as a result of congenital defects in thyroid hormone synthesis. If hypothyroidism (Box 49-3) is recognized and corrected within 7 to 14 days after birth, development—including mental development—can proceed almost normally. Once the clinical signs of congenital hypothyroidism become apparent, the developmental abnormalities in the CNS are irreversible. For this reason, all U.S. states and territories conduct laboratory screening of newborns for hypothyroidism. This screening has shown that the overall rate of congenital hypothyroidism is ~0.3% and varies considerably across racial and ethnic groups, being lower in African Americans (~0.1%) and higher in Hispanic infants (~0.6%).

BOX 49-3  Hypothyroidism

H

ypothyroidism is one of the most common of all endocrine illnesses, affecting between 1% and 2% of all adults at some time in their lives. Women are much more commonly affected than men. Although hypothyroidism has several causes, the most common cause worldwide is iodine deficiency. In the United States, by far the most common cause is an autoimmune disorder called Hashimoto thyroiditis. Like Graves disease, Hashimoto thyroiditis is caused by an abnormal immune response that includes the production of antithyroid antibodies—in this case, anti­ bodies against the thyroid follicular cells, microsomes, and TSH receptors. Unlike in Graves disease, the antibodies in Hashimoto thyroiditis are not stimulatory, but rather are part of an immune process that blocks and destroys thyroid function. The titers of these autoantibodies can reach colossal proportions. Typically, hypothyroidism in Hashimoto thyroiditis is an insidious process that develops slowly; indeed, many patients are diagnosed long before striking clinical manifestations are apparent when routine blood tests reveal an elevated TSH level despite normal levels of T3 and T4. These individuals, although not yet clinically hypothyroid, are sometimes treated with thyroid hormone replacement, so the clinical manifestations of hypothyroidism are never given a chance to develop. In patients in whom the disease does evolve, the classical presentation consists of painless goiter, skin changes, peripheral edema, constipation, headache, joint aches, fatigue, and, in women, anovulation. The TSH level should be checked in any female patient with secondary amenorrhea. A subset of these Hashimoto thyroiditis patients may also develop other autoimmune endocrine deficiency disorders. Those with multiple endocrine deficiency type 1 have insufficient production of parathyroid, adrenal, and thyroid hormones. Those with multiple endocrine deficiency type 2 have insufficiencies in pancreatic islet β-cell (i.e., insulin), adrenal, and thyroid hormones. Other nonendocrine autoimmune diseases (e.g., pernicious anemia, myasthenia gravis) also are associated with autoimmune thyroid disease. Like patients with hyperthyroidism, who may be threatened by thyroid storm, those with hypothyroidism have their own severe, life-threatening variant, in this case called myxedema coma. This malady is quite rare and occurs most commonly in elderly patients with established hypothyroidism. Hypothermia and coma evolve slowly in these patients, and the usual causes are failure to take prescribed thyroid hormone replacement drugs, cold exposure, sepsis, heart failure, and alcohol abuse.

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SECTION VIII  •  The Endocrine System

regard to its effect on growth; other signs and symptoms of an overactive thyroid predominate. Cellular explanations of the effects of thyroid hormones on human development are incomplete. In rats, thyroid hormone induces the secretion of pituitary growth hormone (GH); thus, the growth retardation in thyroid-deficient rats may be partly the result of decreased GH secretion. However, in humans, who have no thyroid hormone response element in the promoter region of the GH gene, plasma [GH] is normal in hypothyroidism. Thus, the growth failure of hypothyroid human infants is not as readily explained. In humans, changes in the growth of long bones are more or less characteristic of thyroid hormone deficiency. These changes include a delay in formation of centers of ossification at the growth plate, followed by the appearance of several ossification centers, which eventually merge. Short stature in human juvenile or infantile hypothyroidism may be in part related to these abnormalities of cartilage growth and development as well as to resistance to the normal action of GH to promote growth.

9 Thyroxine treatment started

8

Normal

7 Bone age

6 5

Thyroid-deficient child

Developmental age 4

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3 2

ge

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1 0 0

1

2

3 4 5 6 Chronological age

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9

Figure 49-7  Effect of thyroid hormone on growth and development. The graph shows developmental age—that is, the age that the child appears to be based on height, bone radiography, and mental function—versus chronological age. For a normal child, the relationship is the straight line (red), for which developmental and chronological age are identical. The three green curves are growth curves for a child with thyroid hormone deficiency. Notice that at age 4.5 years, before initiation of therapy, height age, bone age, and mental age are all substantially below normal. Initiating replacement therapy with thyroid hormone at age 4.5 causes a rapid increase in both height age and bone age (“catch-up”) but has no effect on mental age, which remains infantile. Treatment can help mental development only if the therapy is begun within a few days of birth. (Data from Wilkins L: The Diagnosis and Treatment of Endocrine Disorders of Childhood and Adolescence. Springfield, IL, Charles C Thomas, 1965.)

Typically overshadowed by the impaired cognitive development that occurs in cretinism is the dwarfism that results from the effects of thyroid hormone deficiency on human growth (Fig. 49-7). In children with normal thyroid function at birth, development of hypothyroidism at any time before the fusion of the epiphyses of the long bones leads to growth retardation or arrest. Much of the loss in height that occurs can be recovered after thyroid hormone treatment is begun, a phenomenon called catch-up growth. If the diagnosis and treatment of hypothyroidism are delayed, loss of potential growth may occur, as indicated in Figure 49-7. However, as noted above, mental development does not catch up unless the treatment is begun within 7 to 14 days of birth. In general, the longer the duration of congenital hypothyroidism, the more profound is the mental retardation. In rodents, thyroid hormone regulates the induction of expression of several neural proteins, including myelin basic protein (MBP; see Table 11-4). How deficiencies in these proteins result in the generalized cortical atrophy seen in human infantile hypothyroidism is not clear. The growth curve (i.e., a plot of the child’s height and weight versus age) can provide a particularly sensitive early indicator of hypothyroidism in children who develop hypothyroidism after the neonatal period. An overactive thyroid is much less a problem than is an underactive thyroid with

HYPOTHALAMIC-PITUITARY-THYROID AXIS The pituitary regulates the synthesis and secretion of thyroid hormones through the release of thyrotropin—also known as thyroid-stimulating hormone (TSH)—from the anterior pituitary. The hypothalamus, in turn, stimulates the release of TSH through thyrotropin-releasing hormone (TRH). Finally, circulating thyroid hormones exert feedback control on both TRH and TSH secretion.

TRH from the hypothalamus stimulates thyrotrophs of the anterior pituitary to secrete TSH, which stimulates T4/T3 synthesis Thyrotropin-Releasing Hormone  TRH is a tripeptide pyroGlu-His-Pro containing the modified amino acid pyro-Glu. It is found in many tissues, including the cerebral cortex, multiple areas of the GI tract, and the β cells of the pancreas. However, the arcuate nucleus and the median eminence of the hypothalamus appear to be the major sources of the TRH that stimulates TSH synthesis and secretion (Fig. 49-8). TRH released by neurons in the hypothalamus travels to the anterior pituitary through the hypophyseal portal system (see p. 978). Hypothalamic lesions that interrupt TRH release or delivery cause a fall in basal TSH levels. Conversely, administering TRH intravenously can cause a rapid, dosedependent release of TSH from the anterior pituitary. However, it is not clear that such bursts of TRH release and TSH secretion occur physiologically. TRH Receptor  Once it reaches the thyrotrophs in the anterior pituitary, TRH binds to the TRH receptor, a G protein– coupled receptor on the cell membranes of the thyrotrophs. TRH binding triggers the phospholipase C pathway (see p. 58). The formation of diacylglycerols (DAGs) stimulates protein kinase C and leads to protein phosphorylation. The simultaneous release of inositol trisphosphate (IP3) triggers Ca2+ release from internal stores, raising [Ca2+]i. The result is an increase in both the synthesis and release of TSH, which is stored in secretory granules. TRH produces some of its

Small-bodied neurons in the arcuate nucleus and median eminence synthesize and secrete thyrotropinreleasing hormone (TRH).

Periventricular region

Somatostatin

Arcuate nucleus TRH

T3, T4

Median eminence

Hypothalamus

Dopamine

TRH

Long portal vessels carry TRH to the anterior pituitary.

Anterior lobe of the pituitary

TRH receptor

TRH

γ

TSH

β

G protein

α

IP3

[Ca2+]i

Thyrotrophs

DAG

PLC

PKC

Ca2+ stores 2+

Ca

Thyrotroph

TSH

MCT8 (SLC16A2)

T3, T4

T4

T3 AC

TSH receptor

cAMP

Follicular cell cytosol

THYROID

Gαs

TSH

Colloid

Figure 49-8  Hypothalamic-pituitary-thyroid axis. Small-bodied neurons in the arcuate nucleus and median eminence of the hypothalamus secrete TRH, a tripeptide that reaches the thyrotrophs in the anterior pituitary via the long portal veins. TRH binds to a G protein–coupled receptor on the thyrotroph membrane, triggering the DAG/IP3 pathway; stimulation of this pathway leads to protein phosphorylation and a rise in [Ca2+]i. These pathways stimulate the thyrotrophs to synthesize and release TSH, which is a 28-kDa glycoprotein stored in secretory granules. The TSH binds to receptors on the basolateral membrane of thyroid follicular cells, stimulating Gαs, which in turn activates adenylyl cyclase and raises [cAMP]i. As outlined in Figure 49-3, TSH stimulates a number of steps in the synthesis and release of T4 and T3. Inside the pituitary, the type 2 form of 5′/3′-monodeiodinase converts T4 to T3, which negatively feeds back on the thyrotrophs as well as on the TRH-secreting neurons. Somatostatin and dopamine—released by hypothalamic neurons—inhibit TSH release and thus can influence the set-point at which TSH is released in response to a given amount of T3 in the pituitary. AC, adenylyl cyclase; MCT8, monocarboxylate transporter 8; PKC, protein kinase C; PLC, phospholipase C.

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SECTION VIII  •  The Endocrine System

effects by activating phospholipase A2, a process leading to the release of arachidonic acid and the formation of a variety of biologically active eicosanoids (see pp. 62–64). In healthy humans, administering TRH also raises plasma [prolactin] by stimulating lactotrophs in the anterior pituitary (see p. 1150). However, no evidence indicates a regulatory role for TRH in prolactin secretion or milk production. Thyrotropin  The thyrotrophs represent a relatively small number of cells in the anterior pituitary. The TSH that they release is a 28-kDa glycoprotein with α and β chains. The α chain of TSH is identical to that of the other glycoprotein hormones: the gonadotropins luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG). The β chain is unique to TSH and confers the specificity of the hormone. Once secreted, TSH acts on the thyroid follicular cell via a specific receptor. TSH Receptor  The TSH receptor on the thyroid follicular cells is a G protein–coupled receptor. Like receptors for the other glycoprotein hormones (LH, FSH, and hCG), the TSH receptor, via Gαs, activates adenylyl cyclase (see p. 53). The rise in [cAMP]i stimulates a diverse range of physiological processes or events, summarized in Figure 49-3: 1. Iodide uptake by NIS on the basolateral membrane of the thyroid follicular cell. Stimulation of this cotransporter allows for trapping of dietary iodine within the thyroid gland (including follicular cells and colloid). The ratio of thyroid to serum iodine (the so-called thyroid/serum or T/S ratio) is 30 : 1 in euthyroid individuals. The T/S ratio decreases under conditions of low TSH (e.g., hypophysectomy), and increases under conditions of high TSH (e.g., a TSH-secreting pituitary adenoma). 2. Iodination of thyroglobulin in the follicular lumen. 3. Conjugation of iodinated tyrosines to form T4 and T3 within the thyroglobulin molecule. 4. Endocytosis of iodinated thyroglobulin into the follicular cells from thyroid colloid. 5. Proteolysis of the iodinated thyroglobulin in the follicular cell. 6. Secretion of T4 and T3 into the circulation. 7. Hyperplasia of the thyroid gland because of the growthpromoting effects of TSH.

Figure 49-9 illustrates the goiter that occurs when TSH concentrations are elevated for a prolonged period and stimulate an otherwise normal thyroid gland (see Box 49-1). Hyperplasia of the thyroid gland also occurs in Graves disease (Box 49-4) because of stimulation of the TSH receptor by a thyroid-stimulating immunoglobulin (see p. 1017). In contrast, the chronic elevation of TSH typically seen when the thyroid gland undergoes autoimmune destruction of follicular cells (Hashimoto thyroiditis) does not lead to follicular hypertrophy, but the gland may increase modestly in size from infiltration by immune cells.

T3 exerts negative feedback on TSH secretion Circulating free T4 and T3 inhibit both the synthesis of TRH by hypothalamic neurons and the release of TSH by the thyrotrophs in the anterior pituitary. Plasma [TSH] is very sensitive to alteration in the levels of free T4 and T3; a 50%

Figure 49-9  Goiter in iodine deficiency. A young woman from a region in Central Africa where iodine deficiency is prevalent exhibits a large goiter secondary to iodine deficiency and the growth-promoting effects of TSH, the levels of which are part of a feedback mechanism for achieving a sufficient amount of thyroid hormone.

decline in free T4 levels can cause plasma [TSH] to increase 50- to 100-fold. Conversely, as may be expected of a wellfunctioning feedback system, an excess of thyroid hormone leads to a decrease in plasma [TSH]. At the level of the thyrotroph, the sensor in this feedback system monitors the concentration of T3 inside the thyrotroph (see Fig. 49-8). As noted above, either T3 can enter directly from the blood plasma, or T3 can form inside the thyrotroph by deiodination of T4. The negative feedback of T4 and T3 on TSH release occurs at the level of the pituitary thyrotroph by both indirect and direct mechanisms. In the indirect feedback pathway, intracellular T3 decreases the number of TRH receptors on the surface of the thyrotroph. As a result, thyroid hormones indirectly inhibit TSH release by reducing the sensitivity of the thyrotrophs to TRH. In the direct feedback pathway, intracellular T3 inhibits the synthesis of both the α and the β chains of TSH. Indeed, both the α and β TSH genes have T3 response elements in their promoter regions. These response elements, which are inhibitory, differ from those found in genes that are positively regulated by T3 (e.g., Na-K pump). Free T4 and T3 concentrations in the plasma, which determine intracellular T3 levels in the thyrotroph, are relatively constant over the course of 24 hours, a finding reflecting the long half-lives of both T4 and T3 (see p. 1009). Given that the levels of T4 and T3 are the primary triggers in the afferent limb of the negative feedback for the hypothalamic-pituitarythyroid axis, the feedback regulation of TSH secretion by

Chapter 49  •  The Thyroid Gland

BOX 49-4  Graves Disease

S

urprisingly, it is not uncommon for B lymphocytes to synthesize immunoglobulins that bind to and activate the TSH receptor, thereby reproducing all the actions of TSH on the thyroid. Unfortunately, these errant lymphocytes do not regulate the production of these immunoglobulins in a manner analogous to the regulation of TSH secretion by the pituitary. As a result, iodine trapping by the thyroid increases, the synthesis and secretion of both T3 and T4 increase, and the thyroid enlarges to produce a goiter. Untreated, the affected individual becomes increasingly hyperthyroid.   N49-4 The clinical manifestations of hyperthyroidism include an increased metabolic rate with associated weight loss, sweating and heat intolerance, a rapid and more forceful heartbeat, muscle weakness and wasting, tremulousness, difficulty concentrating, and changes in hair growth and skin texture. Because TSH stimulates all areas of the thyroid, the thyroid is symmetrically enlarged, and even the isthmus is frequently palpable and visible on clinical examination. The abnormal immunoglobulin is designated thyroidstimulating immunoglobulin (TSI). The constellation of symptoms noted previously, together with a symmetrical goiter, is called Graves disease after Robert Graves, who provided one of the first detailed descriptions of the disorder in the early 19th century. In some patients these antibodies are also able to stimulate connective tissue in the extraocular muscles and in the dermis of the lower extremity to synthesize mucopolysaccharides, which leads to thickening of both the muscle and the dermis. Therefore, in addition to the abnormalities of thyroid growth and hyperfunction, a minority of individuals with Graves disease develop a peculiar infiltrative abnormality in the extraocular muscles. When severe, this infiltrative ophthalmopathy impairs muscle function and causes diplopia (double vision) and a forward protrusion of the eyes (exophthalmos). Even less frequently, patients with Graves disease develop infiltrating dermopathy in the skin over the lower legs called pretibial myxedema. This thickening of the skin occurs in localized patches and is pathologically distinct from the generalized thickening and coarsening of the skin seen in hypothyroidism (generalized myxedema).

thyroid hormones appears to be a slow process—essentially integrating thyroid hormone levels over time. Indeed, T3 feeds back on the thyrotroph by modulating gene transcription, which by its very nature is a slow process. The feedback of T4 and T3 on the release of TSH may also be under the control of somatostatin and dopamine, which travel from the hypothalamus to the thyrotrophs through the portal vessels (see Fig. 49-8). Somatostatin and dopamine both inhibit TSH secretion, apparently by making the thyrotroph more sensitive to inhibition by intracellular T3—that is, shifting the set-point for T3. Thus, somatostatin and dopamine appear to counterbalance the stimulatory effect of TRH. Although these inhibitory effects are readily demonstrated with pharmacological infusion of these agents, their physiological role in the regulation of TSH secretion appears small. In particular, with long-term administration of somatostatin or dopamine, compensatory mechanisms appear to override any inhibition. A special example of feedback between T3 and TSH is seen in neonates of mothers with abnormal levels of T3. If the mother is hyperthyroid, both she and the fetus will have low TSH levels because T3 crosses the placenta. After

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BOX 49-5  Clinical Assessment of Thyroid Function Plasma TSH Levels Direct measurements of T4/T3 provide a measure of total circulating hormone (i.e., the sum of free T4 and T3, as well as T4 and T3 bound to TBG, TTR, and albumin; see pp. 1008– 1009). However, these direct measurements do not allow one to distinguish between bound and free T4/T3. The sensitive response of TSH to changes in thyroid hormone levels provides an extremely valuable tool for assessing whether the free T4/T3 levels in the circulation are deficient, sufficient, or excessive. Indeed, the level of TSH reflects the amount of free, biologically active thyroid hormone in the target tissue. As a result, in recent years, measurements of plasma TSH, using very sensitive immunoassay methods, have come to be regarded as the single best measure of thyroid hormone status. Obviously, this approach is valid only if the thyrotrophs themselves are able to respond to T3/T4—that is, if patients show no evidence of pituitary dysfunction. The health of the thyrotrophs themselves can be tested by injecting a bolus of synthetic TRH and monitoring changes in plasma [TSH]. In hypothyroid patients, the subsequent rise in plasma [TSH] is more dramatic than in physiologically normal individuals. This test was of great value in confirming the diagnosis of hypothyroidism before the advent of today’s ultrasensitive assays, but it has largely been abandoned.

Radioactive Iodine Uptake Determining the amount of a standard bolus of radioactive iodine—123I (half-life, 13 hours) or 131I (half-life, 8 days)—that the thyroid can take up was also once widespread as a measure of thyroid function. A hyperactive gland will take up increased amounts of the tracer, whereas an underactive gland will take up subnormal amounts. Today, the test is used mostly for three other purposes. First, radioactive iodine uptake can show whether a solitary thyroid nodule, detected on physical examination, is “hot” (functioning) or “cold” (nonfunctioning). Cold nodules are more likely than hot ones to harbor a malignancy. Second, radioactive iodine uptake can show whether hyperthyroidism is the result of thyroid inflammation (i.e., thyroiditis), in which tracer uptake is minimal because of TSH suppression, or Graves disease, in which tracer uptake is increased because thyroid-stimulating immunoglobulin (see p. 1017) mimics TSH. Third, higher doses of radioactive iodine are commonly used to treat patients with hyperthyroidism. In this circumstance, the use of 131I causes radiolytic destruction of the overactive thyroid tissue. In the setting of thyroid cancer, therapists give very high doses of 131 I to deliver sufficient radiation to tumors that retain only a little of the iodine-concentrating ability of the normal thyroid.

birth, the newborn rapidly metabolizes its T3, but its TSH remains suppressed, so that the infant temporarily becomes hypothyroid. Conversely, if the mother’s thyroid gland has been removed and she is hypothyroid because she is not receiving sufficient thyroid hormone replacement therapy, both she and the fetus will have high levels of circulating TSH. Immediately after birth, the newborn will be temporarily hyperthyroid (Box 49-5).

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 49  •  The Thyroid Gland 1017.e1

N49-4  Thyroid Storm Contributed by Eugene Barrett Some patients with hyperthyroidism become extremely ill and are said to be in thyroid storm. These individuals usually have a severe illness superimposed on their hyperthyroidism, and they develop high fevers, a profound tachycardia, sweating, and restlessness. Altered mental status is common. If untreated, these patients can develop severe circulatory collapse resulting in death. Thyroid storm can be the initial presentation of hyperthyroidism or it can occur in patients already known to be hyperthyroid and treated appropriately. However, when these latter individuals experience the severe stress of a major operation, trauma, or illness, they can develop thyroid storm. This condition is a true emergency. Treatment consists of giving sodium iodide, which over the long term encourages thyroid hormone synthesis but in the short term blocks the release of already-synthesized thyroid hormones; a β blocker to inhibit the β adrenoceptors, whose expression is increased by the elevated levels of thyroid hormones in the blood; and a drug such as propylthiouracil (PTU), which blocks the manufacture of additional thyroid hormone by inhibiting the iodination and conjugation steps. Fluid replacement and stress doses of corticosteroids are also given to support the circulation.

1017.e2 SECTION VIII  •  The Endocrine System

REFERENCES Books and Reviews Alper SL, Sharma AK: The SLC26 gene family of anion transporters and channels. Mol Aspects Med 34:494–515, 2013. Bassett JH, Harvey CB, Williams GR: Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Mol Cell Endocrinol 213:1–11, 2003. Bates JM, St Germain DL, Galton VA: Expression profiles of the three iodothyronine deiodinases, D1, D2, and D3, in the developing rat. Endocrinology 140(2):844–851, 1999. Brent GA, Moore DD, Larsen PR: Thyroid hormone regulation of gene expression. Annu Rev Physiol 53:17–35, 1991. Cavalieri RR: Iodine metabolism and thyroid physiology: Current concepts. Thyroid 7:177–181, 1997. Dumont JE, Lamy F, Roger P, Maenhaut C: Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol Rev 72:667–697, 1992. Gershengorn MC, Osman R: Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev 76:175– 191, 1996. Larsen PR: Update on the human iodothyronine selenodeiodinases, the enzymes regulating the activation and inactivation of thyroid hormone. Biochem Soc Trans 25:588–592, 1997. Orban Z, Bornstein SR, Chrousos GP: The interaction between leptin and the hypothalamic-pituitary-thyroid axis. Horm Metab Res 30:231–235, 1998. Samuels HH, Forman BM, Horowitz ZD, Ye Z-S: Regulation of gene expression by thyroid hormone. Annu Rev Physiol 51:623–639, 1989.

Wright EM: Glucose transport families SLC5 and SLC50. Mol Aspects Med 34:183–196, 2013. Journal Articles Arvan P, Kim PS, Kuliawat R, et al: Intracellular protein transport to the thyrocyte plasma membrane: Potential implications for thyroid physiology. Thyroid 7:89–105, 1997. Dai G, Levy O, Carrasco N: Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460, 1996. Di Cosmo C, Liao X-H, Dumitrescu AM, et al: Mice deficient in MCT8 reveal a mechanism regulating thyroid hormone secretion. J Clin Invest 120:3377–3388, 2010. Friesema ECH, Ganguly S, Abdalla A, et al: Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J Biol Chem 278:40128–40135, 2003. Friesema EC, Grueters A, Biebermann H, et al: Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation. Lancet 364:1435–1437, 2004. Koenig RJ: Thyroid hormone receptor coactivators and corepressors. Thyroid 8:703–713, 1998. Lazar MA: Thyroid hormone action: A binding contract. J Clin Invest 112(4):497–499, 2003. Ohye H, Sugawara M: Dual oxidase, hydrogen peroxide and thyroid diseases. Exp Biol Med 235:424–433, 2010. Paroder-Belenitsky M, Maestas MJ, Dohán O, et al: Mechanism of anion selectivity and stoichiometry of the Na+/I− symporter (NIS). Proc Natl Acad Sci U S A 108:17933–17938, 2011.

C H A P T E R 50  THE ADRENAL GLAND Eugene J. Barrett

The human adrenal glands, each weighing only ~4 g, are located above the upper pole of each kidney in the ret­ roperitoneal space. They produce four principal hormones: cortisol, aldosterone, epinephrine (adrenaline), and nor­ epinephrine. Each adrenal gland is composed of an inner medulla and an outer cortex (Fig. 50-1). Embryologically, the cortex is derived from mesoderm, whereas the medulla is derived from neural crest cells (see p. 261) that migrate into the developing cortex. The cortex produces two principal steroid hormones, cortisol and aldosterone, as well as several androgenic steroids. The medulla produces epinephrine and norepinephrine. The adrenal cortex can be further divided into three cellular layers: the glomerulosa layer near the surface, the fasciculata layer in the midcortex, and the reticularis layer near the cortical-medullary junction. Aldosterone, the main mineralocorticoid in humans, is made in the glo­ merulosa cell layer. Cortisol, the principal glucocorticoid, is made in the fasciculata and to a small extent in the reticu­ laris layer. The adrenal androgens—dehydroepiandrosterone (DHEA) and its sulfated form DHEAS—are made in the reticularis layer. Although both cortisol and aldosterone are made by enzymatic modification of cholesterol and are structurally quite similar, their actions on the body differ dramatically. Cortisol is considered a glucocorticoid because it was recognized early on to increase plasma glucose levels; deficiency of cortisol can result in hypoglycemia. Aldosterone is considered a mineralocorticoid because it promotes salt and water retention by the kidney. The activities of these two hormones overlap, particularly at high hormone levels, but this distinction is still very useful in identifying their most obvious functions. DHEA and DHEAS are weak androgens (compared to testosterone or dihydrotestosterone) and little is known about the regula­ tion of their secretion. Plasma DHEA concentrations follow a diurnal pattern like that of cortisol. DHEAS circu­ lates at much higher concentrations and shows no diurnal fluctuation. In the adrenal medulla, chromaffin cells produce epinephrine (or adrenaline), a catecholamine that is synthe­ sized from the amino acid tyrosine. Although the primary product of the medulla is epinephrine, it also produces vari­ able amounts of the epinephrine precursor norepinephrine. These catecholamines are distinct from the steroid hormones both structurally and functionally. 1018

THE ADRENAL CORTEX: CORTISOL Cortisol is the primary glucocorticoid hormone in humans Steroid hormones are divided into three major classes based on their actions: glucocorticoids, mineralocorticoids, and sex steroids. Cortisol is the prototypical naturally occurring glucocorticoid. The ability of cortisol to increase plasma [glucose] largely results from its ability to enhance mobilization of amino acids from proteins in many tissues and to enhance the ability of the liver to convert these amino acids into glucose and glycogen by activating gluconeogenesis. The structures of cortisol and aldosterone (Fig. 50-2) differ only slightly: aldosterone lacks the –OH group at posi­ tion 17 and has an aldehyde (aldo) group at position 18. Despite the seemingly minor chemical difference, aldoste­ rone at physiological concentrations has virtually no gluco­ corticoid activity. Target Tissues  Although classified as a glucocorticoid, cortisol affects more than the principal glucose-regulatory tissues, namely, the liver, fat, and muscle. Most body tissues, including bone, skin, other viscera, hematopoietic and lym­ phoid tissue, and the central nervous system (CNS), are target sites for glucocorticoid action. Although cortisol is the primary glucocorticoid in humans, in other species (e.g., the rat), corticosterone is the major glucocorticoid. Actions  Glucocorticoids have numerous actions other than their ability to raise plasma glucose levels. These actions are described below and include potent immunosuppressive and anti-inflammatory activity, effects on protein and fat metabolism, behavioral effects due to actions on the CNS, and important effects on calcium and bone metabolism. Some of the diverse physiological effects of the glucocorti­ coids can be appreciated from clinical studies of excess glu­ cocorticoid secretion (Cushing syndrome; Box 50-1) and glucocorticoid deficiency (Addison disease; see Box 50-1). The multiple actions of glucocorticoids, in particular, their “anti-inflammatory” action on leukocytes, has led to the development of numerous synthetic analogs that are more potent, have a longer half-life, and are more selective in their specific glucocorticoid actions than are either cortisol or corticosterone. Table 50-1 lists some of these compounds

Chapter 50  •  The Adrenal Gland

Adrenal gland

BOX 50-1  Cushing Syndrome and Addison Disease

Capsule Zona glomerulosa Zona fasciculata

Cortex

Zona reticularis Capsular artery

Medulla

HORMONES

Capsule

Mineralocorticoid (aldosterone)

Zona glomerulosa

Glucocorticoids (e.g., cortisol)

Zona fasciculata

Androgens (DHEA and androstenedione)

Zona reticularis

Medulla

Medullary vein

1019

Epinephrine Preganglionic sympathetic terminal

Figure 50-1  Anatomy of the adrenal gland. An adrenal gland—actually

two glands, cortex and medulla—sits upon each kidney. The adrenal cortex comprises three layers that surround the medulla: glomerulosa, fasciculata, and reticularis. The blood supply enters the cortex in the subcapsular region and flows through anastomotic capillary beds while coursing through first the cortex and then the medulla. The adrenal medulla contains chromaffin cells.

and indicates their relative potency as mineralocorticoids and glucocorticoids. Most of the well-characterized actions of glucocorticoids result from their genomic actions to influence (either posi­ tively or negatively) the transcription of a variety of genes through glucocorticoid response elements (see p. 986). However, glucocorticoids also exert nongenomic actions (see p. 989) that occur promptly (0 to 3 hours) and are not inhibited by blockade of gene transcription.

The adrenal zona fasciculata converts cholesterol to cortisol Synthesis of cortisol, as for all steroid hormones, starts with cholesterol (see Fig. 50-2). Like other cells producing steroid hormones, the adrenal gland has two sources of cholesterol (see p. 985): (1) it can import cholesterol from circulating cholesterol-containing low-density lipoprotein (LDL) cho­ lesterol by means of LDL receptor–mediated endocytosis

G

lucocorticoid excess is most commonly seen clinically in individuals receiving glucocorticoids for treatment of a chronic inflammatory or neoplastic disorder. Less commonly, individuals overproduce cortisol either because of a primary cortisol-producing adrenal tumor or secondary to a pituitary tumor that produces ACTH, which in turn stimulates excess cortisol production by normal adrenal glands. In either case, the cortisol excess causes a constellation of symptoms and signs including adiposity of the trunk, neck, and facies; hypertension; loss of subcutaneous adipose and connective tissue in the extremities with associated easy bruising; loss of bone mineral; muscle weakness and wasting; and hyperglycemia. This constellation is referred to as Cushing syndrome after the famous American neurosurgeon who characterized this disorder. The specific therapy is based upon identifying whether the clinical picture arises from a tumor in the adrenal or in the pituitary gland, and then removing the culprit. When the pituitary gland is responsible, the disorder is referred to as Cushing disease. In the case of patients receiving glucocorticoid therapy, the signs and symptoms of Cushing syndrome are carefully monitored, and efforts are made to minimize these side effects. Unfortunately, all glucocorticoid drugs with anti-inflammatory actions also produce these other effects. Glucocorticoid deficiency—which occurs in primary adrenal insufficiency, also called Addison disease, and affects both glucocorticoid and mineralocorticoid levels—can produce an array of symptoms and signs. Although tuberculosis was once a common cause of primary adrenal insufficiency, today autoimmune adrenal disease is the most common cause. Failure of adrenal cortical hormone secretion leads to increases in circulating concentrations of ACTH as well as other products of POMC (see p. 1023). Two of these products (αMSH and γ-MSH) as well as ACTH (see p. 1023) cause skin hyper­pigmentation. The lack of glucocorticoid predisposes to hypoglycemia. The combined absence of glucocorticoid and mineralocor­ticoid leads to hypotension and hyponatremia,  N50-1  whereas aldosterone deficiency leads to hyperkalemia. Before the development of glucocorticoid and mineralocorticoid therapy, this disorder was uniformly fatal.

TABLE 50-1  Relative Potency* of Glucocorticoid and Mineralocorticoid Analogs COMPOUND Cortisol

GLUCOCORTICOID MINERALOCORTICOID EFFECT EFFECT 1

1.5

Prednisone

3–4

0.5

Methylprednisone

10

0.5

Dexamethasone

20

1

Fludrocortisone

12

125

*Relative potency is determined by a combined consideration of the compound’s biological half-life and its affinity for the glucocorticoid or mineralocorticoid receptor.

Chapter 50  •  The Adrenal Gland 1019.e1

N50-1  Hyponatremia in Primary Adrenal Insufficiency Contributed by Emile Boulpaep and Walter Boron Whereas aldosterone deficiency causes renal salt wasting, hypovolemia and hypotension do not directly cause hyponatremia (i.e., a low plasma [Na+]). Rather, reduced effective circulating volume triggers the release of AVP (see pp. 846– 847); AVP in turn provokes thirst and H2O retention in the collecting ducts, which dilutes plasma Na+ and creates hyponatremia.

1020

SECTION VIII  •  The Endocrine System

Acetate Cholesterol (27-carbon compound)

10

A HO

C

19 9

1 2

5

4

21 20 22 24 25 26 17 23 16 27

12 18

11

B

13

D

15

14

8

21-CARBON CORTICOSTEROIDS

7

6

MITOCHONDRIA Side-chain cleavage enzyme

CH3

B

A

CH3

D

C

CH3

CH3 A

B

17 α-Hydroxyprogesterone CH3

C O

C O

A

B

HO

SER 17,20-Desmolase Dehydroepiandrosterone (DHEA) O CH3

C

CH3 A

3β-Hydroxysteroid Dehydrogenase

CH3

D

C

CH3

CH3

A

SER 17 α-Hydroxylase 11-Deoxycortisol CH2OH C

CH3

OH CH3

B

A

O

O

CH3

HO

O

D

C

CH3

C

B

A O

D

C

CH3

C

D

O

SER 17 α-Hydroxylase Cortisol

Glomerulosa cells only H

OH

C

CH3 A

B

D

CH3 A O

O OH

B

O

SER 11 β-Hydroxysteroid dehydrogenase

Androstenedione Cortisone CH3

B

A

CH2OH

D

C

CH3

O

C CH3

O C

CH3

B

O

A

D

O OH

B

O

Androstenediol CH3 C

CH3 A HO

Testosterone OH

CH3

D

C

CH3

B

A

O

HO

CH2OH C CH3

HO

SER 17,20-Desmolase

D

HO

B

CH2OH

11 β-Hydroxylase

17 α-Hydroxypregnenolone

21α-Hydroxylase

SER 17 α-Hydroxylase

OH

D

C

O

SER 17 α-Hydroxylase

CH3

O

Aldosterone Synthase

O

HO

CH2OH C

O

Corticosterone

MITOCHONDRIA

C

MITOCHONDRIA

SER

A

CH3

D

C

CH3

O

C

SER

CH3 CH3

11-Deoxycorticosterone

Progesterone

Pregnenolone

MINERALOCORTICOSTEROID

GLUCOCORTICOSTEROIDS

OH

D

B

O

Figure 50-2  Biosynthesis of adrenal steroids. This schematic summarizes the synthesis of the adrenal steroids—the mineralocorticoid aldosterone and the glucocorticoid cortisol—from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; they are located in either the SER or the mitochondria. The SCC enzyme that produces pregnenolone is also known as 20,22-desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. If the synthesis of cortisol is prevented by any one of several dysfunctional enzymes, other steroid products might be produced in excess. For example, a block in 21α-hydroxylase will diminish production of both cortisol and aldosterone and increase production of the sex steroids. Certain of these pathways are shared in the biosynthesis of the androgens (see Fig. 54-6) as well as the estrogens (see Fig. 55-8).

C C

CH2OH C

O

D

B

Aldosterone

Glomerulosa cells only

Chapter 50  •  The Adrenal Gland

TABLE 50-2  Cytochrome P-450 Enzymes Involved in Steroidogenesis* ENZYME

SYNONYM

GENE

Cholesterol side-chain cleavage

P-450SCC

CYP11A1

11β-hydroxylase

P-450c11

CYP11B1

17α-hydroxylase

P-450c17

CYP17

17,20-desmolase

P-450c17

CYP17

21α-hydroxylase

P-450c21

CYP21A2

Aldosterone synthase

P-450aldo

CYP11B2

Aromatase*

P-450arom

CYP19

*P-450arom catalyzes a reaction essential for the production of estrogens (see p. 1117).

(see p. 42), or (2) it can synthesize cholesterol de novo from acetate (see Fig. 46-16). Although both pathways provide the steroid nucleus needed for cortisol and aldosterone synthe­ sis, circulating LDL is quantitatively more important. In the adrenal gland, cholesterol is metabolized through a series of five reactions to make either cortisol or aldo­ sterone. All relevant enzymes are located in either the mitochondria or smooth endoplasmic reticulum (SER), and except for 3β-hydroxysteroid dehydrogenase (3β-HSD), belong to the family of cytochrome P-450 oxidases (Table 50-2). 1. The pathway for cortisol and aldosterone synthesis begins in the mitochondria, where the cytochrome P-450 side-chain-cleavage (SCC) enzyme (also called 20,22desmolase or P-450SCC) removes the long side chain (carbons 22 to 27) from the carbon at position 20 of the cholesterol molecule (27 carbon atoms). This enzyme, or the supply of substrate to it, appears to be the ratelimiting step for the overall process of steroid hormone synthesis. 2. The product of the SCC-catalyzed reaction is pregnenolone (21 carbon atoms), which exits the mitochondrion. The SER enzyme 3β-HSD (not a P-450 enzyme) oxidizes the hydroxyl group at position 3 of the A ring to a ketone to form progesterone. 3. A P-450 enzyme in the SER, 17α-hydroxylase (P-450c17), then adds a hydroxyl group at position 17 to form 17αhydroxyprogesterone. However, as shown in Figure 50-2, an alternative path to 17α-hydroxyprogesterone exists: the 17α-hydroxylase might first add a hydroxyl group at position 17 of pregnenolone and form 17αhydroxypregnenolone, which the aforementioned 3βHSD can then convert to 17α-hydroxyprogesterone. 4. In the SER, 21α-hydroxylase (P-450c21) adds a hydroxyl at carbon 21 to produce 11-deoxycortisol. 5. In the mitochondria, 11β-hydroxylase (P-450c11) adds yet another hydroxyl, this time at position 11, to produce cortisol. The enzymes represented by the vertical bars in Figure 50-2, as well as SCC, are present in all three cellular layers of the adrenal cortex. However, 17α-hydroxylase is not substantially present in the glomerulosa layer. Thus, the

1021

fasciculata and, to a much lesser extent, the reticularis layers synthesize cortisol. The cells of the reticularis layers are principally respon­ sible for androgen synthesis. These cells convert 17αhydroxypregnenolone and 17α-hydroxyprogesterone into the adrenal androgens dehydroepiandrosterone and androstenedione. The enzyme that catalyzes this reaction is called 17,20-desmolase; however, it turns out to be the same SER enzyme as the 17α-hydroxylase that produced the 17αhydroxypregnenolone and 17α-hydroxyprogesterone in the first place. The androgens formed by the adrenal are far less potent than either testosterone or dihydrotestosterone. However, other tissues (e.g., liver, kidney, adipose) can use 17β-hydroxysteroid dehydrogenase to convert androstenedi­ one to testosterone (see p. 1097). In this manner, the adrenal can contribute significant amounts of circulating androgen, particularly in women. Increases in adrenal androgen pro­ duction precede by 1 to 2 years the increases in gonadal steroid production that occur with puberty. This androgen production promotes growth of pubic and axillary hair and is referred to as adrenarche (see pp. 1088–1090). The cortisol synthesized by the adrenal cortex diffuses out of the cells and into the blood plasma. There, ~90% of the cortisol is transported bound to corticosteroid-binding globulin (CBG), also known as transcortin, which is made in the liver. Transcortin is a 383–amino-acid glycoprotein whose affinity for cortisol is ~30-fold higher than that for aldosterone. An additional ~7% of the circulating cortisol is bound to albumin. Thus, only 3% to 4% of the circulating cortisol is free. The clearance of cortisol from the body depends princi­ pally on the liver and kidney. An early step is the formation of an inactive metabolite, cortisone, by the action of either of two 11β-hydroxysteroid dehydrogenases (11β-HSDs). 11β-HSD1 is highly expressed in certain glucocorticoid target tissues, including liver and both subcutaneous and visceral adipose tissue. The enzymatic reaction is reversible. Indeed, when glucocorticoids were first developed as phar­ maceutical agents, it was cortisone that was used to treat patients with a variety of inflammatory disorders (e.g., rheu­ matoid arthritis). For some time, investigators thought that cortisone was the active principle. Only later did it become apparent that the body must convert cortisone to cortisol, which is the biologically active agent. Because excess cortisol produces insulin resistance and many features of metabolic syndrome (e.g., glucose intolerance, hypertension, dyslipid­ emia; see Box 51-5)—and 11β-HSD1 is expressed abun­ dantly in adipose tissue—an interesting hypothesis is that increased 11β-HSD1 activity in adipose tissue locally pro­ duces cortisol and thus promotes the development of insulin resistance. The second 11β-HSD isozyme (11β-HSD2) is expressed in the adrenal cortex (see Fig. 50-2; Box 50-2), although the adrenal gland does not normally make a significant contri­ bution to the formation of cortisone. 11β-HSD2 is highly expressed in the renal distal tubule and collecting duct (see Fig. 35-13C), where it catalyzes an essentially irreversible conversion of cortisol to cortisone. This breakdown of cor­ tisol allows aldosterone to regulate the relatively nonspecific mineralocorticoid receptor (MR) without interference from cortisol.

1022

SECTION VIII  •  The Endocrine System

BOX 50-2  21α-Hydroxylase Deficiency

M

utations can affect one or more of the enzyme steps in steroid hormone synthesis and can produce unique clinical syndromes that are a direct result of failure to manufacture a particular hormone, accumulation of excessive amounts of precursor steroids, or both. The most common of these enzymatic disorders is 21α-hydroxylase deficiency. From Figure 50-2 we would predict that deficiency of this enzyme would lead to inadequate production of both glucocorticoid and mineralocorticoid hormones, which is indeed what occurs. Affected infants are ill with symptoms of “salt losing” (hypotension, dehydration) and glucocorticoid deficiency (hypoglycemia). The natural reaction of the body is to attempt to overcome this deficiency by increasing the secretion of ACTH, which stimulates the synthesis of cortisol and aldosterone. ACTH also causes growth of the adrenal gland. However, if the mutant enzyme is totally inactive, no cortisol or aldosterone synthesis will occur, although all other enzymes of the pathway involved in glucocorticoid and mineralocorticoid synthesis will be expressed in increased amounts. The result is greater than normal activity of SCC, 3β-HSD, 17α-hydroxylase, and 11β-hydroxylase, and the net effect is increased synthesis of both precursor molecules and adrenal androgens. The combination of inadequate production of glucocorticoids and mineralocorticoids, excessive production of androgens, and enhanced growth of the adrenal gland is the classical clinical syndrome of salt-losing, virilizing congenital adrenal hyperplasia. In female infants, the presence of excessive adrenal androgen in utero results in ambiguous genitalia at birth, a condition that should alert the pediatrician to the potential diagnosis. No such clue occurs in the male infant, in whom dehydration, hypotension, and hyperkalemia are the major manifestations.

Cortisol binds to a cytoplasmic receptor that translocates to the nucleus and modulates transcription in multiple tissues The multiple hydroxylation reactions that convert choles­ terol to cortisol result in a hydrophilic compound that, unlike cholesterol, is soluble in plasma, yet lipophilic enough to cross the plasma membrane of target tissue without requiring a membrane transporter. Cortisol, like all steroid hormones, binds to intracellular receptors within target cells (see pp. 71–72). Virtually all nucleated tissues in the body contain receptors for glucocorticoids. The glucocorticoid receptor (GR) is primarily located in the cytoplasm, where in its unbound form it is complexed to a chaperone protein (i.e., the heat shock protein hsp90, among others; see Fig. 4-15A). Binding of cortisol causes the chaperone to dissoci­ ate from the GR and this allows the cortisol-GR complex to translocate to the nucleus. There, the cortisol-receptor complex associates with glucocorticoid response elements (GREs) on the 5′ untranslated region of multiple genes to either enhance or diminish gene expression (see p. 90). GRs are structurally similar to the receptors for mineralo­ corticoids, sex steroids, vitamin D, vitamin A, and thyroid hormone. These receptors, either homodimers or heterodi­ mers, belong to the superfamily of nuclear receptors that contains domains A through F (see Fig. 3-14). Activity of the glucocorticoid-receptor complex requires dimerization of

BOX 50-3  Therapy with Glucocorticoids

T

he variety of glucocorticoid actions on body tissues is well illustrated by considering some of the clinically observed effects of hypercortisolism in patients receiving glucocorticoid drugs. Most strikingly, the entire body habitus changes. Body fat redistributes from the extremities to the face and trunk, producing (1) increased supraclavicular and dorsal interscapular fat (buffalo hump), (2) a rounded abdomen, and (3) a rounding of the face called moon facies, caused by increased subcutaneous fat in the cheeks and submandibular region. Conversely, the wasting of fat (and some supporting tissues) in the extremities produces thinning of the skin and fragility of cutaneous blood vessels. In bone, glucocorticoids reduce mineral density (osteopenia), which can lead to osteoporosis and bone fractures. The interference with normal immune function increases both the frequency and severity of infections. Rare malignancies can develop. Wasting of muscle tissue leads to a generalized weakness that is usually most prominent in the proximal muscles of the lower extremities. Finally, as would be expected from a glucocorticoid, patients become insulin resistant and even glucose intolerant (see p. 1038) or frankly diabetic (see Box 51-5). When cortisol is overproduced endogenously (from tumors producing either ACTH or cortisol), hypertension is common. The hypertension most likely results from the weak mineralocorticoid action of cortisol. Exogenous synthetic glucocorticoid therapy rarely produces hypertension because most of these drugs lack the mineralocorticoid activity of the endogenous hormone.

two identical receptor complexes (i.e., the GR functions as a homodimer) at the near-palindromic nucleotide site of the GRE on the chromatin. Glucocorticoids mainly act by mod­ ulating gene transcription. One exception is the acute feed­ back effect of cortisol to block the release of preformed adrenocorticotropic hormone (ACTH) in the secretory granules of pituitary corticotrophs. This glucocorticoid effect is demonstrable within seconds to minutes and may relate to an as-yet undefined effect of glucocorticoid on membrane trafficking. Although glucocorticoids are named for their ability to increase hepatic glucose and glycogen synthesis, they affect many somatic tissues. In liver, cortisol induces the synthesis of enzymes involved in gluconeogenesis and amino-acid metabolism in support of gluconeogenesis, thus enhancing hepatic glucose production. In muscle, cortisol stimulates the breakdown of muscle protein, which releases amino acids for uptake by the liver. Similarly, cortisol promotes lipolysis in adipose tissue. The fatty acids thus released provide an alternative fuel to glucose, whereas the accom­ panying glycerol provides another substrate for gluconeo­ genesis, thereby increasing the availability of glucose. For unknown reasons, although fat is mobilized from the extremities, some is also deposited centrally (see description of moon facies in Box 50-3). Cortisol has effects unrelated to its glucocorticoid actions that lead to its extensive clinical use in disorders like vascu­ litis, arthritis, malignancies, and in prevention of organ transplant rejection. Glucocorticoids also act on the cellular elements of trabecular bone (see pp. 1068–1069), decreasing the ability of osteoblasts to synthesize new bone. They also

Chapter 50  •  The Adrenal Gland

interfere with absorption of Ca2+ from the gastrointestinal tract. As a result, long-term glucocorticoid use causes osteo­ porosis. In addition, glucocorticoids act on the CNS and can cause a variety of effects, including alterations in mood and cognition.

Corticotropin-releasing hormone from the hypothalamus stimulates anterior pituitary corticotrophs to secrete ACTH, which stimulates the adrenal cortex to synthesize and secrete cortisol As summarized in Figure 50-3, regulation of the synthesis and secretion of cortisol begins with the release of corticotropin-releasing hormone (CRH) from hypotha­ lamic neurons as part of either a normal daily circadian rhythm or a centrally driven stress response. CRH stimulates the release of ACTH, also called corticotropin, from the anterior pituitary. ACTH directly stimulates the adrenal fas­ ciculata layers to synthesize and secrete cortisol. Circulating cortisol exerts negative-feedback control on the release of both ACTH and CRH. Corticotropin-Releasing Hormone  Small-bodied neurons in the paraventricular nucleus of the hypothalamus (see Fig. 47-3) secrete CRH, a 41–amino-acid neuropeptide. The structure of CRH is highly conserved among species. In humans, CRH is also present in several tissues, including the pancreas and testes, as well as throughout the CNS, where it serves as a neurotransmitter. The hypothalamic neurons syn­ thesize and release CRH via the classic secretory pathway (see pp. 34–35). Neurons store CRH in secretory vesicles located in synaptic terminals in the median eminence of the hypo­ thalamus and can release CRH acutely in the absence of new synthesis. After release into the interstitial fluid of the median eminence, CRH enters the hypophyseal portal venous plexus (see p. 978) and travels to the anterior pituitary. CRH Receptor  CRH arriving in the anterior pituitary binds to CRH-R1, a G protein–coupled receptor (GPCR) on the cell membrane of corticotroph cells. Hormone binding activates Gαs, which in turn stimulates adenylyl cyclase and raises [cAMP]i (see p. 53). Subsequent stimulation of protein kinase A (PKA) activates L-type Ca2+ channels, leading to an increase in [Ca2+]i, which stimulates the exocytosis of pre­ formed ACTH. Over a much longer time, CRH receptor activation also leads to increased gene transcription and syn­ thesis of the ACTH precursor (discussed later). Arginine Vasopressin  Although CRH is the major regula­ tor of ACTH secretion, the paraventricular nuclei also make another hormone, arginine vasopressin (AVP; see Fig. 40-8). AVP is also a potent ACTH secretagogue and probably plays a physiological role in the regulation of ACTH secretion during stresses like dehydration or trauma. Adrenocorticotropic Hormone  A 39–amino-acid peptide hormone secreted by the corticotroph cells of the anterior pituitary, ACTH can also be produced by ectopic sources, particularly by small-cell carcinomas of the lung. Pituitary corticotrophs synthesize ACTH by complex post-translational processing of a large precursor protein (i.e., a preprohor­

1023

mone) called pro-opiomelanocortin (POMC). POMC is the precursor not only for ACTH but also for a variety of peptide hormones (Fig. 50-4). In the anterior pituitary, POMC yields a long N-terminal peptide, a joining (J) peptide, ACTH, and β-lipotropin (β-LPH). During fetal life and pregnancy, the intermediate pituitary lobe—a small wedge of tissue between the more familiar anterior and pos­ terior lobes—processes the same POMC in a very different manner to yield a different array of peptides: a short N-terminal peptide, γ-melanocyte–stimulating hormone (γ-MSH), J peptide, α-MSH, corticotropin-like intermediatelobe peptide (CLIP), γ-LPH, and β endorphin. Other cells—such as the appetite-controlling POMC neurons in the hypothalamus (see p. 1002)—can also synthesize POMC. The melanocortins include ACTH as well as α-, β-, and γ-MSH and bind to a family of five GPCRs, the melanocortin receptors (MC1R to MC5R). α-MSH, γ-MSH, and ACTH act on MC1R receptors in melanocytes to increase the dispersion of pigment granules. In some patients who greatly overproduce ACTH, hyperpigmentation is a promi­ nent clinical finding. Whether this hyperpigmentation is the result of increased production of MSH, increased production of β-LPH (which also has MSH activity), or the melanotropic action of ACTH per se remains uncertain. β-LPH and γLPH mobilize lipids from adipocytes in animals, although their physiological role in humans is unclear. β endorphin has potent opioid actions in the CNS (see p. 315), but its physiological actions (if any) in the systemic circulation are not known. ACTH Receptor  In the adrenal cortex, ACTH binds to MC2R on the plasma membranes of all three steroidsecreting cell types. However, because only the cells in the fasciculata and reticularis layers have the 17α-hydroxylase needed for synthesizing cortisol (see Fig. 50-2), these cells are the only ones that secrete cortisol in response to ACTH. ACTH appears to have few other actions at physiological concentrations. MC2R is coupled to a heterotrimeric G protein and stimulates adenylyl cyclase (see p. 53). The resulting increase in [cAMP]i activates PKA, which phos­ phorylates a variety of proteins. A rapid effect of ACTH is to stimulate the rate-limiting step in cortisol formation; that is, the conversion of cholesterol to pregnenolone via the SCC enzyme. In addition, ACTH—over a longer time frame—increases the synthesis of several proteins needed for cortisol synthesis: (1) each of the P-450 enzymes involved in cortisol synthesis (see Fig. 50-2), (2) the LDL receptor required for the uptake of cholesterol from blood (see p. 42), and (3) the 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase that is the rate-limiting enzyme for cholesterol synthesis by the adrenal (see p. 968). Thus, ACTH promotes the acute synthesis of cortisol— and, as discussed later, aldosterone to a lesser extent—by the adrenal and increases the content of adrenal enzymes involved in steroidogenesis. In the absence of pituitary ACTH, the fasciculata and reticularis layers of the adrenal cortex atrophy. The glomerulosa layer does not atrophy under these conditions because in addition to ACTH, angio­ tensin II (ANG II) and high levels of K+ are trophic factors that act on the glomerulosa layer. The atrophy of the fascicu­ lata and reticularis layers occurs routinely in people treated

1024

SECTION VIII  •  The Endocrine System

CEREBRAL CORTEX STRESS

HYPOTHALAMUS

Diurnal rhythms

Physical

Emotional

Biochemical

Small-bodied neurons in the hypothalamus synthesize and secrete corticotropinreleasing hormone (CRH).

Paraventricular nucleus

Hypothalamus

Long portal vessels carry CRH to the anterior pituitary. CRH Short feedback (ACTH)

Anterior lobe of pituitary

CRH receptor

Corticotroph γ

ACTH

α

β

G protein

AC

cAMP

Corticotrophs 2+

Ca

PKA 2+

Ca

ACTH

Long feedback (cortisol)

ACTH

Adrenal cortex cell

Melanocortin-2 receptor γ

G protein Adrenal medulla

β

α

AC cAMP

PKA

Activity of P-450SCC Synthesis of several enzymes

Chapter 50  •  The Adrenal Gland

1025

Figure 50-3  Hypothalamic-pituitary-adrenocortical axis. Small-bodied neurons in the paraventricular nucleus of the hypothalamus secrete CRH, a 41–amino-acid peptide that reaches the corticotrophs in the anterior pituitary via the long portal veins. CRH binds to a GPCR on the corticotroph membrane, triggering the adenylyl cyclase (AC)–cAMP–PKA pathway. The activation of L-type Ca2+ channels results in an increase in [Ca2+]i that rapidly leads to the release of preformed ACTH. CRH also increases gene transcription and synthesis of the ACTH precursor, POMC. After its release by corticotrophs, ACTH binds to melanocortin-2 receptors on the cell membranes in all three layers of the adrenal cortex. This receptor triggers the AC–cAMP–PKA pathway, rapidly enhancing the conversion of cholesterol to pregnenolone and more slowly increasing the synthesis of several proteins that are needed for cortisol synthesis. The cerebral cortex can stimulate the hypothalamic neurons to increase their secretion of CRH. Cortisol exerts negative feedback at the level of both the pituitary and hypothalamus. In addition, ACTH produced by the corticotrophs negatively feeds back on the hypothalamic neurons in a “short loop.”

with glucocorticoid drugs and leaves the person with an iatrogenic form of adrenal insufficiency when use of the drug is abruptly discontinued. Conversely, chronic stimulation of the adrenals by ACTH, such as can occur with pituitary tumors (Cushing disease) or with the simple physiological ACTH excess that can occur with chronic stress, can increase the weight of the adrenals several-fold.

Cortisol exerts negative feedback on CRH and ACTH secretion, whereas stress acts through higher CNS centers to stimulate the axis Cortisol exerts negative-feedback control on the very axis that stimulates its secretion (see Fig. 50-3), and it does so at the level of both the anterior pituitary and hypothalamus. Feedback to the Anterior Pituitary  In the corticotrophs of the anterior pituitary, cortisol acts by binding to a cytosolic receptor, which then moves to the nucleus where it binds to GREs and modulates gene expression and thus inhibits the synthesis of both the CRH receptor and ACTH. Even though, as seen above, the POMC gene yields multiple secretory products, cortisol is the main regulator of the

N

Feedback to the Hypothalamus  The negative feedback of cortisol on the CRH-secreting neurons of the hypothala­ mus is less important than that on the corticotrophs dis­ cussed above. Plasma cortisol decreases the mRNA and peptide levels of CRH in paraventricular hypothalamic neurons. Cortisol also inhibits the release of presynthesized CRH. Synthetic glucocorticoids have a similar action. Control by a Higher CNS Center  CRH-secreting neurons in the hypothalamus are under higher CNS control, as illus­ trated by two important features of the hypothalamicpituitary-adrenocortical axis: (1) the circadian and pulsatile nature of ACTH and cortisol secretion, and (2) integration of signals from higher cortical centers that modulate the body’s responses to a variety of stressors. The pituitary secretes ACTH with a circadian rhythm. The suprachiasmatic nucleus of the hypothalamus, which lies above the optic chiasm and receives input from the retina, controls the circadian rhythms of the body. Indeed, blind

POMC

Signal peptide N

transcription of POMC. In addition, elevated levels of corti­ sol in plasma inhibit the release of presynthesized ACTH stored in vesicles.

N-terminal peptide

J-Peptide

ACTH (1–39)

β-LPH (1–89)

N-terminal peptide (1–76)

J-Peptide (1–30)

ACTH (1–39)

β-LPH (1–89)

C

Anterior lobe of pituitary

POMC γ-MSH (51–76)

J-Peptide (1–30)

γ-MSH (51–76)

J-Peptide (1–30)

α-MSH (1–13)

α-MSH (1–13)

CLIP (18–39)

γ-LPH (1–56)

CLIP (18–39)

γ-LPH (1–56)

β-Endorphin (59–89)

β-Endorphin (59–89)

Figure 50-4  Processing of POMC. The primary gene transcript is a preprohormone called pro-opiomelanocortin (POMC). The processing of POMC yields a variety of peptide hormones. This processing is different in the anterior and intermediate lobes of the pituitary. In the anterior pituitary, POMC yields a long N-terminal peptide, a joining (J) peptide, ACTH, and β-LPH. In the intermediate pituitary, the same POMC yields a short N-terminal peptide, γ-MSH, J peptide, α-MSH, CLIP, γ-LPH and β-endorphin. Metabolism by the intermediate lobe is important only during fetal life and pregnancy. (Data from Wilson JD, et al (eds): Williams Textbook of Endocrinology. Philadelphia, WB Saunders, 1998.)

Intermediate lobe of the pituitary (fetal life and pregnancy)

1026

SECTION VIII  •  The Endocrine System

20 Sleep

Awake Cortisol

16 12 Cortisol (µg/dL) 8

50 ACTH

4 0

12 Midnight

25 ACTH (pg/mL)

4

8

12 Noon Time elapsed (hr)

4

8

0 12 Midnight

Figure 50-5  Rhythm of ACTH and cortisol release. The corticotrophs release ACTH in a circadian rhythm, more in the early morning hours and less late in the afternoon and early evening. Superimposed on the circadian rhythm is the effect on the corticotrophs of the pulsatile secretion of CRH by the hypothalamus. Thus, ACTH levels exhibit both circadian and pulsatile behavior. Notice that, although both ACTH and cortisol are secreted episodically, the duration of the ACTH bursts is briefer, which reflects the shorter half-life of ACTH in plasma. (Data from Wilson JD, et al (eds): Williams Textbook of Endocrinology. Philadelphia, WB Saunders, 1998.)

people lose their circadian rhythms. Input from hypotha­ lamic nuclei to the corticotrophs—via both CRH and AVP— appears to modulate the circadian secretion of ACTH and thus the circadian secretion of cortisol as well. As is the case for other hypothalamic releasing hormones, CRH is released in pulses. As a result, superimposed on the circadian rhythm of ACTH is the pulsatile secretion of ACTH, as shown in Figure 50-5. ACTH secretory activity is greatest in the early morning and diminishes late in the afternoon and early evening. The mechanism by which hypothalamic neurons generate pulses of secretory activity is not understood. Other evidence of higher CNS control is the enhanced CRH secretion—and thus the enhanced ACTH secretion— that occurs in response to physical, psychological, and biochemical stress. An example of biochemical stress is hypoglycemia, which stimulates the secretion of both CRH and ACTH and thus leads to an increased release of cortisol that tends to raise blood glucose levels. The increase in ACTH secretion that occurs nocturnally and with stress appears to result from an increased ampli­ tude of the secretory CRH burst rather than an increased frequency of secretion episodes. Because the half-life of cortisol is much longer than that of ACTH, the period of the pulsatile changes in cortisol is longer and the magni­ tude of the excursions is damped in comparison with those of ACTH.

THE ADRENAL CORTEX: ALDOSTERONE The mineralocorticoid aldosterone is the primary regulator of salt balance and extracellular volume Aldosterone determines extracellular volume by controlling the extent to which the kidney excretes or reabsorbs the Na+ filtered at the renal glomerulus. Na+ in the extracellular space

retains water—it is the primary osmotically active particle in the extracellular space—and thus the amount of Na+ that is present determines the volume of extracellular fluid (see pp. 135–136). The extracellular volume is itself a prime determi­ nant of arterial blood pressure (see pp. 554–555), and there­ fore aldosterone plays an important role in the maintenance of blood pressure. The effects of aldosterone on salt balance determine the extracellular volume and should not be confused with the effects of AVP (also known as antidiuretic hormone, or ADH). AVP regulates the free-water balance of the body (see p. 844). Water freely passes across cell membranes and thus affects the concentration of Na+ and other solutes throughout the body (see pp. 135–136). Unlike aldosterone, AVP makes only a small contribution to the maintenance of extracellular volume; instead, AVP regulates serum osmolality and hence the Na+ concentration. Thus, to a first approximation, one can think of aldosterone as the primary regulator of extracel­ lular volume because of its effect on renal Na+ reabsorption, and AVP as the primary regulator of plasma osmolality because of its effect on free-water balance.

The glomerulosa cells of the adrenal cortex synthesize aldosterone from cholesterol via progesterone As is the case for cortisol, the adrenal cortex synthesizes aldosterone from cholesterol by using P-450 enzymes in a series of five steps. The initial steps in the synthesis of aldosterone from cholesterol follow the same synthetic pathway that cortisol-secreting cells use to generate progesterone (see Fig. 50-2). Because glomerulosa cells are the only ones that contain aldosterone synthase, these cells are the exclusive site of aldosterone synthesis. 1. The cytochrome P-450 SCC enzyme (P-450SCC) produces pregnenolone from cholesterol. This enzyme—or the supply of substrate to it—appears to be the

Chapter 50  •  The Adrenal Gland

rate-limiting step for the overall process of steroid hormone synthesis. 2. The SER enzyme 3β-HSD, which is not a P-450 enzyme, oxidizes pregnenolone to form progesterone. 3. Because glomerulosa cells have minimal 17α-hydroxylase (P-450c17), they do not convert progesterone to 17αhydroxyprogesterone. Instead, glomerulosa cells use a 21α-hydroxylase (P-450c21) in the SER to further hydrox­ ylate the progesterone at position 21 and to produce 11-deoxycorticosterone (DOC). 4. In the mitochondria, 11β-hydroxylase (P-450c11) adds an –OH at position 11 to produce corticosterone. This pair of hydroxylations in steps 3 and 4 are catalyzed by the same two enzymes that produce cortisol from 17α-hydroxyprogesterone. 5. The glomerulosa cells—but not the fasciculata and reticu­ laris cells—also have aldosterone synthase (P-450aldo), which first adds an –OH group to the methyl at position 18 and then oxidizes this hydroxyl to an aldehyde group, hence the name aldosterone. This mitochondrial P-450 enzyme, also called 18-methyloxidase, is an isoform of the same 11β-hydroxylase (P-450c11) that catalyzes the DOC-to-corticosterone step. In fact, aldosterone synthase can catalyze all three steps between DOC and aldoste­ rone: 11β-hydroxylation, 18-methyl hydroxylation, and 18-methyl oxidation. As with cortisol, no storage pool of presynthesized aldo­ sterone is available in the glomerulosa cell for rapid secre­ tion. Thus, secretion of aldosterone by the adrenal is limited by the rate at which the glomerulosa cells can synthesize the hormone. Although ACTH also stimulates the pro­ duction of aldosterone in the glomerulosa cell, increases in extracellular [K+] and the peptide hormone ANG II are physiologically more important secretagogues. These secre­ tagogues enhance secretion by increasing the activity of enzymes acting at rate-limiting steps in aldosterone syn­ thesis. These enzymes include the SCC enzyme, which is common to all steroid-producing cells, and aldosterone syn­ thase, which is unique to glomerulosa cells and is responsible for formation of the C-18 aldehyde. Once secreted, ~37% of circulating aldosterone remains free in plasma. The rest weakly binds to CBG (~21%) or albumin (~42%).

Aldosterone stimulates Na+ reabsorption and K+ excretion by the renal tubule The major action of aldosterone is to stimulate the kidney to reabsorb Na+ and water and enhance K+ secretion. Aldoste­ rone has similar actions on salt and water transport in the colon, salivary glands, and sweat glands. MRs are also present in the myocardium, liver, brain, and other tissues, but the physiological role of mineralocorticoids in these latter tissues is unclear. Aldosterone, like cortisol and all the other steroid hor­ mones, acts principally by modulating gene transcription (see pp. 90–92). In the kidney, aldosterone binds to both lowand high-affinity receptors. The low-affinity receptor appears to be identical to the GR. The high-affinity receptor is a distinct MR; it has homology to the GR, particularly in the

1027

zinc-finger region involved in DNA binding. Surprisingly, MR in the kidney has a similar affinity for aldosterone and cortisol. Because cortisol normally circulates at much higher concentrations than does aldosterone (5 to 20 µg/dL versus 2 to 8 ng/dL), the biological effect of aldosterone on any potential target would be expected to be greatly over­ shadowed by that of cortisol. (Conversely, aldosterone has essentially no significant glucocorticoid action because aldo­ sterone binds only weakly to its low-affinity receptor—that is, the GR.) How then do the renal-tubule cells avoid sensing cor­ tisol as a mineralocorticoid? As noted on page 1021, the cells that are targets for aldosterone—particularly in the initial collecting tubule and cortical collecting tubule of the kidney (see p. 766)—contain 11β-HSD2, which converts cortisol to cortisone, a steroid with a very low affinity for MR (see Fig. 35-13C). Unlike 11β-HSD1, which reversibly interconverts cortisone and cortisol, 11β-HSD2 cannot convert cortisone back to cortisol. As a result, locally within the target cell, the cortisol-to-aldosterone ratio is much smaller than the cortisol dominance seen in plasma. In fact, 11β-HSD2 is so effective at removing cortisol from the cytosol of aldosterone target tissues that cortisol behaves as only a weak mineralocorticoid despite the high affinity of cortisol for the so-called MR. Thus, the presence of 11β-HSD2 effectively confers aldosterone specificity on the MR. In the target cells of the renal tubule, aldosterone increases the activity of several key proteins involved in Na+ transport (see pp. 765–766). It increases transcription of the Na-K pump, thus augmenting distal Na+ reabsorption. Aldoste­ rone also raises the expression of apical Na+ channels and of an Na/K/Cl cotransporter. The net effect of these actions is to increase Na+ reabsorption and K+ secretion. The enhanced K+ secretion (see p. 799) appears to occur as a secondary effect to the enhanced Na+ reabsorption. However, the stoi­ chiometry between Na+ reabsorption and K+ secretion in the distal tubule is not fixed. Aldosterone regulates only that small fraction of renal Na+ reabsorption that occurs in the distal tubule and collect­ ing duct. Although most Na+ reabsorption occurs in the proximal tubule by aldosterone-independent mechanisms, loss of aldosterone-mediated Na+ reabsorption can result in significant electrolyte abnormalities, including lifethreatening hyperkalemia and, in the absence of other compensatory mechanisms, hypotension. Conversely, excess aldosterone secretion produces hypokalemia and hyperten­ sion (see p. 1030). In addition to acting via MR, aldosterone also can exert rapid, nongenomic effects by binding to the GPCR known as GPR30 (see p. 989).

Angiotensin II, K+, and ACTH all stimulate aldosterone secretion Three secretagogues control aldosterone synthesis by the glomerulosa cells of the adrenal cortex. The most important is ANG II, which is a product of the renin-angiotensin cascade. An increase in plasma [K+] is also a powerful stimu­ lus for aldosterone secretion and augments the response to ANG II. Third, just as ACTH promotes cortisol secretion, it

1028

SECTION VIII  •  The Endocrine System

Brain

CRH and AVP

Anterior pituitary

ACTH

Three pathways stimulate the glomerulosa cells to synthesize aldosterone. Renin-angiotensin cascade (green)

Angiotensin II

Increased plasma [K+] (red)

ACTH (blue)

Adrenal cortex

ACE in lung

Plasma [K+]

Aldosterone Angiotensin I Kidney Renin Angiotensinogen

Liver

+ Na excretion

H2O excretion [K+] excretion

Plasma [K+] Effective circulating volume Extracellular fluid volume Blood pressure Figure 50-6  Control of aldosterone secretion. Three pathways (shown in three different colors) stimulate the glomerulosa cells of the adrenal cortex to secrete aldosterone.

also promotes the secretion of aldosterone, although this effect is weak. Angiotensin II  We introduced the renin-angiotensinaldosterone axis on pages 841–842. The liver synthesizes and secretes a very large protein called angiotensinogen, which is an α2-globulin (Fig. 50-6). Renin, which is synthesized by the granular (or juxtaglomerular) cells of the juxtaglomeru­ lar apparatus (JGA) in the kidney (see p. 727), is the enzyme

that cleaves this angiotensinogen to form ANG I, a decapep­ tide. Finally, angiotensin-converting enzyme (ACE) cleaves ANG I to form the octapeptide ANG II. ACE is present in both the vascular endothelium of the lung (~40%) and elsewhere (~60%). In addition to acting as a potent secreta­ gogue for aldosterone, ANG II exerts powerful vasoconstric­ tor actions on vascular smooth muscle (see Table 20-8). ANG II has a short half-life (5 times that of the myocardium) and receive both sympathetic and parasympathetic innervation. These cells also can communicate with each other and influence each other’s secretion. We can group these communication links into three categories: 1. Humoral communication. The blood supply of the islet courses outward from the center of the islet toward the periphery, carrying glucose and other secretagogues. In the rat—and less strikingly in humans—β cells are more abundant in the center of the islet, whereas α and δ cells are more abundant in the periphery. Cells within a given islet can influence the secretion of other cells as the blood supply courses outward through the islet carrying the secreted hormonal product of each cell type with it. For example, glucagon is a potent insulin secretagogue, insulin modestly inhibits glucagon release, and somatostatin potently inhibits the secretion of both insulin and glucagon (as well as the secretion of growth hormone and other non-islet hormones). 2. Cell-cell communication. Both gap and tight junctional structures connect islet cells with one another. Cells within an islet communicate via gap junctions, which may be important for the regulation of both insulin and glucagon secretion.

3. Neural communication. Both the sympathetic and parasympathetic divisions of the autonomic nervous system (ANS) regulate islet secretion. Cholinergic stimulation augments insulin secretion. Adrenergic stimulation can have either a stimulatory or inhibitory effect, depending on whether β-adrenergic (stimulatory) or α-adrenergic (in­ hibitory) stimulation dominates (see p. 1033).  N51-1 These three communication mechanisms allow for a tight control over the synthesis and secretion of islet hormones.

INSULIN The discovery of insulin was among the most exciting and dramatic events in the history of endocrine physiology and therapy. In the United States and Europe, insulin-dependent diabetes mellitus (IDDM), or type 1 diabetes, develops in ~1 in every 600 children. However, the prevalence is only ~1 in 10,000 in eastern Asia. Before 1922, all children with diabetes died within 1 or 2 years of diagnosis. It was an agonizing illness; the children lost weight despite eating well, became progressively weaker and cachectic, were soon plagued by infections, and eventually died of overwhelming acidosis. No effective therapy was available, and few prospects were on the horizon. It was known that the blood sugar level was elevated in this disease, but beyond that, there was little understanding of its pathogenesis. In 1889, Minkowski and von Mering demonstrated that removing the pancreas from dogs caused hyperglycemia, excess urination, thirst, weight loss, and death—in short, a syndrome closely resembling type 1 diabetes. Following this lead, a group of investigators in the Department of Physiology at the University of Toronto prepared extracts of pancreas and tested the ability of these extracts to lower plasma [glucose] in pancreatectomized dogs. Despite months of failures, these investigators persisted in their belief that such extracts could be beneficial. Finally, by the winter of 1921, Frederick Banting (a surgeon) and Charles Best (at the time, a medical student) were able to demonstrate that an aqueous extract of pancreas could lower blood glucose level and prolong survival in a pancreatectomized dog.  N51-2  Within 2 months, a more purified extract was shown to lower blood glucose level in a young man with diabetes. By the end of 1923, insulin (as the islet hormone was named) was being prepared from beef and pork pancreas on an 1035

Chapter 51  •  The Endocrine Pancreas

N51-1  Antagonistic Effects of α- and β-Adrenergic Receptors on Insulin Secretion Contributed by Emile Boulpaep and Walter Boron On page 1033, we noted that the general rule for α- and β-adrenergic receptors—first noted by Raymond Ahlquist—is that α activation leads to stimulation, whereas β activation leads to inhibition. The pattern in pancreatic islets is just the opposite, as noted in the text.

1035.e1

N51-2  Frederick Banting and Charles Best Contributed by Emile Boulpaep and Walter Boron In 1923, just 2 years after Frederick Banting (a young faculty member just 5 years out of medical school) and Charles Best (a 22-year-old medical student working in Banting’s laboratory) discovered insulin at the University of Toronto, the Nobel Prize in Physiology or Medicine was awarded to Frederick Banting and the head of the research team and chairman of Banting’s department, John Macleod. The short delay between the discovery and the award of the prize indicates the enormous significance of the discovery. It is interesting that Frederick Banting protested the award of the Nobel Prize to John Macleod and gave half of his portion of the monetary award to Charles Best.

REFERENCES Banting FG, Best CH: Pancreatic extracts, 1922. J Lab Clin Med 115:254–272, 1990. Banting FG, Best CH, Collip JB, et al: Pancreatic extracts in the treatment of diabetes mellitus: Preliminary report, 1922. CMAJ 145:1281–1286, 1991.

1036

SECTION VIII  •  The Endocrine System

industrial scale, and patients around the world were receiving effective treatment of their diabetes. For the discovery of insulin, Frederick Banting and John Macleod received the 1923 Nobel Prize in Physiology or Medicine.  N51-3 Since that time, the physiology of the synthesis, secretion, and action of insulin has been studied more extensively than α Cells produce glucagon.

δ Cells produce somatostatin. β Cells produce insulin.

F cell

F cells produce pancreatic polypeptide.

Blood flows from the center to the periphery.

Islet of Langerhans

Pancreas Common bile duct

Pancreatic duct

that of any other hormone. Now, nearly a century later, much is known about the metabolic pathways through which insulin regulates carbohydrate, lipid, and protein metabolism in its major targets: the liver, muscle, and adipose tissue. However, the sequence of intracellular signals that triggers insulin secretion by pancreatic β cells, as well as the signaltransduction process triggered when insulin binds to a plasma membrane receptor on target tissues, remain areas of intense study.

Insulin replenishes fuel reserves in muscle, liver, and adipose tissue What does insulin do? Succinctly put, insulin efficiently integrates body fuel metabolism both during periods of fasting and during feeding (Table 51-2). When an individual is fasting, the β cell secretes less insulin. When insulin levels decrease, lipids are mobilized from adipose tissue and amino acids are mobilized from body protein stores within muscle and other tissues. These lipids and amino acids provide fuel for oxidation and serve as precursors for hepatic ketogenesis and gluconeogenesis, respectively. During feeding, insulin secretion increases promptly, which diminishes the mobilization of endogenous fuel stores and stimulates the uptake of carbohydrates, lipids, and amino acids by insulin-sensitive target tissues. In this manner, insulin directs tissues to replenish the fuel reserves depleted during periods of fasting. As a result of its ability to regulate the mobilization and storage of fuels, insulin maintains plasma [glucose] within narrow limits. Such regulation provides the central nervous system (CNS) with a constant supply of glucose needed to fuel cortical function. In higher organisms, if plasma [glucose] (normally ≅ 5 mM) declines to 15 mM) TABLE 51-2  Effects of Nutritional States AFTER A 24-hr FAST

2 hr AFTER A MIXED MEAL

Plasma [glucose], mg/dL   mM

60–80

100–140

3.3–4.4

5.6–7.8

Plasma [insulin], µU/mL

3–8

50–150

Plasma [glucagon], pg/mL

40–80

80–200

Liver

↑ Glycogenolysis ↑ Gluconeogenesis

↓ Glycogenolysis ↓ Gluconeogenesis ↑ Glycogen synthesis

Adipose tissue

Lipids mobilized for fuel

Lipids synthesized

Muscle

Lipids metabolized Protein degraded and amino acids exported

Glucose oxidized or stored as glycogen Protein preserved

PARAMETER

Duodenum

Figure 51-1  Islet of Langerhans. The distribution of cell types is representative of islets from ~90% of the human pancreas, which arises embryologically from the dorsal pancreatic bud. In the other islets (not shown), F cells dominate.

TABLE 51-1  Products of Pancreatic Islet Cells CELL TYPE

PRODUCT

α

Glucagon

β

Insulin Proinsulin C peptide Amylin

δ

Somatostatin

F

Pancreatic polypeptide

Chapter 51  •  The Endocrine Pancreas

N51-3  Frederick Banting and John Macleod Contributed by Emile Boulpaep and Walter Boron For more information about Frederick Banting and John Macleod and the work that led to their Nobel Prize, visit http:// nobelprize.org/medicine/laureates/1923/index.html (accessed October 2014).

1036.e1

Chapter 51  •  The Endocrine Pancreas

BOX 51-1  Clinical Manifestations of Hypoglycemia   and Hyperglycemia Hypoglycemia 

N51-4

Early symptoms are principally autonomic and include palpitations, tachycardia, diaphoresis, anxiety, hyperventilation, shakiness, weakness, and hunger. More severe hypoglycemia manifests principally as neuroglycopenia, with confusion, aberrant behavior, hallucinations, seizures, hypothermia, focal neurological deficits, and coma.

5´ UTR

B

C

3´ UTR

A

mRNA encoding preproinsulin

Ribosome 5´

3´

mRNA C

HS

ER S

S H

1037

SH SH

Proinsulin C peptide

SH S

N

H

Insulin

Hyperglycemia

S

A C

N

Early manifestations include weakness, polyuria, polydipsia, altered vision, weight loss, and mild dehydration. For prolonged or severe hyperglycemia (accompanied by metabolic acidosis or diabetic ketoacidosis), manifestations include Kussmaul hyperventilation (deep, rapid breathing; see p. 716), stupor, coma, hypotension, and cardiac arrhythmias.

S

S

S

S

B

Golgi S

produces an osmotic diuresis (see Box 35-1) that, when severe, can lead to dehydration, hypotension, and vascular collapse.

Converting enzymes

S C

N

S

S

S

S

β cells synthesize and secrete insulin The Insulin Gene  Circulating insulin comes only from the β cells of the pancreatic islet. It is encoded by a single gene on the short arm of chromosome 11. Exposing islets to glucose stimulates insulin synthesis and secretion. Though the process is not completely understood, this stimulation requires that the glucose be metabolized. Insulin Synthesis  Transcription of the insulin gene product and subsequent processing produces full-length messenger RNA (mRNA) that encodes preproinsulin. Starting from its 5′ end, this mRNA encodes a leader sequence and then peptide domains B, C, and A. Insulin is a secretory protein (see pp. 34–35). As the preprohormone is synthesized, the leader sequence of ~24 amino acids is cleaved from the nascent peptide as it enters the rough endoplasmic reticulum. The result is proinsulin (Fig. 51-2), which consists of domains B, C, and A. As the trans Golgi packages the proinsulin and creates secretory granules, proteases slowly begin to cleave the proinsulin molecule at two spots and thus excise the 31–amino-acid C peptide. The resulting insulin molecule has two peptide chains, designated the A and B chains, that are joined by two disulfide linkages. The mature insulin molecule has a total of 51 amino acids, 21 on the A chain and 30 on the B chain. In the secretory granule, the insulin associates with zinc. The secretory vesicle contains this insulin, as well as proinsulin and C peptide. All three are released into the portal blood when glucose stimulates the β cell. Secretion of Insulin, Proinsulin, and C Peptide  C peptide has no established biological action. Yet because it is secreted in a 1 : 1 molar ratio with insulin, it is a useful marker for insulin secretion. Proinsulin does have modest insulin-like activities; it is ~ 1 20 th as potent as insulin on a molar basis.

trans-Golgi

Cleavage S

S C

N

S

S

S

S

Cleavage

C peptide

Secretory granule

+ A B

S

S

S

S

S

S

Insulin

Figure 51-2  Synthesis and processing of the insulin molecule. The mature mRNA of the insulin gene product contains a 5′ untranslated region (UTR); nucleotide sequences that encode a 24–amino-acid leader sequence, as well as B, C, and A peptide domains; and a 3′ UTR. Together, the leader plus the B, C, and A domains constitute preproinsulin. During translation of the mRNA, the leader sequence is cleaved in the lumen of the rough endoplasmic reticulum (ER). What remains is proinsulin, which consists of the B, C, and A domains. Beginning in the trans Golgi, proteases cleave the proinsulin at two sites, releasing the C peptide as well as the mature insulin molecule, which consists of the B and A chains that are connected by two disulfide bonds. The secretory granule contains equimolar amounts of insulin and the C peptide, as well as a small amount of proinsulin. These components all are released into the extracellular space during secretion.

Chapter 51  •  The Endocrine Pancreas

1037.e1

N51-4  Hypoglycemia Contributed by Eugene Barrett Hypoglycemia, which can be viewed most simply as the opposite of diabetes mellitus, has many causes. Perhaps the most frequent setting is a patient with type 1 diabetes who skips a meal or fails to adjust the insulin dose when exercising. Many diabetic patients who seek to maintain tight control over their blood sugar experience frequent hypoglycemic reactions, which they quickly learn to abort with a carbohydrate snack. Patients with type 2 diabetes who take an excessive dose of sulfonylureas are subject to severe hypoglycemia, which may require continuous treatment for several days because the half-life of some of these drugs is quite long. We saw in Chapter 50 that epinephrine—acting as a β-adrenergic agonist—is a hyperglycemic agent; that is, it promotes glycogenolysis in liver and muscle (see p. 1033). Thus, β blockers rarely cause hypoglycemia in healthy individuals because these people can appropriately regulate their insulin secretion. However, because β blockers can mask the early adrenergic response to mild hypoglycemia (sweating, tachycardia, tremulousness), diabetic patients taking both insulin and β blockers commonly progress to severe hypoglycemia without warning. Another drug that can induce hypoglycemia is pentamidine, an agent used to treat Pneumocystis jiroveci pneumonia. Pentamidine is a β-cell toxin that leads to an acute, excessive release of insulin, which can be followed by hypoglycemia.

Alcoholic patients are at great risk of hypoglycemia. Ethanol suppresses gluconeogenesis, and hepatic glycogen stores may already be low because of poor nutrition. Other severe illnesses that can produce persistent hypoglycemia include liver disease, renal failure, and some large tumors that produce a hypoglycemia-inducing peptide, usually IGF-2. Rarely, an insulinoma may develop, which is an islet cell tumor (usually benign) that releases high and unregulated concentrations of insulin into the bloodstream. Many individuals complain of postprandial hypoglycemia, frequently called reactive hypoglycemia. Despite long years of skepticism, investigators now believe that at least some of these patients do indeed experience true symptoms of hypoglycemia within a few hours of eating. There is no absolute glucose level at which symptoms occur; many people can tolerate extremely low levels of glucose without any problems. However, a rather high rate of decline in the plasma glucose level after a meal may cause symptoms. One cause of postprandial hypoglycemia may be a delay in the timing of insulin release after a meal. Thus, the β cells release too much insulin too late after a meal, so the blood glucose level initially rises markedly and then falls rapidly. In some patients, this defect may herald the development of diabetes mellitus.

1038

SECTION VIII  •  The Endocrine System

However, the β cell secretes only ~5% as much proinsulin as insulin. As a result, proinsulin does not play a major role in the regulation of blood glucose. Most of the insulin (~60%) that is secreted into the portal blood is removed in a first pass through the liver. In contrast, C peptide is not extracted by the liver at all. As a result, whereas measurements of the insulin concentration in systemic blood do not quantitatively mimic the secretion of insulin, measurements of C peptide do. C peptide is eventually excreted in the urine, and the quantity of C peptide excreted in a 24-hour period is a rough measure of the amount of insulin released during that time.

A

NORMAL SUBJECT UNDERGOING SAME CHANGES IN PLASMA [GLUCOSE] FOLLOWING ORAL VS. IV GLUCOSE CHALLENGES

300 Insulin after oral glucose

Glucose (mg/dL)

Glucose is the major regulator of insulin secretion In healthy individuals, the plasma glucose concentration remains within a remarkably narrow range. After an overnight fast, it typically averages between 4 and 5 mM; the plasma [glucose] rises after a meal, but even with a very large meal it does not exceed 10 mM. Modest increases in plasma [glucose] provoke marked increases in the secretion of insulin and C peptide and hence raise plasma [insulin], as illustrated by the results of an oral glucose tolerance test (OGTT) as shown in Figure 51-3A. Conversely, a decline in plasma [glucose] of only 20% markedly lowers plasma [insulin]. The change in the concentration of plasma glucose that occurs in response to feeding or fasting is the main determinant of insulin secretion. In a patient with type 1 diabetes mellitus caused by destruction of pancreatic islets, an oral glucose challenge evokes either no response or a much smaller insulin response, but a much larger increment in plasma [glucose] that lasts for a much longer time (see Fig. 51-3B). A glucose challenge of 0.5 g/kg body weight given as an intravenous bolus raises the plasma glucose concentration more rapidly than glucose given orally. Such a rapid rise in plasma glucose concentration leads to two distinct phases of insulin secretion (see Fig. 51-3C). The acute-phase or firstphase insulin response lasts only 2 to 5 minutes, whereas the second-phase insulin response persists as long as the blood glucose level remains elevated. The insulin released during the acute-phase insulin response to intravenous glucose arises from preformed insulin that had been packaged in

150

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Insulin after IV glucose 0

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DIABETIC SUBJECT RECEIVING ORAL GLUCOSE

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150 Glucose

Glucose (mg/dL)

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C NORMAL SUBJECT RECEIVING A HIGHER DOSE OF IV GLUCOSE THAN IN PANEL A

300

Glucose (mg/dL)

150

Glucose

Figure 51-3  Glucose tolerance test results. A, When a person ingests a glucose meal (75 g), plasma [glucose] (green curve) rises slowly, reflecting intestinal uptake of glucose. As a result, plasma [insulin] (solid red curve) rises sharply. When a lower glucose dose is given intravenously (IV) over time—in a manner that reproduces the green curve— plasma [insulin] rises only modestly (dashed red curve). The difference between the insulin responses indicated by the solid and dashed red lines is due to the “incretin effect” of oral glucose ingestion. B, In a patient with type 1 diabetes, the same oral glucose load as that in A causes plasma [glucose] to rise to a higher level and to remain high for a longer time. The diagnosis of diabetes is made if the plasma glucose level is above 200 mg/dL at the second hour. C, If a large IV glucose challenge (0.5 g glucose/kg body weight given as a 25% glucose solution) is administered as a bolus, plasma [glucose] rises much more rapidly than it does with an oral glucose load. Sensing a rapid rise in [glucose], the β cells first secrete some of their stores of presynthesized insulin. Following this “acute phase,” the cells secrete both presynthesized and newly manufactured insulin in the “chronic phase.”

Insulin (µU/mL)

200

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Insulin (µU/mL)

Chapter 51  •  The Endocrine Pancreas

1039

secretory vesicles docked at, or residing near, the β-cell plasma membrane. The second-phase insulin response also comes from preformed insulin within the vesicles with some contribution from newly synthesized insulin. One of the earliest detectable metabolic defects that occurs in both type 1 and type 2 diabetes is loss of the first phase of insulin secretion, as determined by an intravenous glucose tolerance test. If a subject consumes glucose or a mixed meal, plasma [glucose] rises much more slowly—as in Figure 51-3A—because the appearance of glucose in plasma depends on gastric emptying and intestinal absorption. Given that plasma [glucose] rises so slowly, the acute-phase insulin response can no longer be distinguished from the chronic response, and only a single phase of insulin secretion is apparent. However, the total insulin response to an oral glucose challenge exceeds the response observed when comparable changes in plasma [glucose] are produced by intravenously administered glucose (see Fig. 51-3A). This difference is referred to as the incretin effect (Box 51-2).

glucose itself is the best secretagogue, some amino acids (especially arginine and leucine) and small keto acids (e.g., α-ketoisocaproate, α-ketoglutarate), as well as ketohexoses (fructose), can also weakly stimulate insulin secretion. The amino acids and keto acids do not share any metabolic pathway with hexoses other than oxidation via the citric acid cycle (see p. 1185). These observations have led to the suggestion that the ATP generated from the metabolism of these varied substances may be involved in insulin secretion. In the laboratory, depolarizing the islet cell membrane by raising extracellular [K+] provokes insulin secretion. From these data has emerged a relatively unified picture of how various secretagogues trigger insulin secretion. Key to this picture is the presence in the islet of an ATP-sensitive K+ channel and a voltage-gated Ca2+ channel in the plasma membrane (Fig. 51-4). The K+ channel (KATP; see p. 198) is an octamer of four Kir6.2 channels (see p. 196) and four sulfonylurea receptors (SURs; see p. 199; Box 51-3), Glucose triggers insulin release in a seven-step process:

Metabolism of glucose by the β cell triggers insulin secretion

Step 1: Glucose enters the β cell via the GLUT2 glucose transporter by facilitated diffusion (see p. 114). Amino acids enter through a different set of transporters. Step 2: In the presence of glucokinase (the rate-limiting enzyme in glycolysis), the entering glucose undergoes glycolysis as well as oxidation via the citric acid cycle (see p. 1185), phosphorylating ADP and raising [ATP]i. Some amino acids also enter the citric acid cycle. In both cases, the following ratios increase: [ATP]i/[ADP]i, [NADH]i/ [NAD+]i, and [NADPH]i/[NADP+]i (NADH and NAD+ are the reduced and oxidized forms of nicotinamide adenine dinucleotide [NAD], and NADPH and NADP+

The pancreatic β cells take up and metabolize glucose, galactose, and mannose, and each can provoke insulin secretion by the islet. Other hexoses that are transported into the β cell but that cannot be metabolized (e.g., 3-O-methylglucose or 2-deoxyglucose) do not stimulate insulin secretion. Although

BOX 51-2  Nonhuman and Mutant Insulin

C

loning of the insulin gene has led to an important therapeutic advance, namely, the use of recombinant human insulin for the treatment of diabetes. Human insulin was the first recombinant protein available for routine clinical use. Before the availability of human insulin, either pork or beef insulin was used to treat diabetes. Pork and beef insulin differ from human insulin by one and three amino acids, respectively. The difference, although small, is sufficient to be recognized by the immune system, and antibodies to the injected insulin develop in most patients treated with beef or pork insulin; occasionally, the reaction is severe enough to cause a frank allergy to the insulin. This problem is largely avoided by using human insulin. Sequencing of the insulin gene has not led to a major understanding of the genesis of the common forms of human diabetes. However, rare patients with diabetes make a mutant insulin molecule with a single amino-acid substitution in either the A or B chain. In each case that has been described, these changes lead to a less-active insulin molecule (typically only ~1% as potent as insulin on a molar basis). These patients have either glucose intolerance or frank diabetes, but very high concentrations of immunoreactive insulin in their plasma. In these individuals, the immunoreactivity of insulin is not affected to the same extent as the bioactivity. In addition to revealing these mutant types of insulin, sequencing of the insulin gene has allowed identification of a flanking polymorphic site upstream of the insulin gene that contains one of several common alleles. In some populations, certain polymorphisms are associated with an increased risk of development of type 1 diabetes mellitus.

BOX 51-3  Sulfonylureas

A

n entire class of drugs—the sulfonylurea agents—is used in the treatment of patients with type 2 diabetes, or non–insulin-dependent diabetes mellitus (NIDDM). Type 2 diabetes arises from two defects: (1) β cells are still capable of making insulin but do not respond adequately to increased blood [glucose], and (2) insulin target tissues are less sensitive or “resistant” to insulin. The sulfonylurea agents were discovered accidentally. During the development of sulfonamide antibiotics after the Second World War, investigators noticed that the chemically related sulfonylurea agents produced hypoglycemia in laboratory animals. These drugs turned out to have no value as antibiotics, but they did prove effective in treating the hyperglycemia of type 2 diabetes. The sulfonylureas enhance insulin secretion by binding to the SUR subunits (see p. 199) of KATP channels, thereby decreasing the likelihood that these channels will be open. This action enhances glucose-stimulated insulin secretion (see Fig. 51-4). By increasing insulin secretion, sulfonylureas overcome insulin resistance and decrease blood glucose in these patients. Unlike insulin, which must be injected, sulfonylureas can be taken orally and are therefore preferred by many patients. However, they have a therapeutic role only in type 2 diabetes; the β cells in patients with type 1 diabetes are nearly all destroyed, and these patients must be treated with insulin replacement therapy.

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SECTION VIII  •  The Endocrine System

Extracellular space

Glucose

Leucine

GLUT2 transporter 1 Glucose enters the cell via a GLUT2 transporter, which mediates facilitated diffusion of glucose into the cell.

β cell cytosol GLYCOLYSIS

2 The increased glucose influx stimulates glucose metabolism, leading to an increase in [ATP]i, [ATP]i / [ADP]i, [NADH]i/[NAD+]i, or [NADPH]i/ + [NADP ]i.

Glucokinase Glucose-6-phosphate Pyruvate

3 The increased [ATP]i, [ATP]i /[ADP]i, [NADH]i / [NAD+]i, or [NADPH]i/ [NADP+]i inhibits the KATP channel.

Mitochondrion Citric acid cycle

H2O CO2

CCK acetylcholine

K+

Gq

4 + Closure of this K channel causes Vm to become more positive (depolarization). Kir 6.2 SUR1

Depolarization

Phospholipase C

PLC

PIP2 IP3

ER

DAG

2+ [Ca ]

Protein kinase C PKC

[Ca2+]i

Protein kinase A Other modulators of secretion act via the adenylyl cyclase-cAMPprotein kinase A pathway and the phospholipase C- phosphoinositide pathway. Gαs

Secretory granules

PKA

cAMP

AC

β-adrenergic Glucagon agonists

5 The depolarization activates a voltage-gated Ca2+ channel in the plasma membrane. Ca2+ Voltage-gated 2+ Ca channel

6 2+ The activation of this Ca channel 2+ promotes Ca influx, and thus 2+ increases [Ca ]i, which also evokes Ca2+-induced Ca2+ release. 7 2+ The elevated [Ca ]i leads to exocytosis and release into the blood of insulin contained within the secretory granules.

Adenylyl cyclase

Gαs

KATP

Gαi

Somatostatin galanin α-adrenergic agonists

Insulin

Figure 51-4  Mechanism of insulin secretion by the pancreatic β cell. Increased levels of extracellular glucose trigger the β cell to secrete insulin in the seven steps outlined in this figure. Metabolizable sugars (e.g., galactose and mannose) and certain amino acids (e.g., arginine and leucine) can also stimulate the fusion of vesicles that contain previously synthesized insulin. In addition to these fuel sources, certain hormones (e.g., glucagon, somatostatin, cholecystokinin [CCK]) can also modulate insulin secretion. ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C.

Chapter 51  •  The Endocrine Pancreas

are the reduced and oxidized forms of NAD phosphate)  N51-5 Step 3: The increase in the ratio [ATP]i/[ADP]i, or [NADH]i/ [NAD+]i, or [NADPH]i/[NADP+]i causes KATP channels (see p. 198) to close. Step 4: Reducing the K+ conductance of the cell membrane causes the β cell to depolarize (i.e., the membrane potential is less negative). Step 5: This depolarization activates voltage-gated Ca2+ channels (see pp. 190–191). Step 6: The increased Ca2+ permeability leads to increased Ca2+ influx and increased intracellular free Ca2+. This rise in [Ca2+]i additionally triggers Ca2+-induced Ca2+ release (see pp. 242–243). Step 7: The increased [Ca2+]i, perhaps by activation of a Ca2+calmodulin phosphorylation cascade, ultimately leads to insulin release. Other secretagogues can also modulate insulin secretion via the phospholipase C pathway (see p. 58) or via the adenylyl cyclase pathway (see p. 53) in addition to the pathway just outlined. For example, glucagon, which stimulates insulin release, may bypass part or all of the glucose/[Ca2+]i pathway by stimulating adenylyl cyclase, thus raising cAMP levels and activating protein kinase A (PKA). Conversely, somatostatin, which inhibits insulin release, may act by inhibiting adenylyl cyclase.

Neural and humoral factors modulate insulin secretion The islet is richly innervated by both the sympathetic and the parasympathetic divisions of the ANS. Neural signals appear to play an important role in the β-cell response in several settings. β-adrenergic stimulation augments islet insulin secretion, whereas α-adrenergic stimulation inhibits it (see Fig. 51-4). Isoproterenol, a synthetic catecholamine that is a specific agonist for the β-adrenergic receptor, potently stimulates insulin release. In contrast, norepinephrine and synthetic α-adrenergic agonists suppress insulin release both basally and in response to hyperglycemia. Because the postsynaptic sympathetic neurons of the pancreas release norepinephrine, which stimulates α more than β adrenoceptors, sympathetic stimulation via the celiac nerves inhibits insulin secretion. In contrast to α-adrenergic stimulation, parasympathetic stimulation via the vagus nerve, which releases acetylcholine, causes an increase in insulin release. Exercise  The effect of sympathetic regulation on insulin secretion may be particularly important during exercise, when adrenergic stimulation of the islet increases. The major role for α-adrenergic inhibition of insulin secretion during exercise is to prevent hypoglycemia. Exercising muscle tissue uses glucose even when plasma [insulin] is low. If insulin levels were to rise, glucose use by the muscle would increase even further and promote hypoglycemia. Furthermore, an increase in [insulin] would inhibit lipolysis and fatty-acid release from adipocytes and would thus diminish the availability of fatty acids, which the muscle can use as an alternative fuel to glucose (see p. 1211). Finally, a rise in [insulin] would decrease glucose production by the liver. Suppression

1041

of insulin secretion during exercise may thus serve to prevent excessive glucose uptake by muscle, which, if it were to exceed the ability of the liver to produce glucose, would lead to severe hypoglycemia, compromise the brain, and abruptly end any exercise! Feeding  Another important setting in which neural and humoral factors regulate insulin secretion is during feeding. Food ingestion triggers a complex series of neural, endocrine, and nutritional signals to many body tissues. The cephalic phase (see pp. 871 and 890) of eating, which occurs before food is ingested, results in stimulation of gastric acid secretion and a small rise in plasma insulin level. This response appears to be mediated by the vagus nerve in both cases. If no food is forthcoming, blood [glucose] declines slightly and insulin secretion is again suppressed. If food ingestion does occur, the acetylcholine released by postganglionic vagal fibers in the islet augments the insulin response of the β cell to glucose. As already discussed, after a subject drinks a glucose solution, the total amount of insulin secreted is greater than when the same amount of glucose is administered intravenously (see Fig. 51-3A). This observation has led to a search for enteric factors or incretins that augment the islet β-cell response to an oral glucose stimulus. Currently, we know of three peptides released by intestinal cells in response to feeding that enhance insulin secretion: cholecystokinin from I cells, glucagon-like peptide 1 (GLP-1) from L cells, and gastric inhibitory polypeptide (GIP, also called glucosedependent insulinotropic peptide) from K cells. GLP-1 (see p. 1051), perhaps the most important incretin yet discovered, has a very short half-life in plasma (80% in the tyrosine kinase region. This similarity is sufficient that very high concentrations of insulin can stimulate the IGF-1 receptor and, conversely, high levels of IGF-1 can stimulate the insulin receptor. The insulin receptor’s extracellular α chains have multiple N-glycosylation sites. The β chains have an extracellular, a membrane-spanning, and an intracellular portion. The β subunit of the receptor is glycosylated on its extracellular domains; receptor glycosylation is required for insulin binding and action. The intracellular domain of the β chain possesses tyrosine kinase activity, which increases markedly when insulin binds to sites on the α chains of the receptor. The insulin receptor can phosphorylate both itself and other intracellular substrates at tyrosine residues (see pp. 68–70). The targets of tyrosine phosphorylation (beyond the receptor itself) include a family of cytosolic proteins known as insulin-receptor substrates (IRS-1, IRS-2, IRS-3, and IRS4) as well as Src homology C terminus (SHC), as illustrated in Figure 51-6. This phosphorylation mechanism appears to be the major one by which insulin transmits its signal across the plasma membrane of insulin target tissues. The IRS proteins are docking proteins to which various downstream effector proteins bind and thus become activated. IRS-1 has at least eight tyrosines within specific motifs that generally bind proteins containing SH2 (Src homology domain 2) domains (see p. 58), so that a single IRS molecule

BOX 51-4  The Insulin Receptor and Rare Forms of Diabetes

T

he ability of insulin to act on a target cell depends on three things: the number of receptors present on the target cell, the receptor’s affinity for insulin, and the receptor’s ability to transduce the insulin signal. Several disorders have been described in which a mutation of the insulin receptor blunts or prevents insulin’s actions. One such mutation markedly affects growth in utero, as well as after birth. This rare disorder is called leprechaunism, and it is generally lethal within the first year of life. Other mutations of the receptor have less devastating consequences. Some individuals make antibodies to their own insulin receptors. Insulin, produced either endogenously or administered to these patients, does not work well because it must compete with these antibodies for sites on the receptor; as a result, the patient is hyperglycemic. Interestingly, other antibodies can be “insulin mimetic”; that is, not only do the antibodies bind to the receptor, but they also actually mimic insulin’s action. This mimicry causes severe hypoglycemia in affected individuals. Neither receptor mutations nor antireceptor antibodies appear to be responsible for any of the common forms of diabetes seen clinically. However, abnormal insulin-receptor signaling may be involved in many patients with type 2 diabetes. Indeed, activation of inflammatory pathways involving the p38 subset of MAPKs (see p. 69) and nuclear factor κB (see pp. 86–87) can lead to phosphorylation of the insulin receptor (and of IRS proteins), principally at serine residues. This serine phosphorylation occurs commonly in animal models of insulin resistance and type 2 diabetes as well as in human diabetes, and can interfere with the normal metabolic actions of insulin.

simultaneously activates multiple pathways. The IGF-1 receptor, which is closely related to the insulin receptor, also acts through IRS proteins. Figure 51-6 illustrates three major signaling pathways triggered by the aforementioned tyrosine phosphorylations.  N51-7  The first begins when phosphatidylinositol 3-kinase (PI3K) binds to phosphorylated IRS and becomes activated. PI3K phosphorylates a membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to form phosphatidylinositol 3,4,5-trisphosphate (PIP3), and it leads to major changes in glucose and protein metabolism. The second signaling pathway begins in one of two ways: (1) the insulin receptor phosphorylates SHC or (2) growth factor receptor–bound protein 2 (GRB2; see p. 69) binds to an IRS and becomes activated. As illustrated in Figure 51-6, both phosphorylated SHC and activated GRB2 trigger the Ras signaling pathway, leading through mitogen-activated protein kinase kinase (MEK) and mitogen-activated protein kinase (MAPK; see pp. 68–69) to increased gene expression and growth. Gene-deletion studies in mice show that IRS-1 deletion does not cause diabetes but results in small mice. In contrast, IRS-2 deletion does cause diabetes, in part because of impaired insulin secretion by the pancreatic β cell! The third signaling pathway begins with the binding of SH2-containing proteins—other than PI3K and GRB2, already discussed—to specific phosphotyrosine groups on either the insulin receptor or IRS proteins. This binding activates the SH2-containing protein (Box 51-4).

Chapter 51  •  The Endocrine Pancreas

N51-6  Insulin and IGF-1 Receptors Contributed by Emile Boulpaep and Walter Boron Activation of the insulin and IGF-1 receptors (see Fig. 51-5) occurs by somewhat different mechanisms, as we discuss on pages 1041–1042 for the insulin receptor and on page 996 for IGF-1 receptor. In brief, these receptors are tetrameric; they are composed of two α and two β subunits. The α subunit contains a cysteine-rich region and functions in ligand binding. The β subunit is a single-pass transmembrane protein with a cytoplasmic tyrosine kinase domain. The α and β subunits are held together by disulfide bonds (as are the two α subunits), forming a heterotetramer. Ligand binding produces conformational changes that appear to cause allosteric interactions between the two α and β pairs, as opposed to the dimerization characteristic of the first class of receptor tyrosine kinases (see Fig. 3-12C). Thus, insulin binding results in the autophosphorylation of tyrosine residues in the catalytic domains of the β subunits. The activated insulin receptor also phosphorylates cytoplasmic substrates such as IRS-1 (see Fig. 51-6), which, once phosphorylated, serves as a docking site for additional signaling proteins.

1042.e1

N51-7  Insulin Signal Transduction Contributed by Eugene Barrett, Emile Boulpaep, and Walter Boron Figure 51-6 shows three major pathways. In the first pathway, activation of phosphatidylinositol 3-kinase (PI3K) phos­ phorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to form phosphatidylinositol 3,4,5-trisphosphate (PIP3; see p. 58), which in turn activates phosphatidylinositol-dependent kinase (PDK). This serine/threonine kinase then activates protein kinase B (PKB), which leads to the insertion of GLUT4 glucose transporters into the plasma membrane. PDK also phosphorylates and thus inactivates glycogen synthase kinase 3 (GSK3); the net effect is reduced inactivation of glycogen synthase (GS) and enhanced glycogen synthesis. Finally PDK activates mTOR (target of rapamycin), a serine/threonine kinase that phosphorylates the binding protein PHAS-1 and thus releases an active initiation factor (IF), promoting translation of mRNA into protein. mTOR also phosphorylates p70-S6 kinase, which phosphorylates the ribosomal S6 protein. In the second pathway, the insulin receptor phosphorylates SHC (which stands for Src homology, C terminus) at tyrosine residues, stimulating SOS. In addition, activation of GRB2 also stimulates SOS. The stimulated SOS activates the Ras pathway, as described in Figure 3-13. The activated Raf-1, which is itself a MEK kinase, activates not only MEK but also other MEK kinases, which in turn activate JNK (a kinase) and p38 kinase. MAPK activates both a transcription factor and p90-S6 kinase. The activated p90-S6 kinase phosphorylates a variety of nuclear proteins as well as phosphoprotein phosphatase 1 (PP1); the latter leads to activation of glycogen synthase. In the third pathway, SH2-containing proteins (shown in blue in Fig. 51-6)—other than PI3K and GRB2, already discussed—bind to specific phosphotyrosine groups on either the insulin receptor or IRS proteins. These SH2-containing proteins have a variety of effects, for example, on enzymes involved in lipid metabolism.

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Chapter 51  •  The Endocrine Pancreas

When insulin binds, the insulin receptor phosphorylates itself and various cytoplasmic proteins, including the IRS family.

GLUT4 transporter PI-3,4,5-P3

Glucose Insertion of GLUT4 into membrane

PI-4,5-P2 (PIP2)

Insulin Extracellular space

P

PDK1,2

P

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P P

P P

Tyrosine kinase

SHC Tyr

SOS

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-1

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P P

Tyr

p70–S6 kinase GSK-3

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Nucleus

Modulation of transcription

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P

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(Inactive)

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(Active)

SOS

IRS-1, 2, 3, and 4 are docking proteins to which a variety of cytosolic SH2-containing proteins bind via phosphorylated tyrosine groups on the IRS proteins. Upon binding, these effector proteins, which are often other kinases and phosphatases, become active.

P

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Glycogen synthesis and glucose oxidation Gluconeogenesis

Enzymes involved in lipid synthesis

Triglyceride synthesis Lipolysis and lipid oxidation

MAPK P

By reducing levels of active FOXO1, Akt and mTOR reduce G6Pase and PEPCK levels, and raise levels of enzymes involved in lipid metabolism.

Protein synthesis Proteolysis

P

Active transcription factor Inactive transcription factor

Translation of mRNA

FOXO1 (Degraded)

By inhibiting the inactivating kinase, insulin stimulates glycogen synthase. G6Pase levels PEPCK

P

Nuclear proteins

Gene expression and growth

Figure 51-6  Insulin signal-transduction system. When insulin binds to its receptor—which is a receptor

tyrosine kinase (RTK)—tyrosine kinase domains on the intracellular portion of the β chains become active. The activated receptor transduces its signals to downstream effectors by phosphorylating at tyrosine residues on the receptor itself, the IRS family (IRS-1, IRS-2, IRS-3, IRS-4), and other cytosolic proteins (e.g., SHC). SH2-containing proteins dock onto certain phosphorylated tyrosine groups on the IRSs and thus become activated. Not all of the signaling pathways are active in all of insulin’s target cells. For example, the liver cell does not rely on the GLUT4 transporter to move glucose in and out of the cell. Likewise, the liver is a very important target for regulation of the gluconeogenic enzymes by insulin, whereas muscle and adipose tissue are not. GS, glycogen synthase; GSK-3, glycogen synthase kinase 3; IF, initiation factor; PDK, phosphatidylinositol-dependent kinase.

P

1044

SECTION VIII  •  The Endocrine System

In controls, the maxium response is reached when only approximately 5% of the receptors are occupied by insulin.

In the cells from the type 2 diabetic, the same maximal response is reached, but only at a much higher insulin concentration.

Controls Glucose transport

0.1

Downregulation of receptors and impairment of postreceptor signaling in type 2 diabetes 1

10 100 1000 Concentration of insulin (µU/mL)

10,000

Figure 51-7  Response to insulin of normal and downregulated adipocytes.

High levels of insulin lead to downregulation of insulin receptors The number of insulin receptors expressed on the cell surface is far greater than that needed for the maximal biological response to insulin. For example, in the adipocyte, the glucose response to insulin is maximal when only ~5% of the receptors are occupied; that is, the target cells have many “spare” receptors for insulin. The number of insulin receptors present on the membrane of a target cell is determined by the balance among three factors: (1) receptor synthesis, (2) endocytosis of receptors followed by recycling of receptors back to the cell surface, and (3) endocytosis followed by degradation of receptors. Cells chronically exposed to high concentrations of insulin have fewer surface receptors than do those exposed to lower concentrations. This dynamic ability of cells to decrease the number of specific receptors on their surface is called downregulation. Insulin downregulates insulin receptors by decreasing receptor synthesis and increasing degradation. Such downregulation is one mechanism by which target tissues modulate their response to hormones. Downregulation of insulin receptors results in a decrease in the sensitivity of the target tissue to insulin without diminishing insulin’s maximal effect. One example of how downregulation can affect insulin’s action is shown in Figure 51-7, which illustrates the effect of increases in insulin concentration on glucose uptake in adipocytes from normal individuals and individuals with type 2 diabetes. Adipocytes from patients with type 2 diabetes (Box 51-5) have fewer insulin receptors per unit of surface area than do adipocytes from normal individuals. The markedly lower glucose transport across the entire physiological range of insulin concentrations in diabetic adipocytes is characteristic of insulin resistance. In healthy control adipocytes, glucose transport is maximal when only a few (~5%) of the receptors are occupied. In diabetic adipocytes, a much higher concentration of insulin is required, and a larger frac­ tion of the insulin receptors is occupied. However, the major effects in type 2 diabetes apparently are not the result of

a decrease in receptor number, but rather are caused by impairment in signaling downstream from the receptor. This impairment includes diminished activity of the insulin receptor tyrosine kinase, PI3K activity, and perhaps other steps along the pathway to GLUT4 recruitment to the plasma membrane (see Fig. 51-6). It is the summation of these multiple defects, only some of which have been identified, that leads to insulin resistance.

In liver, insulin promotes conversion of glucose to glycogen stores or to triacylglycerols Insulin’s actions on cellular targets frequently involve numerous tissue-specific enzymatic and structural processes. As we will see in this and the next two sections, the three principal targets for insulin action are liver, muscle, and adipose tissue. Because the pancreatic veins drain into the portal venous system, all hormones secreted by the pancreas must traverse the liver before entering the systemic circulation. For insulin, the liver is both a target tissue for hormone action and a major site of degradation. The concentration of insulin in portal venous blood before extraction by the liver is three to four times greater than its concentration in the systemic circulation. The hepatocyte is therefore bathed in a relatively high concentration of insulin and is thus well positioned to respond acutely to changes in plasma [insulin]. After feeding, the plasma [insulin] rises, triggered by glucose and by neural and incretin stimulation of β cells. In the liver, this insulin rise acts on four main processes involved in fuel metabolism. These divergent effects of insulin entail the use of multiple enzymatic control mechanisms, indicated by numbered boxes in Figure 51-8. Glycogen Synthesis and Glycogenolysis  Physiological increases in plasma [insulin] decrease the breakdown and utilization of glycogen and—conversely—promote the formation of glycogen from plasma glucose. Although moderately increased levels of insulin allow gluconeogenesis to persist, the hepatocytes store the gluconeogenic product— glucose-6-phosphate—as glycogen rather than releasing it as glucose into the bloodstream. At high concentrations, insulin can inhibit the gluconeogenic conversion of lactate/pyruvate and amino acids to glucose-6-phosphate. Glucose enters the hepatocyte from the blood via GLUT2, which mediates the facilitated diffusion of glucose. GLUT2 is present in abundance in the liver plasma membrane, even in the absence of insulin, and its activity is not influenced by insulin. Insulin stimulates glycogen synthesis from glucose by activating glucokinase (numbered box 1 in Fig. 51-8) and glycogen synthase (box 2). The latter enzyme contains multiple serine phosphorylation sites. Insulin causes a net dephosphorylation of the protein, thus increasing the enzyme’s activity. At the same time that glycogen synthase is being activated, increases in both insulin and glucose diminish the activity of glycogen phosphorylase (box 3). This enzyme is rate limiting for the breakdown of glycogen. The same enzyme that dephosphorylates (and thus acti­ vates) glycogen synthase also dephosphorylates (and thus inhibits) phosphorylase. Thus, insulin has opposite effects

Chapter 51  •  The Endocrine Pancreas

1045

BOX 51-5  Diabetes Mellitus

D

iabetes is the most common serious metabolic disease in humans. The hallmark of diabetes is an elevated blood glucose concentration, but this abnormality is just one of many biochemical and physiological alterations. Diabetes is not one disorder, but can arise as a result of numerous defects in regulation of the synthesis, secretion, and action of insulin. The type of diabetes that most commonly affects children is called type 1 IDDM. The diabetes that generally begins in adulthood and is particularly common in obese individuals is called type 2 or NIDDM.

Type 1 Diabetes 

N51-4

Type 1 diabetes is caused by an immune-mediated selective destruction of the β cells of the pancreas. The other cell types present in the islet are spared. The consequence of the loss of insulin, with the preservation of glucagon, can be viewed as an accelerated form of fasting or starvation. A healthy person who is fasting for several days continues to secrete insulin at a low rate that is sufficient to balance the action of glucagon in modulating the production of glucose and ketones by the liver. However, in type 1 diabetes, insulin deficiency is severe, and glucose and ketone production by the liver occur at a rate that greatly exceeds the rate at which they are being used. As a result, the concentration of these substances in blood begins to rise. Even when glucose concentrations reach levels 5 to 10 times normal, no insulin is secreted because β cells are absent. The increased glucose and ketones provide an immense solute load to the kidney that causes osmotic diuresis. In addition, the keto acids that are produced are moderately strong organic acids (pK < 4.0), and their increased production causes severe metabolic acidosis (see p. 635). If these patients are not treated with insulin, the acidosis and dehydration lead to death from diabetic ketoacidosis. With appropriate diagnosis and the availability of insulin as an effective treatment, persons with type 1 diabetes can lead full, productive lives. Indeed, some patients have been taking insulin successfully for treatment of type 1 diabetes for >75 years. As technology has improved, patients have been able to monitor their blood glucose levels themselves and adjust their insulin dosages accordingly, using specifically designed insulin analogs that have either short or very long half-lives, or insulin pumps that

on the opposing enzymes, with the net effect that it promotes glycogen formation. Insulin also inhibits glucose-6phosphatase (G6Pase; box 4), which otherwise converts glucose-6-phosphate (derived either from glycogenolysis or gluconeogenesis) to glucose. Glycogen is an important storage form of carbohydrate in both liver and muscle. The glycogen stored during the postprandial period is then available for use many hours later as a source of glucose. Glycolysis and Gluconeogenesis  Insulin promotes the conversion of some of the glucose taken up by the liver into pyruvate and—conversely—diminishes the use of pyruvate and other three-carbon compounds for gluconeogenesis. Insulin induces transcription of the glucokinase gene (numbered box 1 in Fig. 51-8) and thus results in increased synthesis of this enzyme, which is responsible for phosphorylating glucose to glucose-6-phosphate and initiating the metabolism of glucose. In acting to promote glycolysis and

continuously deliver insulin via a subcutaneous catheter. Thus, individuals with type 1 diabetes can avoid not only severe, lifethreatening episodes of ketoacidosis but also the long-term consequences of diabetes—namely, blood vessel injury that can lead to blindness, kidney failure, and accelerated atherosclerosis.

Type 2 Diabetes In type 2 diabetes, the cause of hyperglycemia is more complex. These individuals continue to make insulin. β cells not only are present but also are frequently hyperplastic (at least early in the course of the disease). For reasons still being defined, the β cells do not respond normally to increases in plasma glucose level by increasing insulin secretion. However, altered insulin secretion is only part of the problem. If we administered identical doses of insulin to the liver, muscle, and adipose tissue of a person with type 2 diabetes and a healthy control, we would find that the patient with type 2 diabetes is resistant to the action of insulin. Thus, both the secretion of insulin and the metabolism of glucose in response to insulin are abnormal in type 2 diabetes. Which problem—decreased insulin release or insulin resistance—is more important in provoking development of the diabetic state likely varies among individuals. Usually, these patients make enough insulin—and it is sufficiently active—that the severe ketoacidosis described above in patients with type 1 diabetes does not develop. The insulin resistance seen in individuals with type 2 diabetes appears to bring with it an increase in the prevalence of hypertension, obesity, and a specific dyslipidemia characterized by elevated TAGs and depressed high-density lipoproteins (see Fig. 46-15). Insulin resistance (along with one or more of these other metabolic abnormalities) is frequently found in individuals before the development of type 2 diabetes and is referred to as metabolic syndrome. This constellation of abnormalities is estimated to affect >45 million individuals in the United States alone. Because each component of this syndrome has adverse effects on blood vessels, these individuals are at particularly increased risk of early atherosclerosis. Tight control of glucose concentrations in both type 1 and type 2 diabetes, together with careful management of blood pressure and plasma lipids, can retard the development of many of the long-term complications of diabetes.

diminish gluconeogenesis, insulin induces the synthesis of a glucose metabolite, fructose-2,6-bisphosphate. This compound is a potent allosteric activator of phosphofructokinase (box 5), a key regulatory enzyme in glycolysis. Insulin also stimulates pyruvate kinase (box 6), which forms pyruvate, and stimulates pyruvate dehydrogenase (box 8), which catalyzes the first step in pyruvate oxidation. Finally, insulin promotes glucose metabolism by the hexose monophosphate shunt (box 7).  N51-5 In addition, insulin also inhibits gluconeogenesis at several steps. Insulin diminishes transcription of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK; numbered box 9 in Fig. 51-8), thus reducing the synthesis of a key regulatory enzyme required to form phosphoenolpyruvate from oxaloacetate early in the gluconeogenic pathway. The increased levels of fructose-2,6-bisphosphate also inhibit the activity of fructose-1,6-bisphosphatase (box 10), which is also part of the gluconeogenic pathway.

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SECTION VIII  •  The Endocrine System

Blood Liver

Extracellular space (of Disse) Non–insulinsensitive transporter (GLUT2)

Blood sinusoid

Hepatocyte cytosol 3 GLYCOGEN 2 GLYCOGENOLYSIS Glycogen SYNTHESIS Glucose-1-phosphate 1 Glucose

Glucose

Glucose-6-phosphate

Inhibit Activate

4

Hepatocyte Fructose-6-phosphate 5 10

7

Insulin

Fructose-1,6-bisphosphate

Insulin receptor

Phosphoenolpyruvate

Bile canaliculus

9 6 Pyruvate GLUCONEOGENESIS

GLYCOLYSIS

8 LIPOGENESIS 11

Malonyl CoA

Mitochondrion

Acetyl CoA

12 Fatty acids CoA VLDL

Triacylglycerols Lipid droplets

13 Carnitine carrier protein

Citric acid cycle Ketone bodies

PROTEIN METABOLISM Cellular amino acids

15

14 Protein

Figure 51-8  Effect of insulin on hepatocytes. Insulin has four major effects on liver cells. First, insulin promotes glycogen synthesis from glucose by enhancing the transcription of glucokinase (1) and by activating glycogen synthase (2). Additionally, insulin together with glucose inhibits glycogen breakdown to glucose by diminishing the activity of G6Pase (4). Glucose also inhibits glycogen phosphorylase (3). Second, insulin promotes glycolysis and carbohydrate oxidation by increasing the activity of glucokinase (1), phosphofructokinase (5), and pyruvate kinase (6). Insulin also promotes glucose metabolism via the hexose monophosphate shunt (7). Finally, insulin promotes the oxidation of pyruvate by stimulating pyruvate dehydrogenase (8). Insulin also inhibits gluconeogenesis by inhibiting the activity of PEPCK (9), fructose-1,6-bisphosphatase (10), and G6Pase (4). Third, insulin promotes the synthesis and storage of fats by increasing the activity of acetyl CoA carboxylase (11) and fatty-acid synthase (12) as well as the synthesis of several apoproteins packaged with VLDL. Insulin also indirectly inhibits fat oxidation because the increased levels of malonyl CoA inhibit CAT I (13). The inhibition of fat oxidation helps shunt fatty acids to esterification as TAGs and storage as VLDL or lipid droplets. Fourth, by mechanisms that are not well understood, insulin promotes protein synthesis (14) and inhibits protein breakdown (15).

Chapter 51  •  The Endocrine Pancreas

Lipogenesis  Insulin promotes the storage of fats and inhibits the oxidation of fatty acids (see Fig. 58-10) through allosteric and covalent modification of key regulatory enzymes, as well as by transcription of new enzymes (numbered boxes in Fig. 51-8). The pyruvate that is now available from glycolysis can be used to synthesize fatty acids. Insulin promotes dephosphorylation of acetyl coenzyme A (CoA) carboxylase 2 (ACC2; box 11), the first committed step in fatty-acid synthesis in the liver. This dephosphorylation leads to increased synthesis of malonyl CoA, which allosterically inhibits carnitine acyltransferase I (CAT I; box 13). This enzyme converts acyl CoA and carnitine to acylcarnitine, a reaction necessary for long-chain fatty acids to cross the inner mitochondrial membrane, where they can be oxidized. Thus, malonyl CoA inhibits fatty-acid transport and fat oxidation. At the same time, insulin stimulates fatty-acid synthase (box 12), which generates fatty acids. Thus, because insulin promotes the formation of malonyl CoA and fatty acids but inhibits fatty-acid oxidation, this hormone favors esterification of the fatty acids with glycerol within the liver to form triacylglycerols (TAGs). The liver can either store these TAGs in lipid droplets or export them as very-lowdensity lipoprotein (VLDL) particles (see p. 968). Insulin also induces the synthesis of several of the apoproteins that are packaged with the VLDL particle. The hepatocyte then releases these VLDLs, which leave the liver via the hepatic vein. Muscle and adipose tissue subsequently take up the lipids in these VLDL particles and either store them or oxidize them for fuel. Thus, by regulation of transcription, by allosteric activation, and by regulation of protein phosphorylation, insulin acts to promote the synthesis and storage of fat and diminish its oxidation in liver.  N51-8 Protein Metabolism  Insulin stimulates the synthesis of protein and simultaneously reduces the degradation of pro­ tein within the liver (numbered boxes in Fig. 51-8). The general mechanisms by which insulin stimulates protein synthesis (box 14) and restrains proteolysis (box 15) by the liver are complex and are less well understood than the mechanisms regulating carbohydrate and lipid metabolism. In summary, insulin modulates the activity of multiple regulatory enzymes, which are responsible for the hepatic metabolism of carbohydrates, fat, and protein. Insulin causes the liver to take up glucose from the blood and either store the glucose as glycogen or break it down into pyruvate. The pyruvate provides the building blocks for storage of the glucose carbon atoms as fat. Insulin also diminishes the oxidation of fat, which normally supplies much of the ATP used by the liver. As a result, insulin causes the liver, as well as other body tissues, to burn carbohydrates preferentially.

In muscle, insulin promotes the uptake of glucose and its storage as glycogen Muscle is a major insulin-sensitive tissue and the principal site of insulin-mediated glucose disposal. Insulin has four major effects on muscle. First, in muscle, unlike in the liver, glucose crosses the plasma membrane principally via GLUT4, an insulinsensitive glucose transporter. GLUT4, which is found virtually exclusively in striated muscle and adipose tissue, belongs

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to a family of proteins that mediate the facilitated diffusion of glucose (see p. 114). Insulin markedly stimulates GLUT4 in both muscle (Fig. 51-9) and fat (see below) by a process involving recruitment of preformed transporters from a membranous compartment in the cell cytosol out to the plasma membrane. Recruitment places additional glucose trans­porters in the plasma membrane, thereby increasing the Vmax of glucose transport into muscle and increasing the flow of glucose from the interstitial fluid to the cytosol. As discussed above, a different glucose transporter, GLUT2, mediates glucose transport into hepatocytes (see Fig. 51-8) and β cells, and insulin does not increase the activity of that transporter. The enzymatic steps regulated by insulin are indicated by numbered boxes in Figure 51-9. The second effect of insulin on muscle is to enhance the conversion of glucose to glycogen by activating hexokinase (numbered box 1 in Fig. 51-9)—different from the glucokinase in liver—and glycogen synthase (box 2). Third, insulin increases glycolysis and oxidation by increasing the activity of phosphofructokinase (box 3) and pyruvate dehydrogenase (box 4). Fourth, insulin also stimulates the synthesis of protein in skeletal muscle (box 5) and slows the degradation of existing proteins (box 6). The result is preservation of muscle protein mass, which has obvious beneficial effects in preserving strength and locomotion. The insulin-induced increase in glucose utilization permits the muscle to diminish fat utilization and allows it to store as TAGs some of the fatty acid that it removes from the circulation. The stored TAGs and glycogen are a major sources of energy that muscle can use later when called on to exercise or generate heat. Exercise and insulin have some interesting parallel effects on skeletal muscle. Both increase the recruitment of GLUT4 transporters to the sarcolemma and both increase glucose oxidation; therefore, both increase glucose uptake by muscle. Additionally, exercise and insulin appear to have synergistic effects on the above processes. Clinically, this synergism is manifest as a marked increase in insulin sensitivity induced by exercise and is exploited as part of the treatment of patients with diabetes mellitus. In muscle, as in the liver, insulin directs the overall pattern of cellular fuel metabolism by acting at multiple sites. In both tissues, insulin increases the oxidation of carbohydrate, thus preserving body protein and fat stores. Carbohydrate ingested in excess of that used immediately as an oxidative fuel is either stored as glycogen in liver and muscle or is converted to lipid in the liver and exported to adipose tissue and muscle.

In adipocytes, insulin promotes glucose uptake and conversion to TAGs for storage Adipose tissue is the third major insulin-sensitive tissue involved in the regulation of body fuel. Again, insulin has several sites of action in adipocytes. All begin with the same receptor-mediated action of insulin to stimulate several cellular effector pathways. Insulin has four major actions on adipocytes. First, like muscle, adipose tissue contains the insulinsensitive GLUT4 glucose transporter. In insulin-stimulated

Chapter 51  •  The Endocrine Pancreas

N51-8  Nonalcoholic Fatty Liver Disease Contributed by Fred Suchy There are differences in the hepatic metabolism of glucose and fructose of importance to human health. Hepatic glucose metabolism is tightly regulated by phosphofructokinase, which is inhibited by ATP and citrate. Thus, when energy status is sufficient, hepatic uptake of dietary glucose is inhibited and much of the consumed glucose will bypass the liver and reach the systemic circulation. In contrast, dietary fructose is metabolized to fructose-1-phosphate by fructokinase, which is not regulated by hepatic energy status and the inhibitory effects of high ATP and citrate levels. Thus, fructose uptake and metabolism by the liver is unregulated, and relatively little of ingested fructose reaches the systemic circulation. In the liver, the large fructose load can result in increased de novo lipogenesis and inhibition of fatty-acid oxidation. This process contributes to the development of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Owing to the current epidemic of obesity, NAFLD is now the most common liver disorder in adults and children.

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SECTION VIII  •  The Endocrine System

Insulin-sensitive GLUT4 transporter

Insulin

Insulin receptor activation recruits GLUT4 transporters from vesicles.

Muscle cell cytosol GLYCOGEN 2 GLYCOGENOLYSIS Glycogen SYNTHESIS Glucose-1-phosphate

Muscle fiber Glucose

Glucose

1

GLUT4

Insulin

Glucose-6-phosphate Inhibit Activate Fructose-6-phosphate

3 Fructose-1,6-bisphosphate

Insulin receptor

Phosphoenolpyruvate

Pyruvate

GLYCOLYSIS Lactate

Lactate

Ketones

Ketones

4

LIPOGENESIS

Mitochondrion Acetyl CoA

Malonyl CoA Fatty acids

Fatty acids CoA

Citric acid cycle

Triacylglycerols PROTEIN METABOLISM Cellular amino acids

Amino acids

6

5 Protein

Blood

Extracellular space

Figure 51-9  Effect of insulin on muscle. Insulin has four major effects on muscle cells. First, insulin promotes glucose uptake by recruiting GLUT4 transporters to the plasma membrane. Second, insulin promotes glycogen synthesis from glucose by enhancing the transcription of hexokinase (1) and by activating glycogen synthase (2). Third, insulin promotes glycolysis and carbohydrate oxidation by increasing the activity of hexokinase (1), phosphofructokinase (3), and pyruvate dehydrogenase (4). These actions are similar to those in liver; note that there is little or no gluconeogenesis in muscle. Fourth, insulin promotes protein synthesis (5) and inhibits protein breakdown (6).

cells, preformed transporters are recruited from an intracellular compartment to the cell membrane, which markedly accelerates the entry of glucose into the cell. Second, insulin promotes the breakdown of glucose to metabolites that will eventually be used to synthesize TAGs. Unlike in muscle or liver, little of the glucose taken up is stored as glycogen. Instead, the adipocyte glycolytically

metabolizes much of the glucose to α-glycerol phosphate, which it uses to esterify long-chain fatty acids into TAGs. The glucose not used for esterification goes on to form acetyl CoA and then malonyl CoA and fatty acids. Insulin enhances this flow of glucose to fatty acids by stimulating pyruvate dehydrogenase (numbered box 1 in Fig. 51-10) and acetyl CoA carboxylase (box 2).

Chapter 51  •  The Endocrine Pancreas

1049

Greater omentum Extracellular space

Blood Insulin receptor

Insulin

Glucose

Insulin-sensitive GLUT4 transporter

Adipocyte cytosol

GLYCOLYSIS

GLUT4 Glucose

Glucose-6-phosphate

4 Insulin stimulates the synthesis of lipoprotein lipase (LPL), which moves to the surface of the endothelial cell. Esterification Chylomicrons and VLDL LPL Fatty acids

Insulin

Insulin receptor activation recruits GLUT4 transporters from vesicles.

α-Glycerolphosphate

Fructose-6-phosphate LPL Fructose-1,6-bisphosphate

LIPOGENESIS

Phosphoenolpyruvate

Malonyl CoA Pyruvate Fatty acids Triacylglycerols

3

2

1

Mitochondrion

Acetyl CoA Glycerol Lipid droplets

Citric acid cycle

Inhibit Activate

Figure 51-10  Effect of insulin on adipocytes. Insulin has four major effects on adipocytes. First, insulin promotes glucose uptake by recruiting GLUT4 transporters to the plasma membrane. Second, insulin promotes glycolysis, which leads to the formation of α-glycerol phosphate. Insulin also promotes the conversion of pyruvate to fatty acids by stimulating pyruvate dehydrogenase (1) and acetyl CoA carboxylase (2). Third, insulin promotes the esterification of α-glycerol phosphate with fatty acids to form TAGs, which the adipocyte stores in fat droplets. Conversely, insulin inhibits HSL (3), which would otherwise break the TAGs down into glycerol and fatty acids. Fourth, insulin promotes the synthesis of LPL in the adipocyte. The adipocyte then exports this enzyme to the endothelial cell, where it breaks down the TAGs contained in chylomicrons and VLDL, yielding fatty acids. These fatty acids then enter the adipocyte for esterification and storage in fat droplets as TAGs.

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SECTION VIII  •  The Endocrine System

GLP-1 N—

Proglucagon

GRPP Pancreatic islet α cells

+ GRPP

GLP-2 —C

G IP-1

Primary processing

IP-2 Intestinal L cells

+

Glucagon

+ Major proglucagon fragment

Glicentin

+ GLP-1

+ IP-2

GLP-2

Figure 51-11  Synthesis of the glucagon molecule. The proglucagon molecule includes amino-acid sequences that, depending on how the peptide chain is cleaved, can yield glucagon-related polypeptide (GRPP), glucagon, intervening peptide 1 (IP-1), GLP-1, IP-2, and GLP-2. Proteases in the pancreatic α cells cleave proglucagon at points that yield GRPP, glucagon, and a C-terminal fragment. Proteases in neuroendocrine cells in the intestine cleave proglucagon to yield glicentin, GLP-1, IP-2, and GLP-2.

Third, insulin promotes the formation of TAGs by simple mass action; the increased levels of α-glycerol phosphate increase its esterification with fatty acids (principally C-16 and C-18) to yield TAGs. Some of the fatty acids are a result of the glucose metabolism noted above. Most of the fatty acids, however, enter the adipocyte from chylomicrons and VLDLs (see Table 46-4) in the blood. The cell sequesters these TAGs in lipid droplets, which form most of the mass of the adipose cell. Conversely, insulin restrains the activity of adipose triacylglycerol lipase (ATGL; see p. 1182), which converts TAGs to diacylglycerols (DAGs), and hormonesensitive lipase (HSL), which converts DAGs to monoacylglycerols (MAGs). In fat, these enzymes (numbered box 3 in Fig. 51-10) mediate the conversion of stored TAGs to fatty acids and glycerol for export to other tissues. Fourth, insulin induces the synthesis of a different enzyme—lipoprotein lipase (LPL). This lipase does not act on the lipid stored within the adipose cell. Rather, the adipocyte exports the LPL to the endothelial cell, where it resides on the extracellular surface of the endothelial cell, facing the blood and anchored to the plasma mem­ brane. In this location, the LPL acts on TAGs in chylomicrons and VLDLs and cleaves them into glycerol and fatty acids. These fatty acids are then available for uptake by nearby adipocytes, which esterify them with glycerol phosphate to form TAGs. This mechanism provides an efficient means by which insulin can promote the storage of lipid in adipose tissue.

GLUCAGON Glucagon is the other major pancreatic islet hormone that is involved in the regulation of body fuel metabolism. Ingestion of protein appears to be the major physiological stimulus for secretion of glucagon. Glucagon’s principal target tissue is the liver. Like insulin, glucagon is secreted first into the portal blood and is therefore anatomically well positioned to regulate hepatic metabolism. Although the amino acids released by digestion of a protein meal appear to be the major glucagon secretagogue, glucagon’s main actions on the liver appear to involve the regulation of carbohydrate and lipid metabolism. Glucagon is particularly important in stimulating glycogenolysis,

gluconeogenesis, and ketogenesis. Glucagon does not act solely on the liver but also has glycogenolytic action on cardiac and skeletal muscle, lipolytic action on adipose tissue, and proteolytic actions on several tissues. However, these extrahepatic effects appear to be more prominent at pharmacological concentrations of glucagon. At more physiological concentrations, the liver is the major target tissue. In many circumstances, glucagon’s actions on liver antagonize those of insulin, and the mechanism of glucagon action is understood in considerable detail.

Pancreatic α cells secrete glucagon in response to ingested protein Glucagon is a 31–amino-acid peptide (molecular weight, ~3500 Da) synthesized by α cells in the islets of Langerhans. In humans, the glucagon gene is located on chromosome 2. The initial gene product is the mRNA encoding preproglucagon. As is the case for insulin, a peptidase removes the signal sequence of preproglucagon during translation of the mRNA in the rough endoplasmic reticulum to yield proglucagon. Proteases in the α cells subsequently cleave the proglucagon (molecular weight, ~9000 Da) into the mature glucagon molecule and several biologically active peptides (Fig. 51-11). Neuroendocrine cells (i.e., L cells) within the gut process the proglucagon differently to yield not glucagon but GLP-1—a potent incretin—and other peptides. Pancreatic α Cells  The mature glucagon molecule is the major secretory product of the α cell. As with insulin, the fully processed glucagon molecule is stored in secretory vesicles within the cell’s cytosol. Although amino acids are the major secretagogues, the concentrations of amino acids required to provoke secretion of glucagon in vitro are higher than those generated in vivo. This observation suggests that other neural or humoral factors amplify the response in vivo, in a manner analogous to the effects of incretin on insulin secretion. However, the best studied incretin (GLP-1) inhib­ its glucagon secretion. Whereas both glucose and several amino acids stimulate insulin secretion by β cells, only amino acids stimulate glucagon secretion by α cells; glucose inhibits glucagon secretion. The signaling mechanism by which α cells recognize either amino acids or glucose is not known.

Chapter 51  •  The Endocrine Pancreas

Glucagon, like the incretins, is a potent insulin secretagogue. However, because most of the α cells are located downstream from the β cells (recall that the circulation of blood proceeds from the β cells and then out past the α cells), it is unlikely that glucagon exerts an important paracrine effect on insulin secretion. Intestinal L Cells  Proteases in neuroendocrine cells in the intestine process proglucagon differently than do α cells (see Fig. 51-11). L cells produce four peptide fragments: glicentin, GLP-1, intervening peptide 2 (IP-2), and GLP-2. Glicentin contains the amino-acid sequence of glucagon but does not bind to glucagon receptors. Both GLP-1 and GLP-2 are glucagon-like in that they cross-react with some antisera directed to glucagon, but GLP-1 and GLP-2 have very weak biological activity as glucagon analogs. However, GLP-1— released by the gut into the circulation in response to carbohydrate or protein ingestion—is one of the most potent incretins, stimulating insulin secretion. GLP-2 is not an incretin, and its biological actions are not known.

Glucagon, acting through cAMP, promotes the synthesis of glucose by the liver Glucagon is an important regulator of hepatic glucose production and ketogenesis in the liver. As shown in Figure 51-12, glucagon binds to a receptor that activates the heterotrimeric G protein Gαs, which stimulates membrane-bound adenylyl cyclase (see p. 53). The cAMP formed by the cyclase in turn activates PKA, which phosphorylates numerous regulatory enzymes and other protein substrates, thus altering glucose and fat metabolism in the liver. Whereas insulin leads to the dephosphorylation of certain key enzymes (i.e., glycogen synthase, acetyl CoA carboxylase, phosphorylase), glucagon leads to their phosphorylation. A particularly clear example of the opposing actions of insulin and glucagon involves the activation of glycogenolysis (see p. 1182). PKA phosphorylates the enzyme phosphorylase kinase (see Fig. 58-9), thus increasing the activity of phosphorylase kinase and allowing it to increase the phosphorylation of its substrate, glycogen phosphorylase b. The addition of a single phosphate residue to phosphorylase b converts it to phosphorylase a. Liver phosphorylase b has little activity in breaking the one to four glycosidic linkages of glycogen, but phosphorylase a is very active. In addition to converting phosphorylase b to the active phosphorylase a form, PKA also phosphorylates a peptide called inhibitor 1 (see Fig. 3-7). In its phosphorylated form, inhibitor I decreases the activity of phosphoprotein phosphatase 1 (PP1), which otherwise would dephosphorylate both phosphorylase kinase and phosphorylase a (converting them to their inactive forms). PP1 also activates glycogen synthase. Thus, via inhibitor I, glucagon modulates several of the enzymes involved in hepatic glycogen metabolism to provoke net glycogen breakdown. As a result of similar actions on the pathways of gluconeogenesis and lipid oxidation, glucagon also stimulates these processes. Conversely, glucagon restrains glycogen synthesis, glycolysis, and lipid storage. Glucagon also enhances gluconeogenesis by genomic effects, acting synergistically with glucocorticoids (see p. 1022). The genomic effects of glucagon occur as PKA

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phosphorylates the transcription factor cAMP response element–binding protein (CREB; see p. 89), which interacts with the cAMP response elements (CREs; see p. 89), increasing the expression of key gluconeogenic enzymes (e.g., G6Pase and PEPCK). Phosphorylated CREB also increas­ es the expression of peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), which also enhances the expression of key gluconeogenic enzymes. Insulin restrains the transcription of these two enzymes in two ways, both via the PI3K/Akt pathway (see Fig. 51-6). First, insulin increases the release of the transcription-factor domain of sterol regulatory element–binding protein 1 (SREBP-1; see pp. 87–88), which antagonizes the transcription of mRNA encoding the two enzymes. Second, insulin increases the phosphorylation of the transcription factor FOXO1, thereby promoting its movement out of the nucleus and subsequent degradation; this action prevents FOXO1 from binding to the promoter regions of G6Pase and PEPCK. These actions of glucagon can be integrated with our understanding of insulin’s action on the liver in certain physiological circumstances. For example, after an overnight fast, when insulin concentrations are low, glucagon stimulates the liver to produce the glucose that is required by the brain and other tissues for their ongoing function.  N51-9  With ingestion of a protein meal, absorbed amino acids provoke insulin secretion, which can inhibit hepatic glucose production and promote glucose storage by liver and muscle (see above). If the meal lacked carbohydrate, the secreted insulin could cause hypoglycemia. However, glucagon secreted in response to a protein meal balances insulin’s action on the liver and thus maintains glucose production and avoids hypoglycemia.

Glucagon promotes oxidation of fat in the liver, which can lead to ketogenesis Glucagon plays a major regulatory role in hepatic lipid metabolism. As we saw in our discussion of insulin (see Fig. 51-8), the liver can esterify fatty acids with glycerol to form TAGs, which it can store or export as VLDL particles. Alternatively, the liver can partially oxidize fatty acids—and form ketone bodies (see p. 1185)—or fully oxidize them to CO2. Whereas fatty-acid esterification and storage occur in the liver cytosol, oxidation and ketogenesis occur within the mitochondria. Glucagon stimulates fat oxidation indirectly by increasing the activity of CAT I (see pp. 1183–1185), which mediates the transfer of fatty acids across the outer mitochondrial membrane. Glucagon produces this stimulation by inhibiting acetyl CoA carboxylase, which generates malonyl CoA, the first committed intermediate in the synthesis of fatty acids by the liver. Malonyl CoA is also an inhibitor of the CAT system. By inhibiting acetyl CoA carboxylase, glucagon lowers the concentration of malonyl CoA, releases the inhibition of CAT I, and allows fatty acids to be transferred into the mitochondria. These fatty acids are oxidized to furnish ATP to the liver cell. If the rate of fatty-acid transport into the mitochondria exceeds the need of the liver to phosphorylate ADP, the fatty acids will be only partially oxidized; the result is the accumulation of the keto acids β-hydroxybutyric acid and acetoacetic acid, which are two of the three ketone

Chapter 51  •  The Endocrine Pancreas

N51-9  Maintaining Plasma Glucose Levels during Starvation Contributed by Fred Suchy Hepatic gluconeogenesis is critical to maintaining normal plasma glucose levels during starvation. Glucagon and glucocorticoids positively regulate gluconeogenesis through synergistic signaling pathways. Glucagon promotes the interaction of cAMP response element–binding protein (CREB; see p. 89) with CREB-binding protein (CBP; see p. 84) and CREB-regulated transcription coactivator 2 (CRTC2). Both CBP and CRTC2 facilitate the binding of CREB to cAMP response elements (CREs). The hepatocyte nuclear factors forkhead O box (FOXO) and peroxisome proliferator–activated receptor-γ (PPARγ) coactivator 1α (PGC-1α) also acts synergistically to increase transcription of gluconeogenic genes. The response to glucocorticoids is mediated by the glucocorticoid receptor, which binds to glucocorticoid response elements (GREs) in the promoters of gluconeogenic genes. Sirtuin 1, an NAD-dependent deacetylase, is another energy sensor and modifier of the transcriptional activity of some of these transcription factors. For example, it deacetylates and affects the activity of PGC-1α. In contrast, insulin secreted postprandially represses transcription of gluconeogenic enzymes through activation of the Akt signaling pathway.

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SECTION VIII  •  The Endocrine System

Blood

Extracellular space (of Disse) Glucagon receptor

Liver

Gαs

Hepatocyte cytosol

Blood sinusoid

2

Hepatocyte

GLUT2

AC

G protein

GLYCOGEN SYNTHESIS

PKA phosphorylates key enzymes in glycolysis and gluconeogenesis.

Adenylyl cyclase

Glycogen

PKA

cAMP

3

Glucose-1-phosphate GLYCOGENOLYSIS

1 Glucose

Inhibit Activate

Glucose-6-phosphate 4

Fructose-6-phosphate 5 10 Fructose-1,6-bisphosphate

Bile canaliculus

Phosphoenolpyruvate 6 Pyruvate GLUCONEOGENESIS

GLYCOLYSIS LIPID METABOLISM Malonyl CoA

Fatty acids

Mitochondrion 11

Carnitine carrier protein

Fatty acids CoA 13

Acetyl CoA

Citric acid cycle

9

Ketone bodies

Figure 51-12  Glucagon signal transduction. Glucagon generally antagonizes the effects of insulin in the liver. Glucagon binds to a Gαs-coupled receptor, activating the adenylyl cyclase–cAMP–PKA cascade. Glucagon has three major effects on liver cells. First, glucagon promotes net glycogen breakdown. Glucagon inhibits glycogen synthesis by reducing the activity of glucokinase (1) and glycogen synthase (2). However, glucagon promotes glycogen breakdown by activating glycogen phosphorylase (3) and G6Pase (4). Second, glucagon promotes net gluconeogenesis. The hormone inhibits glycolysis and carbohydrate oxidation by reducing the activity of glucokinase (1), phosphofructokinase (5), and pyruvate kinase (6). Glucagon also stimulates gluconeogenesis by increasing the transcription of PEPCK (9), fructose-1,6-bisphosphatase (10), and G6Pase (4). Third, glucagon promotes the oxidation of fats. The hormone inhibits the activity of acetyl CoA carboxylase (11). Glucagon indirectly stimulates fat oxidation because the decreased levels of malonyl CoA relieve the inhibition of malonyl CoA on CAT (13). The numbering scheme for these reactions is the same as that in Figure 51-8.

bodies. These keto acids can exit the mitochondria and the liver to be used by other tissues as oxidative fuel. During fasting, the decline in insulin and the increase in glucagon promote ketogenesis (see pp. 1185–1187); this process is of vital importance to the CNS, which can use keto acids but not fatty acids as fuel. In the adaptation to fasting, glucagon therefore plays the important role of stimulating

the conversion of fatty acids to ketones and provides the brain with the fuel that is needed to allow continued function during a fast. We discuss fasting in more depth beginning on pages 1188–1192.  N51-10 In addition to its effects on hepatic glucose and lipid metabolism, glucagon also has the extrahepatic actions of accelerating lipolysis in adipose tissue and proteolysis in

Chapter 51  •  The Endocrine Pancreas

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N51-10  Fasting Contributed by Eugene Barrett During fasting, falling insulin levels and rising glucagon levels promote the conversion of stored fat to ketone bodies and promote gluconeogenesis from muscle protein. Insulin and glucagon are the principal regulators of body fuel metabolism. The integrated secretion and action of insulin and glucagon are vital to fuel homeostasis. In humans, after an overnight fast, plasma [insulin] is low. The low [insulin] and the availability of glucagon result in the production of glucose by the liver from the breakdown of endogenous glycogen as well as from the conversion of noncarbohydrate substrates into glucose (i.e., gluconeogenesis). These processes are highly regulated so that the rate at which glucose is produced by the liver matches the rate at which it is used by tissues, especially by the CNS. If the amount of glucose being provided by the liver is inadequate, plasma [glucose] will decline. The β cell, sensing this decrease, reduces the amount of insulin being secreted, whereas the α cell increases glucagon secretion. These two changes will increase glucose production by the liver and correct the plasma [glucose] toward normal. Conversely, if glucose is being overproduced and plasma [glucose] rises, insulin secretion increases and glucagon secretion is suppressed. Hepatic glucose production therefore declines and plasma [glucose] returns toward normal.

If fasting continues for several more days, insulin secretion continues to decline and glucagon secretion increases. The decline in insulin concentration allows an increased rate of proteolysis in muscle and mobilization of fatty acids from adipose tissue. The amino acids released by muscle serve as a substrate for hepatic gluconeogenesis. This response is particularly important after the first several days of fasting, when hepatic glycogen stores have been depleted and gluconeogenesis is the major pathway of hepatic glucose production. At the same time, the increased glucagon concentration stimulates ketogenesis in the liver and provides ketone bodies for the CNS. Ketones provide an alternative fuel source for the brain that allows the brain to decrease its use of glucose. Because most of the glucose comes from gluconeogenesis—and because the building blocks for gluconeogenesis come from accelerated proteolysis—the availability of ketone bodies allows the body to use the energy stored in fat and spare body protein. Because much of this body protein comes from the structural proteins in skeletal muscle and because catabolism of these proteins impairs muscle function (such as strength and mobility), it is a clear survival advantage to have the brain burn fat and not protein for fuel. We discuss fasting beginning on pages 1188–1192.

Chapter 51  •  The Endocrine Pancreas

muscle. However, these effects are generally demonstrable only with high concentrations of glucagon, and although they may be important in certain pathological situations associated with greatly elevated glucagon concentrations (e.g., ketoacidosis or sepsis), they appear less important in the day-to-day actions of glucagon.

SOMATOSTATIN Somatostatin inhibits the secretion of growth hormone, insulin, and other hormones Somatostatin is made in the δ cells of the pancreatic islets (see Fig. 51-1), as well as in the D cells of the gastrointestinal tract (see pp. 868–870), in the hypothalamus, and in several other sites in the CNS (see pp. 993–994). Somatostatin was first described as a hypothalamic peptide that suppresses the release of growth hormone; growth hormone had also been called somatotropin, which accounts for the name somatostatin. In both pancreatic δ cells and the hypothalamus, somatostatin exists as both 14– and 28–amino-acid peptides. In the hypothalamus, the 14–amino-acid form is predominant, whereas in the gastrointestinal tract (including the δ cells), the 28–amino-acid form predominates. The 14– amino-acid form is the C-terminal portion of the 28–aminoacid form. The biological activity of somatostatin resides in these 14 amino acids. Somatostatin inhibits the secretion of multiple hormones, including growth hormone, insulin, glucagon, gastrin, vasoactive intestinal peptide (VIP), and thyroid-stimulating

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hormone. This property has led to therapeutic use of a longacting somatostatin analog (octreotide) in some difficult-totreat endocrine tumors, including those that produce growth hormone (acromegaly), insulin (insulinoma), or serotonin (carcinoid), among others. The concentration of somatostatin found in pancreatic venous drainage is sufficiently high to inhibit basal insulin secretion. Recall that blood flows from the center of each islet—which is where the bulk of the β cells are—to the periphery of the islet—which is where the δ cells tend to be located (see Fig. 51-1). This spatial arrangement minimizes the effect of somatostatin on the islet from which it is secreted. Whether somatostatin has important paracrine actions on some β cells or on α cells remains controversial. The islet cells also make other peptides; for example, pancreatic polypeptide is made in the F cells of the pancreas. As with insulin and glucagon, secretion of pancreatic polypeptide is altered by dietary intake of nutrients. However, whether pancreatic polypeptide has any actions in mammalian fuel metabolism is not clearly understood. Occasionally, islet cell tumors may develop and secrete gastrin, VIP, growth hormone–releasing factor, or other hormones. Although these individual instances prove that these peptides can be made by islet tissue, they have no known normal function in the islet.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 51  •  The Endocrine Pancreas

REFERENCES Books and Reviews Cryer PE: Hypoglycemia. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 12th ed. Philadelphia, Saunders, 2001, pp 1552–1578. DeFronzo RA, Ferrannini E, Keen H, Zimmet P: International Textbook of Diabetes Mellitus, 3rd ed. New York, Wiley, 2004. Holst JJ: The physiology of glucagon-like peptide 1. Physiol Rev 87:1409–1439, 2007. Klip A: The many ways to regulate glucose transporter 4. Appl Physiol Nutr Metab 34:481–487, 2009. Olefsky JM, Glass CK: Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 72:219–246, 2010. Journal Articles Bell GI, Pictet RL, Rutter WJ, et al: Sequence of the human insulin gene. Nature 284:26–32, 1980.

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Cherrington AD: Banting Lecture 1997. Control of glucose uptake and release by the liver in vivo. Diabetes 48:1198–1214, 1999. Dennis MD, Baum JI, Kimball SR, Jefferson LS: Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids. J Biol Chem 286:8287–8296, 2011. Gribble FM, Tucker SJ, Haug T, Ashcroft FM: MgATP activates the beta cell KATP channel by interaction with its SUR1 subunit. Proc Natl Acad Sci U S A 95:7185–7190, 1998. Jensen MV, Joseph JW, Ronnebaum SM, et al: Metabolic cycling in control of glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab 295:E1287–E1297, 2008. Li S, Brown MS, Goldstein JL: Bifurcation of insulin signaling pathway in rat liver: mTORC1 required for stimulation of lipogenesis, but not inhibition of gluconeogenesis. Proc Natl Acad Sci U S A 107:3441–3446, 2010. Miki T, Nagashima K, Tashiro F, et al: Defective insulin secretion and enhanced insulin action in KATP channel–deficient mice. Proc Natl Acad Sci U S A 95:10402–10406, 1998.

C H A P T E R 52  THE PARATHYROID GLANDS AND VITAMIN D Eugene J. Barrett and Paula Q. Barrett

CALCIUM AND PHOSPHATE BALANCE Calcium plays a critical role in many cellular processes, including hormone secretion, muscle contraction, nerve conduction, exocytosis, and the activation and inactivation of many enzymes. As described in Chapter 3, calcium also serves as an intracellular second messenger by carrying information from the cell membrane into the interior of the cell. It is therefore not surprising that the body very carefully regulates the plasma concentration of free ionized calcium, the physiologically active form of the ion, and maintains plasma ionized calcium concentration within a narrow range. Phosphate is no less important. Because it is part of the ATP molecule, phosphate plays a critical role in cellular energy metabolism. It also plays crucial roles in the activation and deactivation of enzymes. However, unlike calcium, the plasma phosphate concentration is not strictly regulated, and its levels fluctuate throughout the day, particularly after meals. Calcium and phosphate homeostasis are intimately tied to each other for two reasons. First, calcium and phosphate are the principal components of hydroxyapatite crystals [Ca10(PO4)6(OH)2], which by far constitute the major portion of the mineral phase of bone. Second, they are regulated by the same hormones, primarily parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (calcitriol) and, to a lesser extent, the hormone calcitonin. These hormones act on three organ systems—bone, kidneys, and gastrointestinal (GI) tract—to control the levels of calcium and phosphate in plasma. However, the actions of these hormones on calcium and phosphate are typically opposed in that a particular hormone may elevate the level of one ion while lowering that of the other. Figures 52-1 and 52-2 depict the overall daily balance of calcium and phosphate for an individual in a steady state.

The gut, kidneys, and bone regulate calcium balance In plasma, calcium exists in three physicochemical forms: (1) as a free ionized species (Ca2+), (2) bound to (more accurately, associated with) anionic sites on serum proteins (especially albumin), and (3) complexed with low-molecularweight organic anions (e.g., phosphate, citrate, and oxalate). The total concentration of all three forms in the plasma is normally 2.2 to 2.6 mM (8.8 to 10.6 mg/dL). In healthy 1054

individuals, ~45% of calcium is free, 45% is bound to protein, and 10% is bound to small anions. The body tightly regulates the ionized form of Ca2+ between 1.0 and 1.3 mM (4.0 and 5.2 mg/dL). The ionized form is the most important with regard to regulating the secretion of PTH and is involved in most of the biological actions of calcium. Most total-body calcium is located within bone, ~1 kg (see Fig. 52-1). The total amount of calcium in the extracellular pool is only a tiny fraction of this amount, ~1 g or 1000 mg. The typical daily dietary intake of calcium is ~800 to 1200 mg. Dairy products are the major dietary source of calcium. Although the intestines absorb approximately one half the dietary calcium (~500 mg/day), they also secrete calcium for removal from the body (~325 mg/ day), and therefore, the net intestinal uptake of calcium is only ~175 mg/day. The second major organ governing calcium homeostasis is bone, which in the steady state deposits ~280 mg/day of calcium and resorbs an equal amount. The third organ system involved, the kidney, filters ~10 times the total extracellular pool of calcium per day, ~10,000 mg/day. The kidneys reabsorb ~99% of this Ca2+, so that the net renal excretion of Ca2+ is ~1% of the filtered load (see Fig. 36-16). In a person in Ca2+ balance, urinary excretion (~175 mg/day) matches net absorption by the GI tract.

The gut, kidneys, and bone also regulate phosphate balance The concentration of total phosphate in adult plasma— predominantly inorganic phosphate in the form of H2 PO−4 and HPO2− 4 —ranges from 0.8 to 1.5 mM, a variation of 80%. It is ~50% higher in children. Laboratories report total plasma phosphate concentration as elemental phosphorus (range in adults, 2.5 to 4.5 mg/dL). Between 85% and 90% of the circulating inorganic phosphate is filterable by the kidneys, either ionized (50%) or complexed to Na+, Ca2+, or Mg2+ (40%); only a small proportion (10% to 15%) is protein bound. Like calcium, most total-body phosphate is present in bone, which contains ~0.6 kg of elemental phosphorus (see Fig. 52-2). A smaller amount of phosphorus (0.1 kg) resides in the soft tissues, mainly as organic phosphates, such as phospholipids, phosphoproteins, nucleic acids, and nucleotides. An even smaller amount (~500 mg) is present in the extracellular fluid (ECF) as inorganic phosphate. The daily

Chapter 52  •  The Parathyroid Glands and Vitamin D

Diet 1000 mg/day

Absorbed 500 mg/day

Gut

ECF 1000 mg (8.8–10.6 mg/dL)

Bone Resorption 280 mg/day

Secreted 325 mg/day Feces 825 mg/day

Formation 280 mg/day

Reabsorbed 9825 mg/day

1000 g

Filtered 10,000 mg/day

Kidneys

Urine 175 mg/day

Figure 52-1  Calcium distribution and balance. Note that all values are examples for a 70-kg human, expressed in terms of elemental calcium. These values can vary depending on factors such as diet.

Soft tissues 100 g Diet 1400 mg/day

Absorbed 1100 mg/day

Gut

ECF phosphate pool 500 mg (2.5–4.5 mg/dL)

Secreted 200 mg/day

Feces 500 mg/day

Formation 210 mg/day

Resorption 210 mg/day

Reabsorbed 6100 mg/day

Bone 600 g

Filtered 7000 mg/day

Kidneys

Urine 900 mg/day

Figure 52-2  Phosphate distribution and balance. Note that all values are examples for a 70-kg human, expressed in elemental phosphorus. These values can vary depending on factors such as diet.

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dietary intake of phosphorus is typically 1400 mg, mostly as inorganic phosphate. Again, dairy products are the major source. The net absorption of phosphate by the intestines is ~900 mg/day. In the steady state, bone has relatively small phosphate turnover, ~210 mg/day. The kidneys filter ~14 times the total extracellular pool of phosphate per day (~7000 mg/day) and reabsorb ~6100 mg/day. Hence, the net renal excretion of phosphorus is ~900 mg/day, the same as the net absorption by the GI tract.

surfaces of bone. Their activity is increased by cytokines, with RANK ligand being particularly important. Osteocytes are found within the bony matrix and are derived from osteoblasts that have encased themselves within bone. In response to mechanical loading, osteocytes produced both stimulatory and inhibitory cues. These cells stimulate the bone-forming activities of osteoblasts and lining cells by secreting growth factors such as osteocalcin and Wnt ligands. Osteocytes inhibit osteoblast activity by secreting antagonists of Wnt signaling, including sclerostin and dickkopf1. Osteocytes also appear to play a role in the transfer of mineral from the interior of bone to the growth surfaces. Bone remodeling consists of a carefully coordinated interplay of osteoblastic, osteocytic, and osteoclastic activities. As shown in Figure 52-3, bone consists of two types of bone tissue. Cortical (also called compact or lamellar) bone represents ~80% of the total bone mass. Cortical bone is the outer layer (the cortex) of all bones and forms the bulk of the long bones of the body. It is a dense tissue composed mostly of bone mineral and extracellular matrix elements, interrupted only by penetrating blood vessels and a sparse population of osteocytes nested within the bone. These osteocytes are interconnected with one another and with the osteoblasts on the surface of the bone by canaliculi, through which the osteocytes extend cellular processes. These

PHYSIOLOGY OF BONE Dense cortical bone and the more reticulated trabecular bone are the two major bone types Bone consists largely of an extracellular matrix composed of proteins and hydroxyapatite crystals, in addition to a small population of cells. The matrix provides strength and stability. The cellular elements continually remodel bone to accommodate growth and allow bone to reshape itself in response to varying loading stresses. Basically, bone has three types of bone cells. Osteoblasts promote bone formation. Osteoblasts and preosteoblasts are the principal target cells for PTH’s action to stimulate bone growth. Osteoclasts promote bone resorption and are found on the growth

Canaliculi Osteoblasts

Osteocytes

Haversian Calcified Osteoid canal bone matrix

Osteon

Haversian canal Periosteum Blood vessels

Osteocytes connected by canaliculi

Lining cells

Haversian canals Figure 52-3  Cortical and trabecular bone. Under the periosteum is a layer

Marrow cavity Trabecular (cancellous), spongy bone

Cortical (compact) bone

of compact cortical bone that surrounds the more reticulated trabecular bone. The fundamental unit of cortical bone is the osteon, a tube-like structure that consists of a haversian canal surrounded by ring-like lamellae. The inset shows a cross section through an osteon. The superficial lining cells surround the osteoblasts, which secrete osteoid, a matrix of proteins that are the organic part of bone. The lining cells are formed from osteoblasts that become quiescent. Osteocytes are osteoblasts that have become surrounded by matrix. Canaliculi allow the cellular processes of osteocytes to communicate, via gap junctions, with each other and with osteoblasts on the surface. Trabecular bone has both osteoblasts and osteoclasts on its surface; this is where most bone remodeling takes place.

Chapter 52  •  The Parathyroid Glands and Vitamin D

connections permit the transfer of Ca2+ from the interior of the bone to the surface, a process called osteocytic osteolysis. Dense cortical bone provides much of the strength for weight bearing by the long bones. Trabecular (or cancellous or medullary) bone constitutes ~20% of the total bone mass. It is found in the interior of bones and is especially prominent within the vertebral bodies. It is composed of thin spicules of bone that extend from the cortex into the medullary cavity (see Fig. 52-3, inset). The lacework of bone spicules is lined in many areas by osteoblasts and osteoclasts, the cells involved in bone remodeling. Trabecular bone is constantly being synthesized and resorbed by these cellular elements. Bone turnover also occurs in cortical bone, but the fractional rate of turnover is much lower. When the rate of bone resorption exceeds that of synthesis over time, the loss of bone mineral produces the disease osteoporosis.

The extracellular matrix forms the nidus for the nucleation of hydroxyapatite crystals Collagen and the other extracellular matrix proteins that form the protein matrix of bone are called osteoid. Osteoid provides sites for the nucleation of hydroxyapatite crystals, the mineral component of bone. Osteoid is not a single compound, but a highly organized matrix of proteins synthesized principally by osteoblasts. Type I collagen accounts for ~90% of the protein mass of osteoid. It comprises a triple helix of two α1 monomers and one α2 collagen monomer. While still within the osteoblast, monomers self-associate into helical structures. After secretion from the osteoblast, helices associate into collagen fibers; cross-linking of collagen occurs both within a fiber and between fibers. Collagen fibers are arranged in the osteoid in a highly ordered manner. The organization of collagen fibers is important for the tensile strength (i.e., the ability to resist stretch or bending) of bone. In addition to providing tensile strength, collagen also acts as a nidus for nucleation of bone mineralization. Within the collagen fibers, the crystals of hydroxyapatite are arranged with their long axis aligned with the long axis of the collagen fibers. Several other osteoblast-derived proteins are important to the mineralization process, including osteocalcin and osteonectin. Osteocalcin is a 6-kDa protein synthesized by osteoblasts at sites of new bone formation. 1,25-Dihydroxyvitamin D induces the synthesis of osteocalcin. Osteocalcin has an unusual structure: it possesses three γ-carboxylated glutamic acid residues. These residues are formed by post-translational modification of glutamic acid by vitamin K–dependent enzymes. Like other proteins with γ-carboxylated glutamic acid, osteocalcin binds Ca2+ avidly. It binds hydroxyapatite, the crystalline mineral of bone, with even greater avidity. This observation has led to the suggestion that osteocalcin participates in the nucleation of bone mineralization at the crystal surface. Osteonectin, a 35-kDa protein, is another osteoblast product that binds to hydroxyapatite. It also binds to collagen fibers and facilitates the mineralization of collagen fibers in vitro. Additional proteins have been identified that appear to participate in the mineralization process. For instance, extracellular glycoproteins present in bone may

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inhibit mineralization and their removal may be necessary for mineralization to occur.

Bone remodeling depends on the closely coupled activities of osteoblasts and osteoclasts In addition to providing the proteins in osteoid, osteoblasts promote mineralization by exporting Ca2+ and PO3− 4 from intracellular vesicles that have accumulated these minerals. Exocytosis of Ca2+ and PO3− 4 raises the local extracellular concentration of these ions around the osteoblast to levels that are higher than in the bulk ECF, which promotes crystal nucleation and growth (Fig. 52-4). Bone formation along spicules of trabecular bone occurs predominantly at sites of previous resorption by osteoclasts. The processes of bone resorption and synthesis are thus spatially coupled within an active basic multicellular unit (BMU). In adults, 1 to 2 million BMUs actively remodel bone. Vitamin D and PTH stimulate osteoblastic cells to secrete factors—such as macrophage colony-stimulating factor (M-CSF; see p. 431)—that cause osteoclast precursors to proliferate (see Fig. 52-4). These precursors differentiate into mononuclear osteoclasts and then, with further stimulation by RANK ligand (also released by PTH-stimulated osteoblasts), fuse to become multinucleated osteoclasts. Osteoclasts resorb bone in discrete areas in contact with the “ruffled border” of the cell (Fig. 52-5). The osteoclast closely attaches to the bone matrix when integrins on its membrane attach to vitronectin in the bone matrix. The osteoclast—in reality a one-cell epithelium—then secretes acid and proteases across its ruffled border membrane into a confined resorption space (the lacuna). The acid secretion is mediated by a V-type H pump (see pp. 118–119) and the ClC-7 Cl− channel at the ruffled border membrane. Abundant intracellular carbonic anhydrase provides the H+. Cl-HCO3 exchangers, located in the membrane that faces the blood, remove the HCO3− formed as a byproduct by the carbonic anhydrase. The acidic environment beneath the osteoclast dissolves bone mineral and allows acid proteases to hydrolyze the exposed matrix proteins. Having reabsorbed some of the bone in a very localized area, the osteoclast moves away from the pit or trough in the bone that it has created. Osteoblastic cells replace the osteoclast and now build new bone matrix and promote its mineralization. RANK ligand (RANKL), previously called osteoprotegerin ligand, appears to be a major stimulator of both the differentiation of preosteoclasts to osteoclasts (see Fig. 52-4) and the activity of mature osteoclasts (see Fig. 52-5). RANKL is a member of the tumor necrosis factor (TNF) cytokine family and exists both as a membrane-bound form (mRANKL) on the surface of stromal cells and osteoblasts, and as a soluble protein (sRANKL) secreted by these same cells. RANKL binds to and stimulates a membrane-bound receptor of the osteoclast called RANK (receptor for activation of nuclear factor κB), a member of the TNF receptor family. The interaction is essential for the formation of mature osteoclasts. The activity of RANKL is under the control of a soluble member of the TNF receptor family called osteoprotegerin (OPG; from the Latin osteo [bone] + protegere [to protect]).

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Osteoclast precursors

Stem cells

Mononuclear osteoclast (preosteoclasts) M-CSF

IL-6R

RANK OPG

Osteoclast (multinucleated)

RANKL Vitamin D

IL-6 PTH

Osteoblasts Ca2+

Pi

Nucleation BONE FORMATION

Collagen fibers

Sealing zone

BO NE

N RESORPTIO

Growth factors Osteocytes

Figure 52-4  Bone formation and resorption. PTH and vitamin D stimulate osteoblastic cells to secrete agents that induce stem cells to differentiate into osteoclast precursors, mononuclear osteoclasts, and finally mature, multinucleated osteoclasts. Thus, PTH indirectly promotes bone resorption. Osteoblasts also secrete Ca2+ and inorganic phosphate (Pi), which nucleate on the surface of bone. IL-6R, interleukin-6 receptor.

Like RANKL, OPG is produced by osteoblastic and stromal cells (see Fig. 52-4). By scavenging RANKL, OPG limits osteoclastogenesis, thereby protecting bone from osteoclastic activity. The precise role of RANKL, RANK, and OPG in the development of various forms of osteoporosis (diminished bone density) and osteopetrosis (increased bone density) is only beginning to be understood. However, the balance between OPG and RANKL production by the osteoblast/stromal cell appears to be a very important factor in the development of osteoporosis from either estrogen deficiency or glucocorticoid excess. In both cases, RANKL production rises and OPG production falls. In 2010, the U.S. Food and Drug Administration approved denosumab, a humanized monoclonal antibody against RANKL, for the treatment of postmenopausal osteoporosis. In rare human monogenic syndromes, defects of the Wnt signaling system are associated with marked increases or decreases in bone mass. Moreover, targeted disruption of specific Wnt antagonists in mice reveals the major role of Wnt in osteoblastogenesis and in modulation of the activity of both osteoblasts and osteocytes. Wnt increases the differentiation of mesenchymal stem cells and preosteoblasts, thereby increasing bone-forming capacity. In addition, Wnt increases the production of OPG, which competes with RANKL and thereby decreases osteoclastogenesis. New therapies to treat or prevent bone loss based on manipulation of Wnt signaling offer substantial promise.

PARATHYROID HORMONE Plasma Ca2+ regulates the synthesis and secretion of PTH Humans have four parathyroid glands, two located on the posterior surface of the left lobe of the thyroid and two more on the right. Combined, these four glands weigh 15 mg/dL) and severe hyperparathyroidism. The condition is life threatening and is characterized by markedly elevated plasma [Ca2+], neuronal malfunction, demineralization of bone, and calcification of soft tissues. These infants die unless the inappropriately regulated parathyroid glands are removed. Like the parathyroid gland, the renal TAL and DCT have abundant plasma-membrane Ca2+ receptors on their basolateral membranes. These receptors respond to changes in plasma Ca2+ and inhibit Ca2+ reabsorption (see p. 789). Thus, with a mutated receptor, renal Ca2+ reabsorption may not be inhibited until plasma [Ca2+] rises to abnormally high levels. The result would be the increased Ca2+ reabsorption and the hypocalciuria characteristic of FHH. The discovery of CaSR led to the development of a CaSR agonist that mimics Ca2+ (a calcimimetic). This drug has now come into clinical use for treating patients with parathyroid cancer or hyperparathyroidism secondary to chronic renal disease. Calcimimetics decrease the secretion of PTH and secondarily decrease plasma [Ca2+].

Chapter 52  •  The Parathyroid Glands and Vitamin D

In bone, PTH can promote net resorption or net deposition The second major target tissue for PTH is bone, in which PTH promotes both bone resorption and bone synthesis. Bone Resorption by Indirect Stimulation of Osteoclasts 

The net effect of persistent increases of PTH on bone is to stimulate bone resorption, thus increasing plasma [Ca2+]. Osteoblasts express abundant surface receptors for PTH; osteoclasts do not. Because osteoclasts lack PTH receptors, PTH by itself cannot regulate the coupling between osteoblasts and osteoclasts. Rather, PTH acts on osteoblasts and osteoclast precursors to induce the production of several cytokines that increase both the number and the activity of bone-resorbing osteoclasts. PTH causes osteoblasts to release agents such as M-CSF and stimulates the expression of RANKL, actions that promote the development of osteoclasts (see Fig. 52-4). In addition, PTH and vitamin D stimulate osteoblasts to release interleukin-6 (IL-6), which stimulates existing osteoclasts to resorb bone (see Fig. 52-5). One of the initial clues that cytokines are important mediators of osteoclastic bone resorption came from observations on patients with multiple myeloma—a malignancy of plasma cells, which are of B-lymphocyte lineage. The tumor cells produce several proteins that activate osteoclasts and enhance bone resorption. These proteins were initially called osteoclast-activating factors. We now know that certain lymphocyte-derived proteins strongly activate osteoclastic bone resorption, including RANKL, lymphotoxin, IL-1, and TNF-α.

Bone Resorption by Reduction in Bone Matrix  PTH also changes the behavior of osteoblasts in a manner that can promote net loss of bone matrix. For example, PTH inhibits collagen synthesis by osteoblasts and also promotes the production of proteases that digest bone matrix. Digestion of matrix is important because osteoclasts do not easily reabsorb bone mineral if the bone has an overlying layer of unmineralized osteoid. Bone Deposition  Whereas persistent increases in PTH favor net resorption, intermittent increases in plasma [PTH] have predominately bone-synthetic effects, inducing higher rates of bone formation and mineral apposition. PTH promotes bone synthesis by three mechanisms. First, PTH promotes bone synthesis directly by activating Ca2+ channels in osteocytes, a process that leads to a net transfer of Ca2+ from bone fluid to the osteocyte. The osteocyte then transfers this Ca2+ via gap junctions to the osteoblasts at the bone surface. This process is called osteocytic osteolysis. The osteoblasts then pump this Ca2+ into the extracellular matrix, which contributes to mineralization. Second, PTH decreases the production of sclerostin by osteocytes. Lower levels of plasma sclerostin and dickkopf1 promote osteoblastic differentiation and also inhibit osteoblastic apoptosis. Third, PTH stimulates bone synthesis indirectly in that osteoclastic bone resorption leads to the release of growth factors trapped within the matrix; these include insulin-like growth factor 1 (IGF-1), IGF-2, fibroblast growth factor 2 (FGF2), and transforming growth factor-β. Finally, PTH stimulates osteoblasts

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to produce OPG and thereby interfere with RANKL activation of osteoclasts. The PTH 1-34 peptide is now available as a pharmacological agent for the treatment of osteoporosis. Clinical data show marked increases in bone density—particularly within the axial skeleton—in response to injections of PTH 1-34 once or twice daily. The effects on trabecular bone are striking, with less positive responses seen in cortical bone— particularly in the limbs.

VITAMIN D The active form of vitamin D is its 1,25-dihydroxy metabolite By the 1920s, investigators recognized that dietary deficiency of a fat-soluble vitamin was responsible for the childhood disease rickets (Box 52-2). This disorder is characterized

BOX 52-2  Rickets and Osteomalacia

D

eficiency of vitamin D in children produces the disease rickets, in which bone has abnormal amounts of unmineralized osteoid. Both cortical and trabecular bone are involved. The lack of mineralization diminishes bone rigidity and leads to a characteristic bowing of the long bones of the legs of affected children. In adults, vitamin D deficiency produces a disorder called osteomalacia. Microscopically, the bone looks very much the same in adult and childhood vitamin D deficiency. However, because the longitudinal growth of the long bones has been completed in adults, bowing of weight-bearing bones does not occur. Instead, the increased unmineralized osteoid content of bone causes a decline in bone strength. Affected individuals are more prone to the development of bone fractures. These fractures may be very small and difficult to see radiographically. As more and more of the bone surface is covered by osteoid and as recruitment of new osteoclasts is diminished, osteoclastic bone resorption is impaired and hypocalcemia develops. Hypocalcemia causes nerves to become more sensitive to depolarization. In sensory nerves, this effect leads to sensations of numbness, tingling, or burning; in motor nerves, it leads to increased spontaneous contractions, or tetany. Although rickets and osteomalacia are very uncommon in developed countries because of vitamin D supplementation, milder degrees of vitamin D deficiency are recognized increasingly in the elderly population, in whom milk consumption and sunlight exposure are frequently inadequate. The resulting fall in plasma [Ca2+] can lead to mild secondary hyperparathyroidism. Such continuous elevations of PTH can lead to further bone resorption and worsening osteoporosis. Rickets or osteomalacia also can occur with impaired ability of the kidney to 1-hydroxylate the 25-hydroxyvitamin D previously synthesized in the liver. An acquired impairment is seen in many patients with chronic renal failure, in which the activity of 1α-hydroxylase is reduced. The genetic form of the 1α-hydroxylase deficiency is a rare autosomal recessive disorder. Either form is called vitamin D–dependent rickets because it can be successfully treated with either 1,25dihydroxyvitamin D or doses of dietary vitamin D2 or D3 that are higher (by ~10- to 100-fold) than the 400 U/day used to prevent nutritional rickets.

Chapter 52  •  The Parathyroid Glands and Vitamin D

N52-2  Metabolism of Vitamin D3 and D2 Contributed by Eugene Barrett Vitamin D3

Vitamin D3—or cholecalciferol—actually can be thought of as a hormone because it can arise entirely from the metab­ olism of an endogenous source (7-dehydroxycholesterol) and because it acts through a specific receptor. Ultraviolet light triggers the cleavage in the skin of the B ring of 7dehydroxycholesterol, creating an unstable intermediate that—over a period of about 2 days—rearranges to form cholecalciferol (vitamin D3). Vitamin D3 can also come from animal sources in the diet. However, vitamin D3 is not active as such. In the liver, a P-450 enzyme hydroxylates vitamin D3 at the 25 position, creating 25-hydroxyvitamin D3. Then in the proximal-tubule cells of the kidney, another P-450 enzyme hydroxylates 25-hydroxyvitamin D3 at position 1, forming 1,25dihydroxyvitamin D3, the active form of vitamin D3.

Vitamin D2

Vitamin D2, which comes exclusively from dietary plant sources, differs from vitamin D3 only in the side chain off carbon 17 in ring D. Like vitamin D3, vitamin D2 undergoes 25-hydroxylation in the liver and 1-hydroxylation in the kidney. Also like vitamin D3, the 1,25-dihydroxylated metabolite of vitamin D2 is about 1000-fold more active than the 25monohydroxylated form.

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SECTION VIII  •  The Endocrine System

clinically by hypocalcemia and multiple skeletal abnormalities. Dietary replacement of vitamin D corrects this disorder and has led to the practice of adding vitamin D to milk, bread, and other products. This practice has greatly reduced the prevalence of this previously common disorder. Vitamin D exists in the body in two forms, vitamin D3 and vitamin D2 (Fig. 52-9).  N52-2  Vitamin D3 can be synthesized from the 7-dehydrocholesterol that is present in the skin, provided sufficient ultraviolet light is absorbed. This observation explains why nutritional rickets was more prevalent in northern countries, where people have reduced skin exposure to sunlight. Vitamin D3 is also available from several natural sources, including cod and halibut liver, eggs, and fortified milk. Vitamin D2 is obtained only from the diet, largely from vegetables. Vitamin D3 (see Fig. 52-9A) and vitamin D2 (see Fig. 52-9B) differ only in the side chains of ring D. The side chain in vitamin D3 (cholecalciferol) is characteristic of cholesterol, whereas that of vitamin D2 (ergocalciferol) is characteristic of plant sterols. Vitamin D (i.e., either D2 or D3) is fat soluble, but water insoluble. Its absorption from the intestine depends on its solubilization by bile salts (see p. 933). In the circulation, vitamin D is found either solubilized with chylomicrons (see pp. 932–933) or associated with a vitamin D–binding protein. Most of the body stores of vitamin D are located in body fat. The body’s pools of vitamin D are large, and only 1% to 2% of the body’s vitamin D is turned over each day. Therefore, several years of very low dietary intake (as well as diminished endogenous synthesis) are required before the endogenous pools are depleted and deficiency develops. The principal active form of vitamin D is not vitamin D2 or D3, but rather a dihydroxylated metabolite of either. Hydroxylation of vitamin D proceeds in two steps (see Fig. 52-9A). When circulating levels of 25-hydroxyvitamin D are low, adipocytes release vitamin D into the blood plasma. A cytochrome P-450 mixed-function oxidase, principally in the liver, creates the first hydroxyl group at carbon 25. The 25-hydroxylation of vitamin D does not appear to be highly regulated, but rather it depends on the availability of vitamin D2 or D3. The second hydroxylation reaction occurs in the renal proximal tubule under the tight control of PTH, vitamin D itself, and FGF23 (see p. 787). PTH stimulates this 1-hydroxylation, whereas FGF23 and 1,25dihydroxyvitamin D (the reaction product) both inhibit the process (see Fig. 52-8). In addition to vitamins D2 and D3 and their respective 25-hydroxy and 1,25-dihydroxy metabolites, >15 other metabolites of vitamin D have been identified in plasma. However, the specific physiological function of these metabolites, if any, is unclear. Although considered a vitamin because of its dietary requirement, vitamin D can also be considered a hormone, both because it is endogenously synthesized and because even the fraction that arises from the diet must be metabolized to a biologically active form. Vitamin D and its metabolites, like the steroid hor­ mones, circulate bound to a globulin, in this case a 52-kDa vitamin D–binding protein. This binding protein appears

A

METABOLISM OF VITAMIN D3 25

7–Dehydrocholesterol

A

B

HO Skin

UV light

21

22 20 17

18 11 9

12

D 16

13

C 14

26 23

24

25

H

27

15

8

Cholecalciferol (Vitamin D3)

7

6

19

4 3

HO

5

A

10

2

CH2

1

Liver

25

OH

25–Hydroxycholecalciferol (25–OHD3) CH2 HO Kidney

25

OH

1,25–(OH)2D3

CH2 1

HO B

OH

VITAMIN D2

H Ergocalciferol (Vitamin D2) CH2 HO

N52-2

D

C 1

A Figure 52-9  Forms of vitamin D. UV, ultraviolet. 

H

Chapter 52  •  The Parathyroid Glands and Vitamin D

particularly important for carriage of vitamins D2 and D3 in plasma because they are less soluble than their hydroxylated metabolites. Vitamin D and its metabolites arrive at target tissues and, once in the cytosol, associate with the VDR, a transcription factor that is in the family of nuclear receptors (see pp. 71–72). Like the thyroid hormone receptor (see Table 3-6), VDR forms a heterodimer with RXR. The VDR specifically recognizes the 1,25-dihydroxyvitamin D with an affinity that is three orders of magnitude higher than that for 25-hydroxyvitamin D. However, because the circulating concentration of 25-hydroxyvitamin D is ~1000-fold higher than that of 1,25-dihydroxyvitamin D, both species probably contribute to the biological actions of the hormone. The biological actions of 1,25-dihydroxyvitamin D appear to be expressed principally, but not exclusively, via regulation of the transcription of a variety of proteins. The VDR/RXR complex associates with a regulatory site in the promoter region of the genes coding for certain vitamin D–regulated proteins. Thus, the occupied VDR alters the synthesis of these vitamin D–dependent proteins. An example is PTH, which stimulates the formation of 1,25-dihydroxyvitamin D. The 5′ regulatory region of the PTH gene has a VDR consensus sequence; when occupied by the VDR complex, this element diminishes transcription of the PTH gene.

Vitamin D, by acting on the small intestine and kidney, raises plasma [Ca2+] and thus promotes bone mineralization The actions of vitamin D can be grouped into two categories: actions on classic target tissues involved in regulating body mineral and skeletal homeostasis, and a more general action that regulates cell growth. The actions of vitamin D on the small intestine, bone, and kidney serve to prevent any abnormal decline or rise in plasma [Ca2+]. Small Intestine  In the duodenum, 1,25-dihydroxyvitamin D increases the production of several proteins that enhance Ca2+ absorption. Figure 52-10A summarizes the intestinal absorption of Ca2+, which moves from the intestinal lumen to the blood by both paracellular and transcellular routes (see p. 938). In the paracellular route, which occurs throughout the small intestine, Ca2+ moves passively from the lumen to the blood; 1,25-dihydroxyvitamin D does not regulate this pathway. The transcellular route, which occurs only in the duodenum, involves three steps. First, Ca2+ enters the cell across the apical membrane via TRPV6 Ca2+ channels (see p. 938). Second, the entering Ca2+ binds to several highaffinity binding proteins, particularly calbindin. These proteins, together with the exchangeable Ca2+ pools in the RER and mitochondria, effectively buffer the cytosolic Ca2+ and maintain a favorable gradient for Ca2+ entry across the apical membrane of the enterocyte. Thus, the intestinal cell solves the problem of absorbing relatively large amounts of Ca2+ while keeping its free cytosolic [Ca2+] low. Third, the enterocyte extrudes Ca2+ across the basolateral membrane by means of both a Ca pump and an Na-Ca exchanger. Vitamin D promotes intestinal Ca2+ absorption primarily by genomic effects that involve induction of the synthesis of epithelial Ca2+ channels and pumps and Ca2+-binding proteins, as well as other proteins (e.g., alkaline phosphatase).

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The effect of PTH to stimulate intestinal Ca2+ absorp­ tion is thought to be entirely indirect and mediated by PTH’s action to increase the renal formation of 1,25dihydroxyvitamin D (see Fig. 52-8), which then enhances intestinal Ca2+ absorption. Vitamin D also stimulates phosphate absorption by the small intestine (see Fig. 52-10B). The initial step—as in the renal proximal tubule (see pp. 785–786)—is mediated by the NaPi cotransporter and appears to be rate limiting for transepithelial transport and subsequent delivery of phosphate to the circulation. 1,25-Dihydroxyvitamin D stimulates the synthesis of this transport protein and thus promotes phosphate entry into the mucosal cell. Kidney  In the kidney, vitamin D appears to act syner­ gistically with PTH to enhance Ca2+ reabsorption in the DCT (see p. 789). High-affinity Ca2+-binding proteins, similar to those found in the intestinal mucosa, have been specifically localized to this region of the kidney. It appears that PTH is a more potent regulator of Ca2+ reabsorption than is vitamin D (see Fig. 52-8). Indeed, parathyroidectomy increases the fractional excretion of Ca2+, and even high doses of vitamin D cannot correct this effect. In addition, as in the intestine, vitamin D promotes phosphate reabsorption in the kidney. The effects of vitamin D on phosphate reabsorption, like its effects on Ca2+, are less dramatic than those of PTH. Finally, 1,25-dihydroxyvitamin D directly inhibits the 1-hydroxylation of vitamin D, establishing a negativefeedback loop. Bone  The actions of vitamin D on bone are complex and are the result of both indirect and direct actions. The overall effect of vitamin D replacement in animals with diet-induced vitamin D deficiency is to increase the flux of Ca2+ into bone. However, as we see below, the major effects of vitamin D on bone are indirect: the action of vitamin D on both the small intestine and the kidneys makes more Ca2+ available to mineralize previously unmineralized osteoid. The direct effect of vitamin D on bone is via both osteoblasts and osteoclast precursor cells, both of which have VDRs. Vitamin D increases both osteoblastic and osteoclastic differentiation; when these activities are balanced, vitamin D simply increas­ es bone turnover. However, when vitamin D is present in excess, it favors bone resorption because osteoblasts produce certain proteins with matrix-destroying properties (e.g., alkaline phosphatase, collagenase, plasminogen activator) as well as proteins that favor osteoclastogenesis (e.g., RANKL rather than OPG; see Fig. 52-4). Thus, because vitamin D can directly increase the number of mature osteoclasts, supplying vitamin D to bone obtained from vitamin D–deficient animals in in vitro experiments mobilizes Ca2+ from bone into the medium. Additional evidence that vitamin D directly promotes bone resorption comes from experiments on rachitic animals who are maintained on a calcium-deficient diet. Treating these animals with vitamin D causes plasma [Ca2+] to rise, an indication of net bone resorption. At the same time, however, the elevated plasma [Ca2+] promotes the mineralization of previously unmineralized osteoid—at the expense of bone resorption from other sites. In summary, the antirachitic action of vitamin D is both indirect and direct. By enhancing the absorption of Ca2+ and phosphate from the intestine and by enhancing the

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SECTION VIII  •  The Endocrine System

A

INTESTINAL Ca2+ ABSORPTION Epithelial cell

Intestinal lumen

Interstitial space

1,25-dihydroxyvitamin D [Ca2+] 100 nM

3 Na+

2+

Free [Ca ] =~1 mM

Free [Ca2+] =1.2 mM

Ca2+

mRNA

Blood

Nucleus

Transcellular Protein synthesis Calbindin

Ca2+

2+

Ca

H+

Calbindin Ca2+Calbindin Ca2+

TRPV6 Ca2+

B

Paracellular

INTESTINAL PHOSPHATE ABSORPTION

Intestinal lumen

Epithelial cell

Interstitial space

Blood

Nucleus 2K

mRNA +

+

3 Na+

2 Na

Protein synthesis 2–

HPO4

H2PO4–

NaPi

2–

HPO4 or H2PO4–

?

Figure 52-10  Intestinal absorption of Ca2+ and phosphate. A, The small intestine absorbs Ca2+ by two mecha-

nisms. Passive paracellular absorption of Ca2+ occurs throughout the small intestine. This pathway is the predominant one but is not under the control of vitamin D. The second mechanism—active transcellular absorption of Ca2+—occurs only in the duodenum. Ca2+ enters the cell across the apical membrane via TRPV6 Ca2+ channels. Inside the cell, the Ca2+ is buffered by binding proteins, such as calbindin, and is also taken up into intracellular organelles, such as the ER. The enterocyte then extrudes Ca2+ across the basolateral membrane via a Ca pump and an Na-Ca exchanger. Thus, the net effect is Ca2+ absorption. The active form of vitamin D—1,25-dihydroxyvitamin D—stimulates all three steps of transcellular Ca2+ absorption. B, Inorganic phosphate (Pi) enters the enterocyte across the apical membrane via an Na/Pi cotransporter (NaPi). Once inside the cell, the Pi is extruded across the basolateral membrane. Thus, the net effect is Pi absorption.

Chapter 52  •  The Parathyroid Glands and Vitamin D

reabsorption of Ca2+ and phosphate from the renal tubules, vitamin D raises the concentrations of both Ca2+ and phosphate in the blood and ECF. This increase in the Ca × PO4 ion product, along with more differentiated osteoblasts, results—indirectly—in net bone mineralization. On the other hand, when vitamin D is in excess, the direct effect of vitamin D predominates, increasing bone mobilization.

Calcium ingestion lowers—whereas phosphate ingestion raises—levels of both PTH and 1,25-dihydroxyvitamin D Calcium Ingestion  When an individual ingests a meal containing calcium, the ensuing rise in plasma [Ca2+] inhibits PTH secretion. The decline in PTH causes a decrease in the resorption of Caprotein 2+ and phosphorus from bone, thus limiting the postprandial increase in plasma Ca2+ and phosphate levels. In addition, the decrease in PTH diminishes Ca2+ reabsorption in the kidney and thus facilitates a calciuric response. If dietary Ca2+ intake remains high, the lower PTH will result in decreased 1-hydroxylation of 25-hydroxyvitamin D, which will eventually diminish the fractional absorption of Ca2+ from the GI tract. If dietary Ca2+ intake is deficient, the body will attempt to restore Ca2+ toward normal by increasing plasma [PTH]. This response will help to mobilize Ca2+ from bone, to promote renal Ca2+ retention, and, over time, to increase the level of 1,25-dihydroxyvitamin D, which will enhance gut absorption of Ca2+. Phosphate Ingestion  If one ingests phosphorus much in excess of Ca2+, the rise in plasma [phosphate] will increase the plasma Ca × PO4 ion product, thereby promoting deposition of mineral in bone and lowering plasma [Ca2+]. The low plasma [Ca2+], in turn, increases PTH secretion, provoking a phosphaturia and thus a fall of plasma [phosphate] toward normal. In addition, the PTH mobilizes Ca2+ and phosphate from bone by its action. Over longer periods, the action of PTH to modulate the 1-hydroxylation of 25-hydroxyvitamin D plays an increasingly important role in defending the plasma [Ca2+] by increasing intestinal Ca2+ absorption. The plasma [phosphate] is thus largely maintained indirectly through the actions of PTH in response to [Ca2+]. Another regulator of plasma [phosphate] is the protein fibroblast growth factor 23 (FGF23; see p. 787), secreted by osteocytes and osteoblasts in response to high plasma [phosphate]. FGF23 acts on the intestine to decrease phosphate absorption and on the kidney to limit phosphate reabsorption. Excessive FGF23 production causes an autosomal form of hereditary hypophosphatemia rickets by impairing bone mineralization, secondary to phosphate deficiency. Conversely, FGF23 deficiency can lead to hyperphosphatemia and ectopic calcification.

CALCITONIN AND OTHER HORMONES Calcitonin inhibits osteoclasts, but its effects are transitory Calcitonin is a 32–amino-acid peptide hormone made by the clear or C cells of the thyroid gland. C cells (also called

1067

parafollicular cells) are derived from neural crest cells of the fifth branchial pouch, which in humans migrate into the evolving thyroid gland. Although it is located within the thyroid, calcitonin’s major, if not sole, biological action relates to the regulation of mineral metabolism and bone turnover. The incidental nature of its relationship with the major functions of the thyroid is emphasized by the finding that, in many nonhuman species, C cells are found in a body called the ultimobranchial gland and not in the thyroid at all. Calcitonin is synthesized in the secretory pathway (see pp. 34–35) by post-translational processing of a large procalcitonin. As illustrated in Figure 52-11, alternative splicing of the calcitonin gene product gives rise to several biologically active peptides. In the C cells, calcitonin is the only peptide made in biologically significant amounts. Within the central nervous system, calcitonin gene–related peptide (CGRP) is the principal gene product, and it appears to act as a neurotransmitter in peptidergic neurons (see Table 13-1). Calcitonin is stored in secretory vesicles within the C cells, and its release is triggered by a rise in the extracellular [Ca2+] above normal. Conversely, a lowering of the extracellular [Ca2+] diminishes calcitonin secretion. The threshold [Ca2+] for enhancing calcitonin secretion is in the midphysiological range. In principle, this secretory profile would leave calcitonin well poised to regulate body Ca2+ homeostasis. The precise role of calcitonin in body Ca2+ homeostasis has been difficult to define. This difficulty was first apparent from the simple clinical observation that, after complete thyroidectomy with removal of all calcitonin-secreting tissue, plasma [Ca2+] remains normal (provided the parathyroid glands are not injured). Conversely, patients with a rare calcitonin-secreting tumor of the C cells frequently have plasma calcitonin concentrations that are 50 to 100 times normal, yet they maintain normal plasma levels of Ca2+, vitamin D, and PTH. Nevertheless, several lines of evidence suggest that calcitonin does have biologically important actions. First, although calcitonin appears to have a minimal role in the minute-to-minute regulation of plasma [Ca2+] in humans, it does play an important role in many nonmammalian species. This role is particularly clear for teleost fish. The [Ca2+] in seawater (and therefore in food) is relatively high, and calcitonin, secreted in response to a rise in plasma [Ca2+], decreases bone resorption, thus returning the plasma [Ca2+] toward normal. Salmon calcitonin, which differs from human calcitonin in 14 of its 32 amino-acid residues, is roughly 10-fold more potent on a molar basis in inhibiting human osteoclast function than is the human hormone. The second line of evidence that calcitonin may have biologically important actions is the presence of calcitonin receptors. Like PTH receptors, the calcitonin receptor is a GPCR that, depending on the target cell, may activate either adenylyl cyclase (see p. 53) or phospholipase C (see pp. 53–56). Within bone, the osteoclast—which lacks PTH receptors—appears to be the principal target of calcitonin. Indeed, the presence of calcitonin receptors may be one of the most reliable methods of identifying osteoclasts. In the osteoclast, calcitonin raises [cAMP]i, which activates effectors such as protein kinases. Calcitonin inhibits the resorptive activity of the osteoclast, thus slowing the rate of bone turnover. It also diminishes osteocytic osteolysis, and this action— together with its effect on the osteoclast—is responsible for

1068

SECTION VIII  •  The Endocrine System

Primary transcript(s)

A

5´

B

C

D

Calcitonin CCP CGRP

3´

RNA processing

Mature mRNA 5´

A or B

C

Thyroid “C” cells D Calcitonin CCP

3´

5´

A or B

Brain D

C

mRNA translation C

N

CGRP

Common region + 6AA

Mature peptides

N

C

Common region + 4AA Proteolytic processing

N-terminal peptide (83 amino acids)

3´

mRNA translation

Calcitonin CCP

N

CGRP

+ Calcitonin N— S

Proteolytic processing N-terminal peptide (81 amino acids)

N

+ CGRP

—S

—Amide

+ CCP

+ C-terminal peptide

Figure 52-11  Synthesis of calcitonin and CGRP. A common primary RNA transcript gives rise to both calcitonin and CGRP. In the thyroid gland, C cells produce a mature mRNA that they translate to procalcitonin. They then process this precursor to produce an N-terminal peptide, calcitonin (a 32–amino-acid peptide), and calcitonin C-terminal peptide (CCP). In the brain, neurons produce a different mature mRNA and a different “pro” hormone. They process the peptide to produce an N-terminal peptide, CGRP, and a C-terminal peptide. AA, amino acids.

the hypocalcemic effect after the acute administration of pharmacological doses of calcitonin. The hypocalcemic action of calcitonin is particularly effective in circumstances in which bone turnover is accelerated, as occurs in rapidly growing young animals and in humans with hyperparathyroidism. The antiosteoclastic activity of calcitonin is also useful in treating Paget disease of bone (Box 52-3). However, within hours of exposure to high concentrations of calcitonin, osteoclasts desensitize. This “escape” from the hypocalcemic effect of calcitonin has limited the use of calcitonin in the clinical treatment of hypercalcemia. The transitory nature of the action of calcitonin appears to result, in part, from rapid downregulation of calcitonin receptors. In the kidney, calcitonin, like PTH, causes a mild phosphaturia by inhibiting proximal-tubule phosphate transport. Calcitonin also causes a mild natriuresis and calciuresis. These actions may contribute to the acute hypocalcemic and hypophosphatemic actions of calcitonin. However, these renal effects are of short duration and do not appear

to be important in the overall renal handling of Ca2+, phosphate, or Na+.

Sex steroid hormones promote bone deposition, whereas glucocorticoids promote resorption Although PTH and 1,25-dihydroxyvitamin D are the principal hormones involved in modulating bone turnover, other hormones participate in this process. For example, the sex steroids testosterone and estradiol are needed for maintaining normal bone mass in males and females, respectively. The decline in estradiol that occurs postmenopausally exposes women to the risk of osteoporosis; that is, a decreased mass of both cortical and trabecular bone caused by a decrease in bone matrix (see Box 52-3). Osteoporosis is less common in men because their skeletal mass tends to be greater throughout adult life and because testosterone levels in men decline slowly with age, unlike the abrupt menopausal decline of estradiol in women.

Chapter 52  •  The Parathyroid Glands and Vitamin D

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BOX 52-3  Osteoporosis

A

pproximately 25 million Americans, mostly postmenopausal women, are affected by osteoporosis, and every year between 1 and 2 million of these individuals experience a fracture related to osteoporosis. The cost in economic and human terms is immense. Hip fractures are responsible for much of the morbidity associated with osteoporosis, but even more concerning is the observation that as many as 20% of women with osteoporotic hip fracture will die within 1 year of their fracture. The major risk factor for osteoporosis is the postmenopausal decline in estrogen levels in aging women. Rarely, other endocrine disorders such as hyperthyroidism, hyperparathyroidism, androgen deficiency, and Cushing disease (hypercortisolism) are responsible. Other risk factors include inadequate dietary Ca2+ intake, alcoholism, cigarette smoking, and a sedentary lifestyle. Strategies to prevent the development of osteoporosis begin in the premenopausal years. High Ca2+ intake and a consistent program of weight-bearing exercises are widely recommended. Pharmacological agents are now available for preventing or at least retarding the development of osteoporosis and for treating the disease once it has become established. These agents can be broadly classified into two groups: antiresorptive drugs and anabolic drugs that stimulate bone formation. Among the antiresorptive drugs, estrogen is the most widely used. It is most effective when started at the onset of menopause, although it may offer benefits even in patients who are 20 or more years past menopause. However, possible increased risk of breast and endometrial cancer from postmenopausal estrogen limit use of this treatment. Another class of drugs—the

Glucocorticoids also modulate bone mass. This action is most evident in circumstances of glucocorticoid excess, which leads to osteoporosis, as suggested by the effects of glucocorticoids on the production of OPG (see pp. 1057– 1058) and RANKL (see p. 1057). The precise cellular mechanisms that mediate the action of androgens, estrogens, or glucocorticoids on bone have not been well defined. Despite the loss of bone that occurs with androgen or estrogen deficiency or glucocorticoid excess, in each case the coupling of bone synthesis to degradation is qualitatively preserved. Synthesis of new bone continues to occur at sites of previous bone resorption, and no excess of unmineralized osteoid is present. Presumably, the decline in bone mass reflects a quantitative shift in which the amount of new bone formed at any site is less than what was resorbed. Because this shift occurs at multiple sites, the result is a decline in overall bone mass.

PTHrP, encoded by a gene that is entirely distinct from that for PTH, can cause hypercalcemia in certain malignancies Unlike PTH, which is synthesized exclusively by the parathyroid gland, a peptide called PTH-related protein (PTHrP) is made in many different normal and malignant tissues. The PTH1R receptor (see p. 1061) in kidney and bone recognizes PTHrP with an affinity similar to that for intact PTH. PTHrP mimics each of the actions of PTH on kidney and bone. Thus, when present in sufficient concentrations, PTHrP causes hypercalcemia. PTHrP exists in three alternatively

bisphosphonates—are effective inhibitors of bone resorption and have become a mainstay for the treatment of osteoporosis in both men and women. The newer bisphosphonates, which have a much greater potency, can be given either orally or as a once-a-year intravenous treatment. Agents that can stimulate bone formation include vitamin D—often given as 1,25-dihydroxyvitamin D (calcitriol)—which is combined with Ca2+ therapy to increase the fractional absorption of Ca2+ and to stimulate the activity of osteoblasts. PTH is also now available as an injectable treatment for osteoporosis (see p. 1063), and when given intermittently, it potently stimulates osteoblast formation and increases bone mass. PTH also appears to decrease the rate of vertebral fractures. As mentioned on pages 1057–1058, denosumab—a monoclonal antibody to RANKL—is another antiresorptive therapy. It is also being used in some cases of bone resorption associated with metastatic disease. Now in clinical trials are agents that promote Wnt signaling by interfering with either sclerostin or dickkopf1; the hope is that these agents will have both prosynthetic and antiresorptive actions. Calcitonin and the bisphosphonates have also been used successfully to treat Paget disease of bone, a disorder characterized by localized regions of bone resorption and reactive sclerosis. The level of bone turnover at sites of active Paget disease can be extremely high. Although it remains asymptomatic in many individuals, the disease can cause pain, deformity, fractures, and (if bony overgrowth occurs in the region of the eighth cranial nerve) vertigo and hearing loss. The cause of Paget disease is not known.

spliced isoforms of a single gene product. The gene encoding PTHrP is completely distinct from that for PTH. The similar actions of PTHrP and PTH arise from homology within the first 13 amino acids of PTHrP and native PTH. Only weak homology exists between amino acids 14 and 34 (three amino acids are identical), and essentially none beyond amino acid 34. This situation is an unusual example of mimicry among peptides that are structurally quite diverse. The normal physiological roles of PTHrP are largely in regulating endochondral bone and mammary-gland development. The lactating breast also secretes PTHrP, and this hormone is present in very high concentrations in milk. PTHrP may promote the mobilization of Ca2+ from maternal bone during milk production. In nonlactating humans, the plasma PTHrP concentration is very low, and PTHrP does not appear to be involved in the day-to-day regulation of plasma [Ca2+]. It appears likely that under most circumstances, PTHrP acts in a paracrine or autocrine, rather than in an endocrine, regulatory fashion. Many tumors are capable of manufacturing and secreting PTHrP, among them the following: squamous cell tumors of the lung, head, and neck; renal and bladder carcinomas; adenocarcinomas; and lymphomas. Patients with any of these tumors are subject to severe hypercalcemia of fairly abrupt onset.  N52-3

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 52  •  The Parathyroid Glands and Vitamin D

N52-3  Parathyroid Hormone–Related Protein Contributed by Emile Boulpaep and Walter Boron PTHrP was discovered at Yale University as the factor responsible for humeral hypercalcemia of malignancy (HHM). Andrew Stewart, Karl Insogna, Arthur Broadus, and their colleagues first demonstrated that this factor stimulates adenylyl cyclase activity. They later showed that this activity was inhibited by PTH antagonists and finally identified the 17-kDa protein.

REFERENCES Burtis WJ, Wu T, Bunch C, et al: Identification of a novel 17,000-dalton parathyroid hormone-like adenylate cyclase– stimulating protein from a tumor associated with humoral hypercalcemia of malignancy. J Biol Chem 262:7151–7156, 1987. Rodan SB, Insogna KL, Vignery AM, et al: Factors associated with humoral hypercalcemia of malignancy stimulate adenylate cyclase in osteoblastic cells. J Clin Invest 72:1511– 1515, 1983. Stewart AF, Insogna KL, Goltzman D, Broadus AE: Identifi­ cation of adenylate cyclase–stimulating activity and cytochemical glucose-6-phosphate dehydrogenase–stimulating activity in extracts of tumors from patients with humoral hypercalcemia of malignancy. Proc Natl Acad Sci U S A 80:1454–1458, 1983.

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REFERENCES Books and Reviews Baron R, Hesse E: Update on bone anabolics in osteoporosis treatment: Rationale, current status and perspectives. J. Clin. Endocrinol. Metab. 97: 311–325, 2012. Baron R, Kneissel M: WNT Signaling in bone homeostasis and disease: From human mutations to treatments. Nature Med 19: 179–192, 2013. Bringhurst FR, Demay MB, Kronenberg HM: Hormones and disorders of mineral metabolism. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia, WB Saunders, 1998, pp 1155–1209. Datta HK, Ng WF, Walker JA, et al: The cell biology of bone metabolism, J. Clin. Pathol 61: 577–587, 2008. DeLuca HF: The transformation of a vitamin into a hormone: The vitamin D story. Harvey Lect 75:333–379, 1979–1980. Habener JF, Rosenblatt M, Potts JT Jr: Parathyroid hormone: Biochemical aspects of biosynthesis, secretion, action, and metabolism. Physiol Rev 64:985–1053, 1984.

Jones G, Strugnell SA, DeLuca HD: Current understanding of the molecular actions of vitamin D. Physiol Rev 78:1193–1231, 1998. Murer H, Forster I, Hilfiker H, et al: Cellular/molecular control of renal Na/Pi-cotransport. Kidney Int Suppl 65:2–10, 1998. Journal Articles Broadus AE, Mangin M, Ikeda K, et al: Humoral hypercalcemia of cancer. Identification of a novel parathyroid hormone–like peptide. N Engl J Med 319:556–563, 1988. Brown EM, Gamba G, Riccardi D, et al: Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366:575–580, 1993. Burgess TL, Qian Y, Kaufman S, et al: The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 145:527–538, 1999. Robertson RP, Khosla S: Osteoporosis update. Transl Endocrinol Metab 1:1–154, 2010.

C H A P T E R 53  SEXUAL DIFFERENTIATION Sam Mesiano and Ervin E. Jones

Reproduction is a fundamental process of life. All living organisms must reproduce either asexually (e.g., bacteria) or sexually (e.g., mammals). Asexual reproduction is highly efficient and produces large numbers of genetically identical offspring in a relative short amount of time. This strategy, however, is vulnerable to environmental changes because genetic and phenotypic variation between individual progeny is minimal and consequently the probability of producing progeny that have beneficial traits in a hostile environment is relatively low. In contrast, sexual reproduction is less efficient but produces progeny with markedly increased genetic and phenotypic variation, which increases the probability of producing individuals with characteristics that may be adaptive to environmental changes. In this context, natural selection favors sexual reproduction, and consequently most extant animals and flowering plants reproduce sexually. In sexual reproduction a new individual is created by combining the genetic material of two individuals. Sexual reproduction involves the evolution of two sexually dissimilar individuals belonging to the same species, one male and one female, and each equipped with its own specific attributes necessary for its particular contribution to the process of procreation. Each sex produces its own type of sex cell or gamete, and the union of male and female gametes generates species-specific progeny. In addition, mechanisms—some simple, some complex—have evolved to ensure the proximity and union of the sex cells, known as syngamy. Thus, within each species, the relevant sexual characteristics of each partner have adapted differently to achieve the most efficient union of these progenitor cells. These differences between the sexes of one species are called sexual dimor­ phism. For example, oviparous species such as frogs release their eggs into a liquid medium only when they are in relative proximity to sperm. As effective as this approach is, it also typifies the wastefulness of reproduction among higher species inasmuch as most gametes go unfertilized. Even among species that normally reproduce sexually, sexual dimorphism is not universal. For example, monoecious (i.e., hermaphroditic) species, such as cestodes and nematodes, have the capacity to produce both sperm and eggs. By definition, the ability to produce just one kind of gamete depends on sexually dimorphic differentiation. Organisms that reproduce sexually normally have a single pair of sex chromosomes that are morphologically distinguishable from other chromosomes, the autosomes. Each of the sex chromosomes carries genetic information that determines the primary and secondary sexual characteristics 1072

of an individual; that is, whether the individual functions and appears as male or female. It has become abundantly clear that genes determine sexual differentiation and sexual expression and, as a result, mechanisms and patterns of reproduction. The sex of the gonad is genetically programmed: Will a female gonad (ovary) or a male gonad (testis) develop? Although germ cells of the early embryonic gonad are totipotent, these cells develop into female gametes or ova if the gonad becomes an ovary, but they develop into male gametes or sperm if the gonad becomes a testis. These two anatomically and functionally distinct gonads determine either “maleness” or “femaleness” and dictate the development of both primary and secondary sexual characteristics. Endocrine and paracrine modulators that are specific for either the ovary or the testis are primarily responsible for female or male sexual differentiation and behavior and therefore the individual’s role in procreation.  N53-1

GENETIC ASPECTS OF SEXUAL DIFFERENTIATION Meiosis occurs only in germ cells and gives rise to male and female gametes Gametes derive from a specialized lineage of embryonic cells—the germline—known as germ cells. They are the only cells that can divide by mitosis and meiosis and differentiate into sperm or ova. Germ cells are therefore the critical link between generations. The process by which cells decide between becoming somatic cells of the body or germ cells occurs in the early embryo and involves factors and processes that prevent the somatic fate and induce germline differentiation. Studies in experimental model systems are beginning to unravel the complex process of germline determination, which involves germline-specific transcription factors (see pp. 81–88) and small noncoding RNAs (see pp. 99–100) and DNA methylation (see pp. 95–96) to control expression of specific genes. The process by which germ cells develop into either sperm or ova is referred to as gametogenesis and involves meiosis. Except for the gametes, all other nucleated cells in the human body—somatic cells—have a diploid number (2N) of chromosomes. Human diploid cells have 22 autosome pairs consisting of two homologous chromosomes, one contributed by the father and one by the mother. Diploid cells also contain a single pair of sex chromosomes comprising

Chapter 53  •  Sexual Differentiation

N53-1  Definitions of Sex and Gender Contributed by Ervin Jones Gender—or, more accurately, gender identification—refers to the concept held by the individual (or by those raising the individual) that the individual is male, female, or ambivalent. Sex refers to biological characteristics that distinguish female from male. The distinction may be made on the basis of chromosomes, gonads, internal and external morphology, and hormonal status.

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1

2

3

4

5

5. Cytokinesis. The cell divides into two genetically identical daughter cells, each containing one of the nuclei.

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22 X

X

If female

X

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Y

If male

Figure 53-1  Normal human karyotype. The normal human has 22 pairs of autosomal chromosomes (autosomes) as well as a pair of sex chromosomes. Females have two X chromosomes, whereas males have one X and one Y chromosome.  N53-7

either XX or XY. Each somatic cell in human females has 44 autosomes (i.e., 22 pairs) plus two X chromosomes, and each somatic cell in males has 44 autosomes plus one X and one Y chromosome. The karyotype is the total number of chromosomes and the sex chromosome combination, and thus in normal females is designated 46,XX, and in normal males, 46,XY (Fig. 53-1). Gametes have a haploid number (N) of chromosomes and contain either an X or Y sex chromosome. Mitosis is the only kind of cell division that occurs in somatic cells. Mitosis results in the formation of two identical daughter cells (Fig. 53-2A), each having the same number of chromosomes (i.e., 46 in humans) and the same DNA content as the original cell. After interphase, during which nuclear DNA in the form of chromatin replicates, mitosis proceeds in a continuum of five phases: 1. Prophase. The replicated chromatin condenses into 46 chromosomes that comprise two identical sister chromatids bound together at the centromere. 2. Metaphase. The nuclear envelope breaks down, the chromosomes align along the midplane of the cell known as the metaphase plate, and microtubules enter the nuclear space and attach to the centromere of each chromosome. 3. Anaphase. The centromeres dissolve and the microtubules pull apart the sister chromatids toward opposite poles of the cell. 4. Telophase. A new nuclear membrane envelopes each cluster of chromatids, which decondense back into chromatin.

Daughter cells produced by mitosis are genetically identical because there is no exchange of genetic material between homologous chromosomes and the sister chromatids of each chromosome split, one going to each daughter cell during anaphase of the single mitotic division. Meiosis occurs only in germ cells—spermatogonia in males and oogonia in females—still with a complement of 2N DNA (N = 23). Germ cells initially multiply by mitosis and then enter meiosis when they begin to differentiate into sperm (see Fig. 53-2B) or ova (see Fig. 53-2C). Gameto­ genesis reduces the number of chromosomes by half, so that each gamete contains one chromosome from each of the original 23 pairs. This reduction in genetic material from the diploid (2N) to the haploid (N) number involves two divisions referred to as meiosis I and meiosis II. Because of this halving of the diploid number of chromosomes, meiosis is often referred to as a reduction division. Meiosis is a continuum composed of two phases: the homologous chromosomes separate during meiosis I, and the chromatids separate during meiosis II. Prior to the start of meiosis I, the chromosomes duplicate so that the cells have 23 pairs of duplicated chromosomes (i.e., each chromosome has two chromatids)—or 4N DNA. During prophase of the first meiotic division, homologous pairs of chromosomes—22 pairs of autosomal chromosomes (autosomes) plus a pair of sex chromosomes—exchange genetic material through a process known as recombination or crossing over at attachment points known as chiasmata. This results in a random, but balanced, exchange of chromatid segments between the homologous maternal and paternal chromatids to produce recombinant homologous chromosomes comprising a mix of maternal and paternal DNA. At the completion of meiosis I, the daughter cells have a haploid number (23) of duplicated, crossed-over chromosomes—or 2N DNA. During meiosis II, no additional duplication of DNA takes place. The chromatids simply separate so that each daughter receives a haploid number of unduplicated chromosomes—1N DNA. Gametes produced by this process are genetically different from each other and from either parent. The genetic diversity that arises from recombination during meiosis and the combining of gametes from different parental lineages causes significant phenotypic variation within the population, providing an efficient mechanism for adaptation and natural selection. A major difference between male and female gametogenesis is that one spermatogonium yields four spermatids (see Fig. 53-2B), whereas one oogonium yields one mature oocyte and two or three polar bodies (see Fig. 53-2C). We discuss the details regarding timing and process for spermatogenesis on page 1100, and for oogenesis on page 1120.

Fertilization of an oocyte by an X- or Y-bearing sperm establishes the zygote’s genotypic sex Fusion of two haploid gametes, a mature spermatozoan from the father and a mature oocyte from the mother—referred to as fertilization—produces a new diploid cell with 2N DNA, a zygote, that will become a new individual.

Chapter 53  •  Sexual Differentiation

N53-7  Karyotype Contributed by Emile Boulpaep and Walter Boron The representation in Figure 53-1 is obtained by taking photomicrographs of chromosomes and then rearranging them as shown. The chromosomes are numbered according to size, the largest chromosomes having the smallest number. Pairs of homologous chromosomes are identified on the basis of size, patterns of banding, and the placement of centromeres. In the case of humans, one generally uses leukocytes (white blood cells) that have been treated with a hypotonic solution to cause swelling and thus help disperse the chromosomes, and with colchicine to arrest mitosis in metaphase. A dye is then applied to visualize the chromosomes better.

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SECTION IX  •  The Reproductive System

B

MITOSIS

C

MALE MEIOSIS Maternal homolog

Replication of DNA

Paternal homolog

Replication of DNA Prophase

Lining up of individual duplicated chromosomes on the spindle Diploid number of duplicated chromosomes.

Anaphase I Haploid number of duplicated chromosomes.

Cell division I

Secondary spermatocytes (2N DNA)

Separation of chromatids

Primary oocyte– arrested (4N DNA)

Metaphase I Anaphase I Haploid number of duplicated chromosomes.

Cell division I 1st polar body

Telophase I Secondary oocyte (2N DNA)

Separation of chromatids

Anaphase II

Anaphase Meiotic division II

Separation of homologous, duplicated chromosomes

Telophase I

Metaphase Separation of chromatids

Prophase I

Lining up of homologous pairs of duplicated chromosomes on the spindle Metaphase I

Separation of homologous, duplicated chromosomes

Primary oocyte (4N DNA)

Pairing and recombination of homologous chromosomes

Prophase I

Lining up of homologous pairs of duplicated chromosomes on the spindle

Diploid oogonium (2N DNA)

Replication of DNA

Primary spermatocyte (4N DNA)

Pairing and recombination of homologous chromosomes Meiotic division I

Cell division

Maternal homolog

Diploid spermatogonium (2N DNA)

Paternal homolog

FEMALE MEIOSIS

Cell division II

Anaphase II

Cell division II

Spermatids (1N DNA)

2nd polar body

Mature oocyte (1N DNA)

Figure 53-2  Mitosis and meiosis. A, In mitosis, the two daughter cells are genetically identical to the mother cell. B, In male meiosis, the four daughter cells are haploid. Cell division I produces both recombination (i.e., crossing over of genetic material between homologous chromosomes) and reduction to the haploid number of chromosomes. Cell division II separates the chromatids of each chromosome, just as in mitosis. C, Female meiosis is similar to male meiosis. A major difference is that instead of producing four mature gametes, female meiosis produces only one mature gamete and two polar bodies (or, if the first polar body divides, three polar bodies).  N53-8

The sex chromosomes that the parents contribute to the offspring determine the genotypic sex of that individual. The genotypic sex determines the gonadal sex, which in turn determines the phenotypic sex that becomes fully established at puberty. Thus, sex-determining mechanisms established at fertilization direct all later ontogenetic processes (processes that lead to the development of an organism) involved in male-female differentiation. Fusion of a sperm and an egg—two haploid germ cells— results in a zygote, which is a diploid cell containing 46 chromosomes (see Fig. 53-1): 22 pairs of somatic chromosomes (autosomes) and a single pair of sex chromosomes. In females, these sex chromosomes are both X chromosomes, whereas males have one X and one Y chromosome.

When the karyotypes of normal females and males are compared, two differences are apparent. First, among the 23 pairs of chromosomes in females, 8 pairs—including the 2 X chromosomes—are of similar size, whereas males have only 7 1 2 such pairs. Second, instead of a second X chromosome, males have a Y chromosome that is small and acrocentric (i.e., the centromere is located at one end of the chromosome): this chromosome is the only such chromosome that is not present in females. In the offspring, 23 of the chromosomes—including 1 of the sex chromosomes—are from the mother, and 23— including the other sex chromosome—come from the father. Thus, the potential offspring has a unique complement of chromosomes differing from those of both the mother and

Chapter 53  •  Sexual Differentiation

N53-8  Meiosis in Males versus Females Contributed by Emile Boulpaep and Walter Boron Figure 53-2B in the text shows meiosis in the male, whereas Figure 53-2C shows meiosis in the female. In both males and females, the primordial germ cell (PGC) enters the gonad and undergoes many rounds of mitotic divisions. At some point, both a spermatogonium (in the case of males) and an oogonium (in the case of females) enter the first of two meiotic divisions (top cell in Fig. 53-2B, C). In the case of males (see Fig. 53-2B), one primary spermatocyte (diploid 4N DNA)—a cell that has just entered prophase I—ultimately gives rise to two secondary spermatocytes (haploid 2N DNA) at the completion of the first meiotic division, and four spermatids (haploid 1N DNA) at the completion of the second meiotic division. Thus, one primary spermatocyte yields four mature gametes. In the case of females (see Fig. 53-2C), one primary oocyte (diploid 4N DNA)—a cell that is arrested in prophase I until shortly before ovulation—ultimately gives rise to one secondary oocyte (haploid 2N DNA) and one diminutive first polar body (haploid 2N DNA) at the completion of the first meiotic division. The polar body is equivalent to one of the two cells at telophase I in Figure 53-2B. In the second meiotic division, which the cell completes at the time of fertilization, the secondary oocyte gives rise to one mature oocyte (haploid 1N DNA) and a diminutive second polar body (haploid 1N DNA). Thus, unlike the situation in males, one primary oocyte yields one mature gamete—equivalent to one of the four cells at the bottom of Figure 53-2B. Note that the first polar body sometimes divides during meiosis II, thereby yielding a total of three polar bodies and one oocyte. This is the same amount of DNA produced in spermatogenesis (i.e., four spermatids from one spermatogonium).

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Chapter 53  •  Sexual Differentiation

the father. The ovum provided by the mother (XX) always provides an X chromosome. Because the male is the heterogenetic (XY) sex, half the spermatozoa are X bearing whereas the other half are Y bearing. Thus, the type of sperm that fertilizes the ovum determines the sex of the zygote. X-bearing sperm produce XX zygotes that develop into females with a 46,XX karyotype, whereas Y-bearing sperm produce XY zygotes that develop into males with a 46,XY karyotype. The genotypic sex of an individual is determined at the time of fertilization. The Y chromosome appears to be the fundamental determinant of sexual development. When a Y chromosome is present, the individual develops as a male; when the Y chromosome is absent, the individual develops as a female. In embryos with abnormal sex chromosome complexes, the number of X chromosomes is apparently of little significance.

Genotypic sex determines differentiation of the indifferent gonad into either an ovary or a testis The indifferent gonad is composed of an outer cortex and an inner medulla. In embryos with an XY sex chromosome complement (i.e., 46,XY), the medulla differentiates into a testis and the cortex regresses. On the other hand, in embryos with an XX sex chromosome complement (i.e., 46,XX), the cortex develops into an ovary and the medulla regresses. Thus, the Y chromosome exerts a powerful testis-determining effect on the indifferent gonad. In the absence of a Y chromosome, the indifferent gonad develops into an ovary. Interestingly, two X chromosomes are necessary for normal ovarian development. In individuals with the karyotype 45,XO—Turner syndrome—the ovaries fail to develop fully and appear as streaks on the pelvic sidewall (Box 53-1).

BOX 53-1  Gonadal Dysgenesis

T

he best-known example of gonadal dysgenesis is a syndrome referred to as Turner syndrome, a disorder of the female sex characterized by short stature, primary amenorrhea, sexual infantilism, and certain other congenital abnormalities. Cells in these individuals have a 45,XO karyotype (i.e., they lack one of the X chromosomes). The gonads of individuals with Turner syndrome appear as firm, flat, glistening streaks (referred to as streak gonads) lying below the fallopian tubes with no evidence of either germinal or secretory elements. Instead, they are largely composed of connective tissue arranged in whorls suggestive of ovarian stroma. Individuals with Turner syndrome have normal female differentiation of both the internal and external genitalia, although these genitalia are usually small and immature. Turner syndrome can also be caused by partial deletion of the X chromosome, particularly if the entire short arm of the X chromosome is missing, or by formation of an X-chromosome ring that develops as a result of a deletion and subsequent joining of the two free ends of the chromosome. In at least half of affected individuals, Turner syndrome is caused by the total absence of one X chromosome. In others, the lesion is structural (i.e., partial deletion or ring chromosome). In at least a third of cases, the genetic lesion appears as part of a mosaicism; that is, some of the cells carry the aberrant or absent chromosome, whereas the rest are normal.

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Even the absence of only some genetic material from one X chromosome in XX individuals (e.g., due to chromosome breakage or deletion) may cause abnormal gonadal differentiation. However, a complete Y chromosome is necessary for development of the testes. Indeed, individuals with the karyotype 47,XXY—Klinefelter syndrome—are not phenotypic females (based on the presence of two X chromosomes) but males. Taken together, the data on 45,XO and 47,XXY individuals tell us that the absence of a Y chromosome causes female phenotypic development. We have just seen that the absence of a Y chromosome and the presence of two complete X chromosomes lead to normal ovarian development. Why? The X chromosome is far larger than the Y chromosome (see Fig. 53-1) and contains nearly 10% of the human genome compared to 2.5 cm is compatible with the onset of normal pubertal development. The testicular volume index is defined as the sum of the product of length × width for the left and right testes. An orchidometer allows direct comparison of the patient’s testes with an ovoid of measured volume. A popular method uses the Prader orchidometer, a set of solid or hollow ovoids encompassing the range of sizes from infancy to adulthood (1 to 25 mL). The volumes of the testes are then recorded; a volume of 3 mL closely correlates with the onset of pubertal development.

Contributed by Ervin Jones Androgens also determine the male secondary sexual characteristics, which include deepening of the voice as well as evolving male patterns of hair growth. The changes in the voice are a result of androgen-dependent effects on the size of the larynx as well as the length and thickness of the vocal cords. In boys, the length of the vocal cords increases by ~50% during puberty, whereas girls have little increase in vocal cord length. The surfaces of the human body that bear secondary sexual hair include the face (particularly the upper lip, chin, and the sideburn areas), the axilla, and the pubic region. Temporal hair recession and male-pattern balding— beginning above the temples (i.e., receding hairline) and at the vertex of the scalp—are also androgen-dependent phenomena.

Chapter 53  •  Sexual Differentiation

A

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PATTERNS OF GONADOTROPIN LEVELS THROUGHOUT THE LIFE OF A FEMALE Day Night Figure 53-11  Gonadotropin function LH secretion patterns Fetus

Childhood

Puberty

Reproductive years

Menopause

Infancy

FSH

100 Plasma gonadotropins (mU/mL) 70

monthly surges LH

10 rth Bi d 3r d 2n t 1s

6 mo

50 yr

10–14 yr

Trimesters

B

NEGATIVE FEEDBACK OF SEX STEROIDS ON GONADOTROPIN RELEASE In an adult, much higher levels occur. Child

Adolescent

C AGE DEPENDENCE OF FEEDBACK SENSITIVITY High sensitivity

Adult Relative sensitivity of gonadotropin release to negative feedback by sex steroids

Relative gonadotropin release In a child, even a low level of steroid blocks the release of gonadotropin.

during life. A, Levels of both LH and FSH peak during fetal life and again during early infancy, before falling to low levels throughout the rest of childhood. At the onset of puberty, LH and FSH levels slowly rise and—in females—begin to oscillate at regular monthly intervals. At menopause, gonadotropin levels rise to very high levels. The four insets show daily changes. B, In childhood, even very low sex steroid levels are sufficient to fully suppress gonadotropin output. In adolescence, higher levels—and, in adults, even higher levels—of sex steroids are required to suppress gonadotropin release. C, This graph is a plot of age versus the midpoints of curves such as those in B.

Concentration of sex steroids

Low sensitivity

Fetus

Infancy and childhood

Puberty Adult

TABLE 53-1  Tanner Stages in Male and Female Puberty STAGE

PUBIC HAIR (BOTH SEXES)

MALE GENITAL DEVELOPMENT

FEMALE BREAST AND GENITAL* DEVELOPMENT

1

Preadolescent. No pubic hair is present, only vellus hair, as on the abdomen.

Preadolescent. The penis, scrotum, and testes are the same size— relative to body size—as in a young child.

Preadolescent. Breasts: only papillae are elevated.

2

Pubic hair is sparse, mainly at the base of the penis (boys) or along labia majora (girls).

Scrotum and testes are enlarged.

Breast buds begin to develop. Breasts and papillae are both elevated, and the diameter of the areolae increases.

3

Pubic hair is darker, coarser, and curlier and spreads above the pubis.

Penis is enlarged, predominantly in length. Scrotum and testes are further enlarged.

Breasts and areolae further enlarge. Vagina enlarges and begins producing a discharge. Menstrual periods may begin.

4

Pubic hair is of the adult type, but covers an area smaller than in most adults.

Penis is further enlarged in length and also in diameter. Scrotum and testes are further enlarged.

Areolae and papillae project out beyond the level of the expanding breast tissue. Menstruation and ovulation begin. Periods will most likely be irregular.

5

Adult pattern.

Adult pattern.

With further enlargement of the breast, the areolae are now on the same level as the rest of the breast. Only the papillae project. Adult pattern.

*The official Tanner stages for females include pubic hair and breast development.

1090 A

B

SECTION IX  •  The Reproductive System

FEMALE PUBERTY (BREAST DEVELOPMENT AND PUBIC HAIR)

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Stage 4

Stage 5

MALE PUBERTY (GENITAL DEVELOPMENT AND PUBIC HAIR)

Stage 1

Stage 2

Stage 3

Figure 53-12  Tanner stages of puberty based on pubic hair, genital, and breast development in boys and girls. Pubic hair and genital/breast development may not be synchronous and are usually scored separately. For example, a girl may be at breast stage 3 but pubic hair stage 2. (Data from Carel JC, Léger J: Clinical practice. Precocious puberty. N Engl J Med 358:2366, 2008.)

The pubertal growth spurt, a marked increase in growth rate (total body size), occurs late in puberty in boys. The acceleration of growth appears due to the combined effects of increased secretion of growth hor­ mone and testosterone. In boys, height increases by an average of 28 cm during the pubertal spurt. The 10-cm mean difference in adult stature between men and women is due to a greater pubertal growth spurt in boys and to greater height at the onset of peak pubertal height velocity in boys compared with girls. Before puberty, boys and girls have the same mean body mass, skeletal mass, and body fat. However, men have 150% of the average woman’s lean and skeletal body mass, and women have 200% of the body fat of men. Men have twice

the number of muscle cells of women, and 1.5 times the muscle mass. Females  The first physical sign of puberty in girls is usually the onset of thelarche that begins between 10 and 11 years of age (see Fig. 53-12A). During the next 3 to 5 years, the breasts continue to develop under the influence of several hormones. Progesterone is primarily responsible for development of the alveoli (see Fig. 56-11C). Estrogen is the primary stimulus for development of the duct system that connects the alveoli to the exterior. Insulin, growth hormone, glucocorticoids, and thyroxin contribute to breast development, but they are incapable of causing breast growth by themselves.

Chapter 53  •  Sexual Differentiation

During puberty, the uterus and cervix enlarge, and their secretory functions increase under the influence of estrogens (mainly estradiol). The uterine glands increase in number and length, and the endometrium and stroma proliferate in response to estrogens. The mucous membranes of the female urogenital tract respond to hormones, particularly estrogens. Menarche usually occurs around 2 years after the initiation of thelarche. In the United States, most girls experience menarche between the ages of 11 and 13 years, the average age is 12.5 years, and the normal range is between 8 and 16 years. During puberty, a girl’s body shape changes in response to rising levels of estradiol. The hips and pelvis widen and the proportion of body fat increases (compared with males) and distributes mainly to the breasts, hips, buttocks, thighs, upper arms, and pubis to produce the typical adult female body shape.

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The appearance of secondary sex characteristics at puberty completes sexual differentiation and development Although at birth humans have the primary and secondary sex organs necessary for procreation, final sexual maturity occurs only at puberty. Profound alterations in hormone secretion during the peripubertal period cause changes in the primary and secondary sex organs. In the following chapters the events occurring in puberty are discussed in more detail for both males (see Chapter 54) and females (see Chapter 55).

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 53  •  Sexual Differentiation

REFERENCES Books and Reviews Donahoe PK, Budzik GP, Trelstad R, et al: Mullerian-inhibiting substance. An update. Recent Prog Horm Res 38:279–330, 1982. Grumbach MM, Conte FA: Disorders of sex differentiation. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR (eds): Williams Textbook of Endocrinology, 9th ed. Philadelphia, WB Saunders, 1998, pp 1303–1425. Haqq CM, Donahoe PK: Regulation of sexual dimorphism in mammals. Physiol Rev 78:1–33, 1998. Jost A, Vigier B, Prepin J, Perchellet JP: Studies on sex differentiation in mammals. Recent Prog Horm Res 29:1–41, 1973. Lee MM, Donahoe PK: Müllerian inhibiting substance: A gonadal hormone with multiple functions. Endocr Rev 14:152–164, 1993. Naftolin F, Ryan KJ, Davie KJ, et al: The formation of estrogens by central neuroendocrine tissues. Recent Prog Horm Res 31:295– 319, 1975. Rebar RW: Normal and abnormal sexual differentiation and pubertal development. In Moore TR, Reiter RC, Rebar RW, Baker VV (eds): Gynecology and Obstetrics: A Longitudinal Approach. New York, Churchill Livingstone, 1993, pp 97–146.

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Journal Articles Beitins IZ, Padmanabhan V, Kasa-Vubu J, et al: Serum bioactive follicle-stimulating hormone concentrations from prepuberty to adulthood: A cross-sectional study. J Clin Endocrinol Metab 71:1022–1027, 1990. Griffin JE, Wilson JD: The syndromes of androgen resistance. N Engl J Med 302:198–209, 1980. Judd HL, Hamilton CR, Barlow JJ, et al: Androgen and gonadotropin dynamics in testicular feminization syndrome. J Clin Endocrinol Metab 34:229–234, 1972. New MI, Dupont B, Pang S, et al: An update of congenital adrenal hyperplasia. Recent Prog Horm Res 37:105–181, 1981. Reiter EO, Beitins IZ, Ostrea TR, Gutai JP: Bioassayable luteinizing hormone during childhood and adolescence and in patients with delayed pubertal development. J Clin Endocrinol Metab 54:155–161, 1982. Sinclair AH, Berta P, Palmer MS, et al: A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346:240–244, 1990. Turner HH: A syndrome of infantilism, congenital webbed neck, and cubitus valgus. Endocrinology 23:566–574, 1938. Wilkins L: Masculinization of the female fetus due to the use of orally given progestins. JAMA 172:1028–1032, 1960.

C H A P T E R 54  THE MALE REPRODUCTIVE SYSTEM Sam Mesiano and Ervin E. Jones

The male reproductive system consists of two essential elements: the gonads (in this case the testes) and the complex array of glands and ducts that constitute the sex accessory organs (Fig. 54-1A, B). The testes are responsible for the production of gametes, the haploid cells—spermatozoa, plural of spermatozoon— necessary for sexual reproduction and for the synthesis and secretion of hormones, including the principal male sex hormone, testosterone. These hormones are necessary for functional conditioning of the sex organs, the male secondary sexual characteristics, feedback control of gonadotropin secretion, and modulation of sexual behavior. The testes (see Fig. 54-1C) are composed mainly of seminiferous tubules (see Fig. 54-1D, E) and interstitial cells of Leydig, located in the spaces between the tubules. A seminiferous tubule is an epithelium made up of Sertoli cells (see Fig. 54-1E) and is also the site of spermatogenesis— the production of the haploid spermatozoa from the diploid germ cells. The seminiferous epithelium rests on a basement membrane, itself supported by a thin lamina propria externa. The male sex accessory organs include the paired epididymides, the vas deferens, the seminal vesicles, the ejaculatory ducts, the prostate, the bulbourethral glands (Cowper’s glands), the urethra, and the penis. The primary role of the male sex accessory glands and ducts is to store and transport spermatozoa to the exterior, and thus enable spermatozoa to reach and fertilize female gametes.

HYPOTHALAMIC-PITUITARY-GONADAL AXIS The hypothalamic-pituitary-gonadal axis (Fig. 54-2) controls two primary functions: (1) production of gametes (spermatogenesis in males and oogenesis in females), and (2) gonadal sex steroid biosynthesis (testosterone in males and estradiol and progesterone in females). In both sexes, the hypothalamus produces gonadotropin-releasing hormone (GnRH), which stimulates the gonadotrophs in the anterior pituitary to secrete the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Although the names of these hormones reflect their function in the female reproductive system (see pp. 1111–1112) they play similar roles in controlling gonadal function in both sexes. The hypothalamic-pituitary axis is therefore the central regulator of male and female reproductive systems. In the 1092

male, LH and FSH control, respectively, the Leydig and Sertoli cells of the testes.

The hypothalamus secretes GnRH, which acts on gonadotrophs in the anterior pituitary Gonadotropin-releasing hormone (GnRH), which is synthesized by small-bodied peptidergic neurons in the hypothalamus, stimulates the synthesis, storage, and secretion of gonadotropins by gonadotroph cells in the anterior pituitary. The hypothalamic-pituitary-portal system (see p. 978) describes the route by which GnRH and other releasing hormones emanating from the hypothalamus reach the anterior pituitary gland. The neurons that synthesize, store, and release GnRH are dispersed throughout the hypothalamus but are principally located in the arcuate nucleus and preoptic area. During embryonic development, GnRH neurons originate in the olfactory placode and migrate to the hypothalamus. Studies involving both rats and primates show that sites of GnRH production other than the hypothalamus (e.g., the limbic system) can also participate in the control of sex behavior. Neuronal systems originating from other areas of the brain impinge on the hypothalamic GnRH-releasing neurons and thus form a functional neuronal network that integrates multiple environmental signals (e.g., diurnal lightdark cycles) and physiological signals (e.g., extent of body fat stores, stress) to control GnRH release and, ultimately, the function of the reproductive system. GnRH is a decapeptide hormone encoded by a single gene on chromosome 8. Like many other peptide hormones, GnRH is synthesized as a prohormone—69 amino acids long in this case. Cleavage of the prohormone yields the decapeptide GnRH (residues 1 to 10), a 56–amino-acid peptide (residues 14 to 69) referred to as GnRH-associated peptide (GAP), and three amino acids that link the two (Fig. 54-3). Via the secretory pathway (see p. 34), the neuron transports both GnRH and GAP down the axon for secretion into the extracellular space. The role of GAP is unknown. GnRH neurons project axons directly to a small swelling on the inferior boundary of the hypothalamus, known as the median eminence, which lies above the pituitary stalk. The axons terminate near portal vessels that carry blood to the anterior pituitary (see p. 978). Consequently, GnRH secreted at the axon terminals in response to neuron activation enters the portal vasculature and is transported directly to gonadotrophs in the anterior pituitary.

Chapter 54  •  The Male Reproductive System

A

SAGITTAL SECTION OF THE MALE PELVIS

B

Ductus (vas) deferens

Ureter

Ampulla of ductus deferens

Pubis Corpus cavernosum

Seminal vesicle

Corpus spongiosum Seminal vesicle

Penis

Ejaculatory duct

Penile urethra

Prostate gland Anus

Prostatic Bulbous urethra urethra

Testis (left)

URINARY BLADDER (DORSAL VIEW) Urinary bladder

Urinary bladder

Scrotum

Prostate

Ejaculatory duct (initial part)

Bulbourethral (Cowper’s) glands Efferent ductules (ductuli efferentes)

C TESTIS Ductus (vas) deferens

Glans penis

Epididymis (head) Rete testis Tunica albuginea

Epididymis

Bulbourethral gland

Seminiferous tubules

Epididymis (body) D TESTICULAR LOBULE

Testicular lobules

Rete testis

Septum Testis

Tubulus rectus

Epididymis (tail) E

Septum

Seminiferous tubule

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CROSS SECTION OF SEMINIFEROUS TUBULE

Mature sperm Lumen

Figure 54-1  Anatomy of the male internal genitalia and accessory sex

organs. A, The two major elements of the male sexual anatomy are the gonads (i.e., testes) and the sex accessories (i.e., epididymis, vas deferens, seminal vesicles, ejaculatory duct, prostate, bulbourethral or Cowper’s glands, urethra, and penis). Note that the urethra can be subdivided into the prostatic urethra, the bulbous urethra, and the penile urethra. B, The vas deferens expands into an ampulla before coursing across the rear of the urinary bladder and merging with the outflow from the seminal vesicle. The merger forms the ejaculatory duct. The left and right ejaculatory ducts penetrate the prostate gland and open into the prostatic urethra. C, The spermatozoa form in the seminiferous tubules and then flow into the rete testis and from there into the efferent ductules, the epididymis, and the vas deferens. E, The seminiferous tubule is an epithelium formed by Sertoli cells, with interspersed germ cells. The most immature germ cells (the spermatogonia) are near the periphery of the tubule, whereas the mature germ cells (the spermatozoa) are near the lumen of the tubule. The Leydig cells are interstitial cells that lie between the tubules.

Spermatogonium

Sertoli cell

Basal lamina surrounding seminiferous tubule

Leydig cell

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SECTION IX  •  The Reproductive System

Processing sites

CNS Behavioral effects

N

–23 –1 10 AA Signal peptide GnRH

1

56 AA

14

GnRH-associated peptide

69 C

10

Figure 54-3  Map of the GnRH gene. The mature mRNA encodes a preprohormone with 92 amino acids. Removal of the 23–amino-acid signal sequence yields the 69–amino-acid prohormone. Cleavage of this prohormone yields GnRH. AA, amino acid.

Hypothalamus

GnRH

Anterior pituitary LH

FSH

Leydig cell Growth factors

Sertoli cell Inhibin

Androgens

Aromatization

Peripheral aromatization

Androgen-binding protein

Spermatogenesis

Estrogens Figure 54-2  Hypothalamic-pituitary-gonadal axis. Small-bodied neurons in the arcuate nucleus and preoptic area of the hypothalamus secrete GnRH, a decapeptide that reaches the gonadotrophs in the anterior pituitary via the long portal veins (see Fig. 47-3). Stimulation by GnRH causes the gonadotrophs to synthesize and release LH, which stimulates Leydig cells, and FSH, which stimulates Sertoli cells. Negative feedback on the hypothalamic-pituitary-gonadal axis occurs by two routes. CNS, central nervous system.

GnRH stimulates the release of both FSH and LH from the gonadotroph cells in the anterior pituitary by interacting with high-affinity membrane receptors on the gonadotroph cell surface (see Fig. 55-5). The GnRH receptor (GnRHR) is a G protein–coupled receptor (GPCR; see pp. 51–52) linked to Gαq, which activates phospholipase C (PLC; see p. 58). PLC acts on membrane phosphati­ dylinositol 4,5-bisphosphates (PIP2) to liberate inositol

1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of Ca2+ from internal stores, which triggers exocytosis LH and FSH. DAG stimulates protein kinase C, which indirectly increases expression of genes encoding LH and FSH. The net effect is an increase in the synthesis and release of LH and FSH from the gonadotrophs. Because secretion of GnRH into the portal system is pulsatile, secretion of LH and FSH by the gonadotrophs is also episodic. The frequency of pulsatile LH discharge in men is ~8 to 14 pulses over a 24-hour period. FSH pulses are not as prominent as LH pulses, both because of their lower amplitude and because of the longer half-life of FSH in the circulation. Upon binding GnRH, the GnRH receptor is internalized and partially degraded in the lysosomes. However, some GnRH receptors are shuttled back to the cell surface, and de novo receptor synthesis continues from GnRH receptor gene expression. Return of the GnRH receptor to the cell membrane is referred to as receptor replenishment. A consequence of receptor internalization, however, is that the responsiveness of gonadotrophs to GnRH can be decreased by prolonged exposure to GnRH. Thus, although pulsatile GnRH discharge elicits a corresponding pulsatile release of LH and FSH, continuous administration of GnRH—or intermittent administration of high doses of GnRH analogs— suppresses the release of gonadotropins. This effect occurs because continuous (rather than pulsatile) exposure to GnRH causes a decrease in the number of GnRH receptors on the surface of the gonadotroph (i.e., receptor internalization exceeds replenishment). The induced desensitization to GnRH can be used therapeutically to control the reproductive function. A clinical application of this principle is chemical castration in prostatic cancer. Here, the administration of long-acting GnRH analogs desensitize the gonadotrophs to GnRH, which leads to low LH and FSH levels and thereby reduces testosterone production (see Box 55-2).

Under the control of GnRH, gonadotrophs in the anterior pituitary secrete LH and FSH Luteinizing hormone (LH) and follicle-stimulating hor­ mone (FSH) are members of the same family of hormones as human chorionic gonadotropin (hCG; see p. 1139) and thyroid-stimulating hormone (TSH; see p. 1010). These glycoprotein hormones are composed of two polypeptide chains designated α and β, both of which are required for full biological activity. The α subunits of LH and FSH, as well as the α subunits of hCG and TSH, are identical. In humans, the common α subunit has 92 amino acids and a molecular

Chapter 54  •  The Male Reproductive System

weight of ~20 kDa. The β subunits differ among these four hormones and thus confer specific functional and immunological characteristics to the intact molecules. Each of the unique β subunits of FSH and LH is 115 amino acids in length. The β subunits of LH and hCG are identical except that the β subunit of hCG has an additional 24 amino acids and additional glycosylation sites at the C terminus.  N54-1  The biological activities of LH and hCG are very similar. Indeed, in most clinical uses (e.g., in an attempt to initiate spermatogenesis in oligospermic men), hCG is substituted for LH because hCG is much more readily available.  N54-2 Differential secretion of FSH and LH is affected by several other hormonal mediators, including sex steroids, inhibins, and activins (see pp. 1113–1115). Thus, depending on the specific hormonal milieu produced by different physiological circumstances, the gonadotroph produces the α and β subunits of FSH and LH at different rates. The specific gonadotropin and the relative proportions of each gonadotropin released from the anterior pituitary depend on the developmental age. The pituitary gland of the male fetus contains functional gonadotrophs by the end of the first trimester of gestation. Thereafter, gonadotropin secretion rises rapidly and then plateaus. Gonadotropin secretion begins to decline in utero during late fetal life and increases again during the early postnatal period. Male primates release LH in response to GnRH administration at 1 to 3 months of age, a finding indicative of functional competence of the anterior pituitary gland. Also during this time, a short-lived postnatal surge of LH and testosterone secretion occurs in males. Although the cause of this short-lived surge of gonadotropins remains to be understood, it is clearly independent of sex steroids. The sensitivity of the gonadotrophs to stimulation subsequently diminishes, and the system remains quiescent until just before puberty. Release of FSH is greater than that of LH during the prepubertal period, a pattern that is reversed after puberty. GnRH preferentially triggers LH release in men. This preferential release of LH may reflect maturation of the testes, which secrete inhibins, a specific inhibitor of FSH secretion at the level of the anterior pituitary gland. Increased sensitivity of the pituitary to increasing gonadal steroid production may also be responsible for the diminished secretion of FSH.

LH stimulates the Leydig cells of the testis to produce testosterone LH derives its name from effects observed in the female, that is, from the ability to stimulate ovulation and the formation and maintenance of the corpus luteum (see p. 1116). The comparable substance in the male was originally referred to as interstitial cell–stimulating hormone (ICSH). Subsequently, investigators realized that LH and ICSH are the same substance, and the common name became LH. LH stimulates the synthesis of testosterone by the testes. Testosterone production decreases in males after hypophysectomy. Conversely, LH (or hCG) treatment of men increases testosterone levels, but only if the testes are intact and functional. The interstitial Leydig cells are the principal

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targets for LH and the primary source of testosterone production in the male. The plasma membranes of Leydig cells have a high-affinity LH receptor, a GPCR coupled to Gαs (Fig. 54-4). Binding of LH to this receptor activates membrane-bound adenylyl cyclase (see p. 53), which catalyzes the formation of cAMP, which in turn activates protein kinase A (PKA). Activated PKA modulates gene transcription (see Fig. 4-13) and increases the synthesis of enzymes and other proteins necessary for the biosynthesis of testosterone (see pp. 1097–1100).

FSH stimulates Sertoli cells to synthesize hormones that influence Leydig cells and spermatogenesis The Sertoli cells are the primary testicular site of FSH action (see Fig. 54-4). FSH also regulates Leydig cell physiology via effects on Sertoli cells. The signaling events after FSH binding are similar to those described above for LH on the Leydig cell. Thus, binding of FSH to a GPCR activates Gαs, causing stimulation of adenylyl cyclase, an increase in [cAMP]i, stimulation of PKA, transcription of specific genes, and increased protein synthesis. These proteins are important for synthesis and action of steroid hormones, including the following: 1. Androgen-binding protein (ABP), which is secreted into the luminal space of the seminiferous tubule, near the developing sperm cells. ABP helps to keep local testosterone levels high. 2. P-450 aromatase (P-450arom; see p. 1117 and Table 50-2), a key steroidogenic enzyme that converts testosterone, which diffuses from the Leydig cells to the Sertoli cells, into estradiol. 3. Growth factors and other products that support sperm cells and spermatogenesis. These substances significantly increase the number of spermatogonia, spermatocytes, and spermatids in the testis. The stimulatory effect of FSH on spermatogenesis is not a direct action of FSH on the spermatogonia; instead, stimulation of spermatogenesis occurs via the action of FSH on Sertoli cells. FSH may also increase the fertility potential of sperm; it appears that this effect of FSH results from stimulation of motility, rather than from an increase in the absolute number of sperm. 4. Inhibins, which exert negative feedback on the hypothalamic-pituitary-testicular axis to inhibit FSH secretion (see below). Inhibins are members of the transforming growth factor-β (TGF-β) superfamily, which also includes the activins and antimüllerian hormone (see p. 1080). Inhibins are glycoprotein heterodimers consisting of one α and one β subunit that are covalently linked. The granulosa cells in the ovary and the Sertoli cells in the testis are the primary sources of inhibins. We discuss the biology of inhibins and activins in more detail on pages 1113–1115. Inhibins are secreted into the seminiferous tubule fluid and into the interstitial fluid of the testicle. In addition to exerting an endocrine effect on the axis, inhibins also have paracrine effects, acting as growth factors on Leydig cells. Leydig cells and Sertoli cells engage in crosstalk (see Fig. 54-4). For example, the Leydig cells make testosterone,

Chapter 54  •  The Male Reproductive System

N54-1  Human Chorionic Gonadotropin Contributed by Ervin Jones, Walter Boron, and Emile Boulpaep hCG is secreted by the placenta, and some reports have described that small amounts of this substance are made in the testes, pituitary gland, and other nonplacental tissues. hCG appears in the urine of pregnant women about 12 to 14 days after conception—the basis for pregnancy tests. In former times, hCG was extracted from the urine of pregnant women.

1095.e1

N54-2  Plasma Lifetime of Luteinizing Hormone, Human Chorionic Gonadotropin, and FollicleStimulating Hormone Contributed by Ervin Jones The disappearance of exogenous LH from the circulation is independent of gonadal function and follows a dual exponential time course. The half-life of the fast component is 40 minutes and that of the slow component is 120 minutes. Because of its increased glycosylation, hCG has an even longer half-life. FSH has a slower turnover rate; its disappearance from the blood is described by two exponentials with half-lives of about 4 hours and 3 days, respectively.

SECTION IX  •  The Reproductive System

Adenylyl cyclase

LH

Gs

FSH

Capillary AC cAMP

Spermatogonium To bloodstream

1096

FSH receptor

Gs cAMP

PKA

AC

Sertoli cell

PKA Growth factors Other products

Cholesterol

New protein synthesis

New protein synthesis Inhibins

Estradiol ABP Aromatase

Enzymes Testosterone Testosterone Leydig cell Extracellular space

Lumen

Figure 54-4  Leydig and Sertoli cell physiology. The Leydig cell (left) has receptors for LH. The binding of LH increases testosterone synthesis. The Sertoli cell (right) has receptors for FSH. (Useful mnemonics: L for LH and Leydig, S for FSH and Sertoli.) FSH promotes the synthesis of androgen-binding protein (ABP), aromatase, growth factors, and inhibin. There is crosstalk between Leydig cells and Sertoli cells. The Leydig cells make testosterone, which acts on Sertoli cells. Conversely, the Sertoli cells convert some of this testosterone to estradiol (because of the presence of aromatase), which can act on the Leydig cells. Sertoli cells also generate growth factors that act on the Leydig cells.

N54-3

Adult Plasma testosterone (ng/mL)

which acts on Sertoli cells. In the rat, β endorphin produced by fetal Leydig cells binds to opiate receptors in Sertoli cells and inhibits their proliferation. Synthesis of β endorphins could represent a local feedback mechanism by which Leydig cells constrain the number of Sertoli cells. Conversely, Sertoli cells affect Leydig cells. For example, Sertoli cells convert testosterone—manufactured by Leydig cells—to estradiol, which decreases the capacity of Leydig cells to produce testosterone in response to LH. In addition, FSH acting on Sertoli cells produces growth factors that may increase the number of LH receptors on Leydig cells during development and thus result in an increase in steroidogenesis (i.e., an increase in testosterone production). What, then, is required for optimal spermatogenesis to occur? It appears that two testicular cell types (Leydig cells and Sertoli cells) are required, as well as two gonadotropins (LH and FSH) and one androgen (testosterone). First, LH and Leydig cells are required to produce testosterone. Thus, LH, or rather its substitute hCG, is used therapeutically to initiate spermatogenesis in azoospermic or oligospermic men. Second, FSH and Sertoli cells are important for the nursing of developing sperm cells and for the production of inhibin and growth factors, which affect the Leydig cells. Thus, FSH plays a primary role in regulating development of the appropriate number of the Leydig cells so that adequate testosterone levels are available for spermatogenesis and the development of secondary sex characteristics. During early puberty in boys, both FSH and LH levels increase while, simultaneously, the Leydig cells proliferate and plasma levels of testosterone increase (Fig. 54-5). 

6

Fertilization Birth

Senescence

Puberty

5 4 3 2 1 0

3

6

Months

9

1

10

20

60

Years

Figure 54-5  Plasma testosterone level as a function of age in human males. (Data from Griffin JE, et al: The testis. In Bondy PK, Rosenberg LE [eds]: Metabolic Control and Disease. Philadelphia, WB Saunders, 1980; and Winter JS, Hughes IA, Reyes FI, Faiman C: Pituitary-gonadal relations in infancy: 2. Patterns of serum gonadal steroid concentrations in man from birth to two years of age. J Clin Endocrinol Metab 42:679– 686, 1976.)

The hypothalamic-pituitary-testicular axis is under feedback inhibition by testicular steroids and inhibins The hypothalamic-pituitary-testicular axis in postpubertal males not only induces production of testosterone and inhibin by the testes but also receives negative feedback from these substances (see Fig. 54-2). Normal circulating levels of testosterone inhibit the pulsatile release of GnRH by the hypothalamus and thereby

Chapter 54  •  The Male Reproductive System

1096.e1

N54-3  Effects of Follicle-Stimulating Hormone on Leydig and Sertoli Cells during Puberty Contributed by Ervin Jones As noted in the upper panel of eFigure 54-1, both FSH and LH levels increase during early puberty in boys, while simultaneously the Leydig cells proliferate. As a result, the Leydig cells increase their production of testosterone, and plasma levels of this steroid hormone increase, as shown in the lower panel of eFigure 54-1. The primary target of FSH in the testis is the Sertoli cell (see Fig. 54-4). Via this action on Sertoli cells, FSH indirectly increases the number of Leydig cells, which is a key part of pubertal development. In hypogonadotropic-hypogonadal men (i.e., individuals who have decreased levels of both LH and FSH), treating with exogenous FSH stimulates the Sertoli cells to release factors that induce differentiation and maturation of Leydig cells.

Subsequent treatment with hCG (i.e., which acts like LH) acts on these Leydig cells to synthesize testosterone and thereby support spermatogenesis. During puberty, a related change is that Sertoli cells become relatively less sensitive to FSH, but at the same time they become more dependent on the testosterone that the Leydig cells produce. Thus, there is a continuum in the development of the Sertoli cells: as proliferation and maturation of the Sertoli cells proceeds during puberty, the responsiveness of the Sertoli cell to FSH declines while its responsiveness to testosterone increases. The mechanism for this switch appears to be that FSH stimulates the synthesis of androgen receptors on Sertoli cells.

12

LH

8 Plasma FSH or LH (ng/mL)

4

FSH

0 6 Plasma testosterone (ng/mL)

4 2

0 Stage of puberty 1 Bone age 8

2 12

3 14

4 5/Adult 16 Adult

eFigure 54-1  Plasma levels of FSH, LH, and testosterone from puberty to adulthood. The upper panel shows how plasma levels of biologically active LH and FSH increase during puberty, expressed in terms of both the stages of puberty and bone age. The lower panel shows the concomitant rise in plasma levels of testosterone. LH stimulates Leydig cells to synthesize testosterone. FSH indirectly promotes testosterone synthesis by stimulating the Sertoli cells to produce factors that act on Leydig cells. (Upper panel, modified from Reiter EO, Beitins IZ, Ostrea TR, et al: Bioassayable luteinizing hormone during childhood and adolescence and in patients with delayed pubertal development. J Clin Endocrinol Metab 54:155–161, 1982; and Beitins IZ, Padmanabhan V, Kasa-Vubu J, et al: Serum bioactive follicle-stimulating hormone concentration from prepuberty to adulthood: A cross-sectional study. J Clin Endocrinol Metab 71:1022–1027, 1990; lower panel, modified from Grumbach MM, Styne DM: Puberty: Ontogeny, neuroendocrinology, physiology, and disorders. In Wilson JD, Foster DW, Kronenberg HM, Larsen PR [eds]: Williams Textbook of Endocrinology, 9th ed. Philadelphia, WB Saunders, 1998, pp 1509–1625.)

Chapter 54  •  The Male Reproductive System

reduce the frequency and amplitude of the LH- and FSHsecretory pulses. Testosterone also has negative-feedback action on LH secretion at the level of the pituitary gonadotrophs. The inhibins also feed back on FSH secretion. Evidence for such negative feedback is that plasma FSH concentrations increase in proportion to the loss of germinal elements in the testis. Thus, FSH specifically stimulates the Sertoli cells to produce inhibin, and inhibin “inhibits” FSH secretion. Inhibin appears to diminish FSH secretion by acting at the level of the anterior pituitary rather than the hypothalamus. The secretion of LH and FSH is under the additional control of neuropeptides, amino acids such as aspartate, corticotropin-releasing hormone (CRH), and endogenous opioids.

TESTOSTERONE Leydig cells convert cholesterol to testosterone Cholesterol is the obligate precursor for androgens and other testicular steroids. The Leydig cell can synthesize cholesterol de novo from acetyl coenzyme A or can take it up as low-density lipoproteins from the extracellular fluid by receptor-mediated endocytosis (see p. 42). The two sources appear to be equally important in humans. Preceding the metabolism of cholesterol is the translo­ cation of this precursor to the mitochondrial inner membrane, which requires two proteins. The first is sterol-carrier protein 2 (SCP-2), a 13.5-kDa protein that translocates cholesterol from the plasma or organellar membranes to other organellar membranes, including the outer mitochondrial membrane. The second protein is the steroidogenic acute regulatory protein (StAR), which belongs to a large family of proteins involved in lipid trafficking and metabolism. The 37-kDa pro-StAR protein—the precursor to StAR—ferries cholesterol from the endoplasmic reticulum to the outer mitochondrial membrane. The 30-kDa mature StAR protein resides in the mitochondrial intermembrane space and extracts cholesterol from the mitochondrial outer membrane and ferries it across to the mitochondrial inner membrane. The Leydig cell uses a series of five enzymes to convert cholesterol to testosterone. Three of these enzymes are P-450 N54-4  As summarized in enzymes (see Table 50-2).  Figure 54-6, because 3β-hydroxysteroid dehydrogenase (3βHSD) can oxidize the A ring of four intermediates, testosterone synthesis from cholesterol can take four pathways. The following is the “preferred” pathway: 1. Cholesterol conversion to pregnenolone. The pathway for testosterone synthesis begins in the mitochondrial inner membrane, where the cytochrome P-450 sidechain-cleavage enzyme (P-450SCC, also called 20,22desmolase)  N54-4  removes the long side chain (carbons 22 to 27) from the carbon at position 20 of the cholesterol molecule (27 carbon atoms), yielding pregnenolone (21 carbon atoms). This reaction is the ratelimiting step in the biosynthesis of testosterone, as it is for other steroid hormones. LH stimulates this reaction in the Leydig cell in two ways. First, LH increases the affinity of P-450SCC for cholesterol. Second, LH has

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long-term actions of increasing the levels of SCP-2, StAR, and P-450SCC via PKA-stimulated gene transcription. 2. Pregnenolone conversion to 17α-hydroxypregnenolone. In the smooth endoplasmic reticulum (SER), 17αhydroxylase (P-450c17)  N54-4  then adds a hydroxyl group at position 17 to form 17α-hydroxypregnenolone. P-450c17, a key branch-point enzyme in the steroido­ genic pathway, also converts progesterone to 17αhydroxyprogesterone (see Fig. 54-6, middle column). 3. 17α-hydroxypregnenolone conversion to dehydroepiandrosterone. In the SER, the 17,20-desmolase (a different activity of the same P-450c17 whose 17α-hydroxylase activity catalyzes the previous step) removes the position-20 side chain from position 17 of 17αhydroxypregnenolone, producing a 19-carbon steroid called dehydroepiandrosterone (DHEA). This so-called delta-5 pathway on the left of Figure 54-6 is the preferred route in Leydig cells to yield DHEA, the precursor for all sex steroids. 4. DHEA conversion to androstenediol. In the SER of the Leydig cell, a 17β-hydroxysteroid dehydrogenase (17βHSD, which is not a P-450 enzyme) converts the ketone at position 17 of DHEA to a hydroxyl group to form androstenediol. 5. Androstenediol conversion to testosterone. Finally, in the SER, 3β-HSD (not a P-450 enzyme) oxidizes the hydroxyl group of androstenediol at position 3 of the A ring to a ketone, forming testosterone.  N54-5 In addition, the testis can also use 5α-reductase, which is located in the SER, to convert testosterone to dihydrotestosterone (DHT). However, extratesticular tissue is responsible for most of the production of DHT. The conversion of testosterone to DHT is especially important in certain testosterone target cells (see pp. 1097–1099). The Leydig cells of the testes make ~95% of the circulating testosterone. Although testosterone is the major secretory product, the testes also secretes pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, androsterone, and DHT. The conversion of testosterone to DHT by Leydig cells is minor compared with its production in certain testosterone target cells (see p. 1085). Androstenedione is of major importance because it serves as a precursor for extraglandular estrogen formation. In men who are between the ages of 25 and 70 years, the rate of testosterone production remains relatively constant (Table 54-1). Figure 54-5 summarizes the changes in plasma testosterone levels as a function of age in human males.  N54-6

Adipose tissue, skin, and the adrenal cortex also produce testosterone and other androgens Several tissues besides the testes—including adipose tissue, skin, adrenal cortex, brain, and muscle—produce testosterone and several other androgens. These substances may be synthesized de novo from cholesterol or produced by peripheral conversion of precursors. Moreover, the peripheral organs and tissues may convert sex steroids to less active forms (see Fig. 54-6). Notable sites of extragonadal conversion include adipose tissue and the skin. Androstenedione is converted to testosterone in peripheral tissues. In this case,

Chapter 54  •  The Male Reproductive System

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N54-4  Cytochrome P-450 Enzymes Contributed by Emile Boulpaep and Walter Boron The term cytochrome P-450 enzymes refers to a family of several hundred heme-containing enzymes that are located primarily in the SER. See Table 50-2 for some examples of these enzymes that play a role in steroidogenesis. We discuss the roles of these P-450 enzymes for the adrenal gland on page 1021, for the testis on page 1097, and for the ovary on page 1117. On page 64, we discuss the role of a P-450 enzyme (i.e., epoxygenase) in the metabolism of arachidonic acid (see also Fig. 3-11). These enzymes are monooxygenases.* That is, they transfer one atom of molecular oxygen to an organic substrate RH to form ROH, whereas the other oxygen atom accepts two protons from the reduced form of the enzyme to form water: P- 450 (H2 −−reduced form) + RH + O2 → P- 450 (oxidized form) + ROH + H2O This monooxygenation reaction is also referred to as a hydroxylation reaction because the enzyme hydroxylates RH to

form ROH. Note that at the end of the reaction, the P-450 monooxygenase is in its reduced form. Another enzyme—a cytochrome P-450 reductase—recycles the P-450 monooxygenase to its reduced form; in the process, this P-450 reductase becomes oxidized. Finally, the oxidized P-450 reductase recycles to its reduced form by oxidizing the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+ or the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD+ or one of the flavin nucleotides (reduced flavin adenine dinucleotide [FADH2] or reduced flavin mononucleotide [FMNH2]). The P-450 enzymes are so named because when the reduced forms of the enzymes bind carbon monoxide, they absorb light strongly at 450 nm.

REFERENCE Nelson DL, Cox MM: Lehninger Principles of Biochemistry, 3rd ed. New York, Worth Publishers, 2000, pp 782–783.

*Enzymes that transfer both oxygen atoms of molecular oxygen to an organic substrate are termed dioxygenases. In contrast, oxidases (e.g., cytochrome oxidase in the electron transport chain of mitochondria) are enzymes that catalyze oxidations in which neither of the atoms of molecular oxygen becomes part of the oxidized product. Instead, the molecular oxygen acts as an electron acceptor to form a molecule such as H2O or H2O2.

N54-5  Delta-5 and Delta-4 Steroids Contributed by Sam Mesiano Pregnenolone is called P5. Progesterone is P4. This is where the terms delta-5 and delta-4 come from. 3β-HSD is a major branch-point enzyme in the steroidogenic pathway (see Fig. 54-6). It converts all delta-5 steroids to delta-4 steroids via an isomerase activity and therefore is essential for the production of mineralocorticoids and glucocorticoids. The competition between 17α-hydroxylase/17,20-desmolase (two enzymatic activities mediated in the same protein, also

known as P-450c17) and 3β-HSD for pregnenolone and 17αhydroxypregnenolone is a major determinant of whether a steroidogenic cell will produce mineralocorticoids, glucocorticoids, or sex steroids. In the Leydig cell, 17α-hydroxylase/17,20desmolase prevails to produce DHEA, which 17β-HSD1 then converts to androstenediol. DHEA can also undergo conversion, via 3β-HSD, to androstenedione, which 17β-HSD1 then converts to testosterone.

N54-6  Testosterone Secretion and Production Rates Contributed by Emile Boulpaep and Walter Boron In the text, we noted that plasma testosterone levels are relatively constant in males between the ages of 25 and 70 years. As for any substance in the blood, the stability of plasma levels of testosterone indicates that the rate of testosterone production is equal to the rate of testosterone removal. However, the stability of plasma testosterone levels says nothing about the individual rates of production and removal. It is important to distinguish between the secretion rate of a hormone and the production rate. Secretion refers to the release of the hormone from a specific organ or gland, and may be determined by selectively catheterizing the artery and vein supplying that tissue and ascertaining the arterial-venous difference in the concentration of that substance. For example, the concentration of testosterone is 400 to 500 µg/L in effluent of the spermatic vein; this level is ~75 times higher than the concentration found in the arterial blood. Thus, if we knew the blood flow out of the spermatic vein, then we could compute the rate of secretion of testosterone by the testis. Production rate refers to the total appearance of the hormone in the circulation as the result of the secretion by all tissues in the body. Thus, the secretion rate for the testes equals the

whole-body production rate only when other tissues make no contribution. In the steady state, the amount of testosterone cleared from the circulation equals the amount produced. Thus,

PR = MCR × [S] (µg day ) (L day) (µg L)

(NE 54-1)

Here, PR is the whole-body production rate, MCR is the metabolic clearance rate, and [S] is the concentration of the substance in the plasma. MCR is defined in the same way as renal clearances (see Table 33-2). That is, MCR is the virtual number of liters per day that are fully cleared of testosterone. This clearance is due to the metabolism of testosterone, which is discussed on pages 1099–1100. Because the mean metabolic clearance rate for testosterone is ~1000 L/day, and the testosterone concentration is about 6.5 µg/L (range, 3 to 10 µg/L), the production rate must be about 6500 µg/day. Evidence for this high clearance rate is the fact that the plasma half-life of testosterone is only 10 to 20 minutes.

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SECTION IX  •  The Reproductive System

Acetate

Cholesterol (27-carbon compound) 21 20 22 24 25 26 12 18 17 23 16 27 13

11 1

19 9

A

10

2

HO

4

8

B

5

D

C

14

15

Figure 54-6  Biosynthesis of testosterone. This

7

scheme summarizes the synthesis of the androgens from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; they are located in either the SER or the mitochondria. The side-chain-cleavage enzyme that produces pregnenolone is also known as 20,22desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. There are four possible pathways from pregnenolone to testosterone; the preferred pathway in the human testis appears to be the delta-5 pathway, the one along the left edge of the figure to androstenediol, followed by oxidation of the A ring to testosterone. Some of these pathways are shared in the biosynthesis of the glucocorticoids and mineralocorticoids (see Fig. 50-2) as well as the estrogens (see Fig. 55-8).

6

MITOCHONDRIA Side-chain-cleavage enzyme Pregnenolone

Progesterone

CH3

A

A

B

B

SER 17 α-Hydroxylase 17 α-Hydroxyprogesterone

17 α-Hydroxypregnenolone CH3

CH3

C

C

O

CH3

A

B

A

HO

D

C

CH3

Same protein.

O

CH3

OH

D

C

CH3

OH

B

O

SER 17,20-Desmolase

SER 17,20-Desmolase

CH3 A

C

D

B

SER 17β-Hydroxysteroid dehydrogenase Androstenediol CH3 C

CH3 A

OH D

CH3

A

Testosterone OH CH3 C

CH3

SER 5 α-Reductase

A

C A

OH CH3

OH D

CH3 A O

Estradiol (E2)

HO

SER 5 α-Reductase

B H

SER 17β-Hydroxysteroid dehydrogenase

B

Dihydrotestosterone

C

CH3

B

D

O

CH3

A HO

SER 17β-Hydroxysteroid dehydrogenase

A

Androsterone

D

C

B

O

O

B

HO

CH3

D

C

CH3

Estrone (E1)

O

Aromatase

HO

3β-Hydroxysteroid dehydrogenase

O CH3

SER

Androstenedione

Dehydroepiandrosterone (DHEA)

HO

O

O

SER 17 α-Hydroxylase

ANDROGENS (19 carbons)

C D

C

CH3

HO

(21 carbons)

CH3 CH3

D

C

CH3

O

C

SER

CH3

B H

C

D

Mainly in testosterone target cells.

B

OH CH3 D

ESTROGENS (18 carbons)

Chapter 54  •  The Male Reproductive System

1099

TABLE 54-1  Androgen Production and Turnover STEROID

BLOOD PRODUCTION RATE—HORMONE DELIVERED TO THE BLOOD (µg/day)

PLASMA CONCENTRATION (µg/L)

6500

6.5

Testosterone Androstenedione Dihydrotestosterone

2000–6000

1.5

300

0.5

androstenedione is the precursor for the hormone testosterone. Testosterone can be converted to estradiol or DHT or go “backward” by reversible interconversion to androstenedione. Thus, a potent hormone such as testosterone may also serve as a precursor for a weaker hormone (androstenedione), a hormone with different activities (estradiol), or a more potent hormone having similar activities (DHT). This last example may be illustrated by the effects of DHT on hair follicles, sebaceous glands, and the sex accessory organs. In these tissues, the androgenic effects of circulating testosterone are amplified by its conversion by 5α-reductase to DHT, which has a much higher affinity for the androgen receptor (AR; see p. 1085). Some tissues, including the brain, aromatize testosterone to estradiol, and thus the action of this metabolite occurs via the estrogen receptor. The adrenal cortex (see p. 1021) is another source of androgen production in both males and females. Normal human adrenal glands synthesize and secrete the weak androgens DHEA, conjugated DHEA sulfate, and androstenedione. Essentially, all the DHEA in male plasma is of adrenal origin. However, 2 mL. The typical ejaculate content varies between 150 and 600 million spermatozoa. Aside from the sperm cells, the remainder of the semen (i.e., 90%) is seminal plasma, the extracellular fluid of semen (Table 54-3). Very little seminal plasma accompanies the spermatozoa as they move through the testes and epididymis. The seminal plasma originates primarily from the accessory glands (the seminal vesicles, prostate gland, and

1103

Acrosomal cap

Nucleus

Proximal centriole

Mitochondrial sheath

Ring centriole (annulus) Axoneme

Figure 54-9  Anatomy of a spermatozoon.

TABLE 54-3  Normal Parameter Values for Semen PARAMETER

VALUE

Volume of ejaculate

2–6 mL

Viscosity

Liquefaction in 1 hr

pH

7–8

Count

≥20 million/mL

Motility

≥50%

Morphology

60% normal

bulbourethral glands). The seminal vesicles contribute ~70% of the volume of semen. Aside from the sperm, the remaining ~20% represents epididymal fluids, as well as secretions of the prostate gland and bulbourethral glands. However, the composition of the fluid exiting the urethral meatus during ejaculation is not uniform. The first fluid to exit is a mixture of prostatic secretions and spermatozoa with epididymal fluid. Subsequent emissions are composed mainly

Chapter 54  •  The Male Reproductive System

1103.e1

N54-8  Sperm Maturation Contributed by Ervin Jones Most of the changes that occur to the sperm within the epididymis have to do with the acquisition of motility. Sperm isolated from the caput of the epididymis exhibit inconsistent patterns of motility, ranging from immotility to highly random movements of the flagellum. As sperm pass through the epididymis, rapid forward progression occurs and random tail flexing is reduced; these changes are first seen in a few spermatozoa obtained from the corpus and comprise the predominant pattern in those obtained from the cauda of the vas deferens. Motility is a prerequisite for fertility; it may be surmised that the acquisition of progressive forward motility is largely a function of maturation of the spermatozoa. Biochemical changes also occur as sperm pass through the epididymis. It has been suggested that membrane-bound enzymes play a role in modifying the surface of spermatozoa during epididymal transit. Total protein concentrations decrease

by more than half in spermatozoa but remain unchanged in epididymal fluid during epididymal transit. Further, it appears that the majority of the testicle-derived proteins are removed from the spermatozoal surface during epididymal maturation, to be replaced by new low-molecular-weight peptides. These new peptides appear almost simul­taneously with the development of forward-progressive motility. Nuclear changes also occur as spermatozoa traverse the epididymis. Nuclear proteins play a role in morphogenesis and in the stabilization of the head of the sperm cell. Protamine staining increases as spermatozoa pass from the rete testes to the corpus of the epididymis and sharply declines thereafter. These observations, taken together, underscore the significance of the epididymis for sperm maturation, a prerequisite for the acquisition of fertilizing capacity.

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SECTION IX  •  The Reproductive System

of secretions derived from the seminal vesicles. The first portion of the ejaculate contains the highest density of sperm; it also usually contains a higher percentage of motile sperm cells. Seminal plasma is isotonic. The pH in the lumen of the epididymis is relatively acidic (6.5 to 6.8) as the result of H+ secretion by clear cells that are analogous to intercalated cells in the nephron (see p. 729). Addition of the relatively alkaline secretions of the seminal vesicles raises the final pH of seminal plasma to between 7.3 and 7.7. Spermatozoa generally tolerate alkalinity better than acidity. A pH near neutrality or slightly higher is optimal for the motility and survival of sperm cells in humans and in other species. Seminal plasma contains a plethora of sugars and ions. Fructose and citric acid are contributed to the seminal plasma by the accessory glands, and their concentrations vary with the volume of semen ejaculated. The fructose is produced in the seminal vesicles. In a man with oligospermia (i.e., a low daily sperm output) and a low ejaculate volume (recall that more than half of the ejaculate comes from the seminal vesicles), the absence of fructose suggests obstruction or atresia of the seminal vesicles. Ascorbic acid and traces of B vitamins are also found in human seminal plasma. The prostate gland releases a factor—which contains sugars, sulfate, and a vitamin E derivative—that acts to prevent the clumping of sperm heads. In addition, human semen also contains high concentrations of choline and spermine, and is also rich in Ca2+, Na+, Mg2+, K+, Cl−, Zn2+, and phosphate. Concentrations of Zn2+ and Ca2+ are higher in semen than in any other fluid and most other tissues. Calcium ions stimulate the motility of immature epididymal spermatozoa, but they inhibit the motility of spermatozoa in ejaculates obtained from humans. It appears that the diminished response of sperm to Ca2+ and the acquisition of progressive motility are functions of epididymal maturation. Semen also contains free amino acids, low-molecularweight polypeptides, and proteins. The free amino acids, which probably arise from the breakdown of protein after the semen is ejaculated, may protect spermatozoa by binding toxic heavy metals or by preventing the agglutination of proteins. Human semen coagulates immediately after ejaculation. Coagulation is followed by liquefaction, which is apparently caused by proteolytic enzymes contained in prostatic secretions. Prostatic secretion is rich in acid phosphatase. The natural substrate for acid phosphatase is phosphorylcholine, which is contributed by the seminal vesicles. Hyaluronidase is also present in human semen, although its functional role remains to be clarified. Hyaluronidase is not a product of the accessory glands; rather, it is contained within the sperm cell cytoplasm and is rapidly released into the seminal plasma. Hyaluronidase may play a role in facilitating penetration of the oocyte by the sperm cell because of its ability to depolymerize hyaluronic acid.  N54-9

MALE SEX ACT Sex steroids influence the central nervous system, even in utero, and play important roles in determining and regulating complex patterns of sexual behavior. However,

reproductive behavior is extraordinarily complex and is influenced by numerous factors other than sex steroids, such as one’s genetic constitution, social contacts, and the age at which hormones exert their effects. In this subchapter we describe the neurophysiology of the male sex act.

The sympathetic and parasympathetic divisions of the autonomic nervous system control the male genital system The testes, epididymis, male accessory glands, and erectile tissue of the penis (corpora cavernosa and corpus spongiosum) receive dual innervation from the sympathetic and parasympathetic branches of the autonomic nervous system (ANS; see pp. 340–341). The penis also receives both somatic efferent (i.e., motor) and afferent (i.e., sensory) innervation via the pudendal nerve (S2 through S4). Sympathetic Division of the ANS  As described in Chapter 14, the preganglionic sympathetic neurons originate in the thoracolumbar segments of the spinal cord (T1 through T12, L1 through L3; see Fig. 14-4). For the lower portion of the sympathetic chain (T5 and below), the preganglionic fibers may pass through the paravertebral sympathetic trunk and then pass via splanchnic nerves to a series of prevertebral plexuses and ganglia (see below). Once within one of these plexuses or ganglia, the preganglionic fiber may either (1) synapse with the postganglionic fiber, or (2) pass on to a more caudal plexus or ganglion without synapsing. The sympathetic efferent (motor) nerve fibers that are supplied to the male sex organs emanate from five primary prevertebral nerve plexuses (Fig. 54-10): the celiac, superior mesenteric, inferior mesenteric, superior hypogastric, and inferior hypogastric or pelvic plexuses. The celiac plexus is of interest in a discussion of male sex organs only because preganglionic sympathetic fibers pass through this plexus on their way to more caudal plexuses. The superior mesenteric plexus lies on the ventral aspect of the aorta. Preganglionic fibers from the celiac plexus pass through the superior mesenteric plexus on their way to more caudal plexuses. Most of the preganglionic sympathetic fibers pass from the superior mesenteric plexus to the inferior mesenteric plexus, although some of the nerves pass directly to the hypogastric plexus. The superior hypogastric plexus is a network of nerves located distal to the bifurcation of the aorta. The inferior hypogastric or pelvic plexus receives sympathetic supply from the hypogastric nerve. In addition to these five plexuses, two other small ganglia are of interest. The spermatic ganglion is located near the origin of the testicular artery from the aorta. The spermatic ganglion receives fibers directly from the lumbar sympathetic nerves and from branches of several other ganglia. The hypogastric (or pelvic) ganglion is located at the junction of the hypogastric and pelvic nerve trunks. Parasympathetic Division of the ANS  The preganglionic parasympathetic neurons relating to the male reproductive system originate in the sacral segments of the spinal cord (S2 through S4; see Fig. 14-4). These fibers pass via the pelvic nerve to the pelvic plexus, where they synapse with the postganglionic parasympathetic neurons.

Chapter 54  •  The Male Reproductive System

1104.e1

N54-9  Congenital and Acquired Ductal Obstruction Contributed by Ervin Jones Genital duct obstruction may be congenital and may result from ductal absence or structural abnormality, or it may be acquired as a result of stricture, infection, or vasectomy. Genital duct obstruction is found in ~7% of infertile men. An uncommon cause of male infertility is congenital absence of the vas deferens, which accounts for as many as 50% of cases of congenital ductal obstruction. These patients generally have azoospermic ejaculates with low volume. Congenital absence of the vas deferens is common in male patients with cystic fibrosis (CF) and is sometimes the only manifestation of CF. Epididymal abnormalities range from the presence of an incomplete epididymis to the presence of only small portions of the epididymis; in addition, the seminal vesicles are often absent. Spermatogenesis is thought to be normal inasmuch as testicular biopsy specimens demonstrate germ cells in several stages of development. Obstruction of the epididymis may also occur as a result of gonococcal or tuberculous epididymitis. Smallpox and

filariasis are common causes of ductal obstruction in areas where these diseases are endemic. Inspissated secretions may occlude the epididymis in men with Young syndrome or CF. Elective vasectomy, a simple surgical procedure in which a small segment of the vas deferens is removed to ensure male infertility, is currently the leading cause of ductal obstruction. Azoospermia in men with normal testes is the hallmark of genital duct obstruction. However, when specimens of testicles from men who have had vasectomies are examined microscopically, interstitial fibrosis is found in as many as 20% of cases. This group exhibits low fertility after elective reversal of their vasectomy. When the seminiferous tubules are examined, increased thickness of the tubule wall, an increase in crosssectional tubular area, and decreased numbers of Sertoli cells are usually noted. Testosterone and gonadotropin levels are normal in most patients with ductal obstruction.

Chapter 54  •  The Male Reproductive System

A

B

SYMPATHETIC INNERVATION

Celiac ganglion

Celiac plexus Superior mesenteric plexus

Right aorticorenal ganglion

Left aorticorenal ganglion

Inferior mesenteric ganglion

SAGITTAL SECTION SHOWING INNERVATION OF MALE GENITAL SYSTEM

Aorta

Superior mesenteric ganglion

Spermatic ganglion

Superior hypogastric plexus

Pelvic nerves (pelvic parasympathetics)

Right and left inferior hypogastric nerves

Renal artery

Rectum

Spermatic artery and plexus

Bladder Small and large cavernous nerves

Inferior mesenteric plexus Aorta

1105

Pelvic plexus Pudendal nerve

Aortic plexus

Dorsal nerve of penis

Superior hypogastric plexus Hypogastric nerve

Figure 54-10  Innervation of the male genital system. A, The sympathetic innervation of the male genital system involves a series of prevertebral nerve plexuses and ganglia. B, Three motor pathways as well as a sensory pathway are involved in erection. (1) Parasympathetic innervation: Preganglionic parasympathetic fibers arise from the sacral spinal cord and from the pelvic nerve, and they synapse in the pelvic plexus. The postganglionic parasympathetic fibers follow the cavernous nerve to the penile corpora and vasculature. (2) Sympathetic innervation: Preganglionic sympathetic fibers exit the thoracolumbar cord and synapse in one of several prevertebral ganglia. Postganglionic fibers reach the genitalia via the hypogastric nerve, the pelvic plexus, and the cavernous nerves. (3) Somatic innervation: Somatic (i.e., not autonomic) motor fibers originate in the sacral spinal cord, forming the motor branch of the pudendal nerve. The fibers innervate the striated penile muscles. In addition to these three motor pathways, there is also an afferent pathway from the penis. The dorsal nerve of the penis is the main terminus of the sensory pudendal nerve and is the sole identifiable root for tactile sensory information from the penis.

Inferior hypogastric (pelvic) plexus Hypogastric (pelvic) ganglion

Visceral Afferents  Sensory fibers are present in all the nerve tracts described (see Fig. 14-2). These fibers either (1) travel with the pelvic nerves to the dorsal root of the spinal cord, (2) travel with the sacral nerves to the sympathetic trunk and then rise in the sympathetic trunk to the spinal cord, or (3) travel with the hypogastric nerve and ascend to more rostral prevertebral plexuses and then to the spinal cord.

The principal functions of motor innervation to the male accessory glands include control of smooth-muscle contraction, vascular tone, and epithelial secretory activity.

Erection is primarily under parasympathetic control During erection, relaxation of the smooth muscles of the corpora cavernosa and the corpus spongiosum allows

1106

SECTION IX  •  The Reproductive System

increased inflow of blood to fill the corporal interstices and results in an increase in volume and rigidity. Vascular actions of the smooth muscles of the corpora and the perineal striated muscles are coordinated. For example, contraction of the striated muscles overlying the vascular reservoirs of the penile bulb increases the pressure of the blood in the corpora and promotes increased rigidity.  N54-10  The three major efferent (i.e., motor) pathways for the regulation of penile erection are parasympathetic (pelvic nerve), sympathetic (hypogastric nerve), and somatic (pudendal nerve). The two corpora cavernosa and the corpus spongiosum are usually coordinated in their erection (i.e., tumescence) and detumescence. However, they may act independently inasmuch as their vascular and neuroeffector systems are relatively independent. Parasympathetic Innervation  The first and most important pathway for erection is the parasympathetic division of the ANS. These fibers derive from the lumbar and sacral portions of the spinal cord and travel via the pelvic nerve, the pelvic plexus, and the cavernous nerve to the penile corpora and vasculature (see Fig. 54-10). This pathway is almost entirely parasympathetic but apparently also carries some sympathetic fibers (see below). The parasympathetic activity results in vasodilatation of the penile blood vessels, which increases blood flow to the cavernous tissue and engorges the organ with blood. In erectile tissue, parasympathetic postganglionic terminals release acetylcholine (ACh) and nitric oxide (NO), similar to the system discussed in Figure 14-11. First, ACh binds to M3 muscarinic receptors on endothelial cells. Via Gαq, these receptors would then lead to stimulation of PLC, increased [Ca2+]i, activation of NO synthase, and local release of NO (see p. 66). Second, the nerve terminals may also directly release NO. Regardless of the source of NO, this gas diffuses to the vascular smooth-muscle cell, where it stimulates guanylyl cyclase to generate cGMP, which in turn causes vasodilation (see Table 20-8; Box 54-3). Sympathetic Innervation  The second pathway, which is thought to be entirely sympathetic, exits the thoracolumbar spinal cord. The preganglionic fibers then course via the least splanchnic nerve, the sympathetic chain, and the inferior mesenteric ganglion. The postganglionic fibers reach the genitalia via the hypogastric nerve, the pelvic plexus, and the cavernous nerves (see above). Tonic sympathetic activity contributes to penile flaccidity. During erection, a decrease in this sympathetic tone allows relaxation of the corpora and thus contributes to tumescence. Somatic Innervation  The third pathway is the motor branch of the pudendal nerve. It has primarily somatic (i.e., not autonomic) fibers, originates in the sacral spinal cord, and innervates the striated penile muscles. Contraction of the striated ischiocavernosus muscle during the final phase of erection increases pressure inside the corpora cavernosa to values that are even higher than systemic arterial pressure. Contraction of the striated bulbospongiosus muscle increases engorgement of the corpus spongiosum, and thus the glans penis, by pumping blood up from the penile bulb underlying this muscle. Humans are apparently less dependent on their striated penile muscle for achieving and maintaining

BOX 54-3  Erectile Dysfunction

S

ildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) are reasonably well tolerated oral medications used to treat erectile dysfunction. Men with erectile dysfunction experience significant improvement in rigidity and duration of erections after treatment with these medications. As indicated in the text, the smooth-muscle tone of the human corpus cavernosum is regulated by the synthesis and release of NO, which raises [cGMP]i in vascular smoothmuscle cells, thereby relaxing the smooth muscle and leading to vasodilatation and erection. Breakdown of cGMP by cGMPspecific phosphodiesterase type 5 limits the degree of vasodilation and, in the case of the penis, limits erection. Sildenafil, vardenafil, and tadalafil are highly selective high-affinity inhibitors of cGMP-specific phosphodiesterase type 5 and thereby raise [cGMP]i in smooth muscle and improve erection in men with erectile dysfunction. The new medications are attractive because they are effective and benefit most men with insufficient erection. These medications stimulate erection only during sexual arousal and thus have a rather natural effect. They can be taken as little as 1 hour before planned sexual activity. One of the side effects of sildenafil is “blue vision,” a consequence of the effect of inhibiting cGMP-specific phosphodiesterase in the retina. In individuals taking other vasodilators, sildenafil can lead to sudden death. In women, sildenafil may improve sexual function by increasing blood flow to the accessory secretory glands (see pp. 1108 and 1127).

erection. However, these muscles are active during ejaculation and contribute to the force of seminal expulsion. Postganglionic neurons release other so-called nonadrenergic, noncholinergic neurotransmitters (see p. 543)— including NO—that also contribute to the erectile process. Afferent Innervation  The penis also has an afferent pathway. The dorsal nerve of the penis is the main terminus of the sensory pudendal nerve and is the sole identifiable root for tactile sensory information from the penis.

Emission is primarily under sympathetic control The term seminal emission refers to movement of the ejaculate into the prostatic or proximal part of the urethra. Under some conditions, seminal fluid escapes episodically or continuously from the penile urethra; this action is also referred to as emission. Emission is the result of peristaltic contractions of the ampullary portion of the vas deferens, the seminal vesicles, and the prostatic smooth muscles. These actions are accompanied by constriction of the internal sphincter of the bladder, which is under sympathetic control (see p. 736); this constriction of the sphincter prevents retrograde ejaculation of sperm into the urinary bladder (see Box 54-3). The rhythmic contractions involved in emission result from contraction of smooth muscle. In contrast to those of other visceral organ systems, the smooth-muscle cells of the male ducts and accessory glands fail to establish close contact with one another and show limited electrotonic coupling. In the male accessory glands, individual smooth-muscle cells

Chapter 54  •  The Male Reproductive System

N54-10  Nonvascular Contributions to Erection Contributed by Ervin Jones Striated muscle also contributes to erection. The contribution of skeletal muscle varies among species but plays a lesser role in humans, where erections are purely vascular.

1106.e1

Chapter 54  •  The Male Reproductive System

are directly innervated and have only limited spontaneous activity (i.e., multiunit smooth muscle; see p. 243). This combination allows a fast, powerful, and coordinated response to neural stimulation. Motor Activity of the Duct System  A gradation between

two forms of smooth-muscle activity occurs along the male duct system. The efferent ducts and proximal regions of the epididymis are sparsely innervated but display spontaneous contractions that can be increased via adrenergic agents acting on α-adrenergic receptors. In contrast, the distal end of the epididymis and the vas deferens are normally quiescent until neural stimulation is received during the ejaculatory process. Contraction of the smooth muscle of the distal epididymis, vas deferens, and accessory sex glands occurs in response to stimulation of the sympathetic fibers in the hypogastric nerve and release of norepinephrine. Indeed, an intravenous injection of epinephrine or norepinephrine can induce seminal emission, whereas selective chemical sympathectomy or an adrenergic antagonist can inhibit seminal emission. The role of parasympathetic innervation of the musculature of these ducts and accessory glands in the male is not entirely clear. Parasympathetic fibers may be preferentially involved in basal muscular activity during erection (i.e., before ejaculation) and during urination. Secretory Activity of the Accessory Glands  The effect of autonomic innervation on the secretory activity of the epithelia of the male accessory glands has been studied extensively. Electrical stimulation of the pelvic nerves (parasympathetic) induces copious secretions. The secretory rate depends on the frequency of stimulation and can be blocked with atropine, a competitive inhibitor of muscarinic ACh receptors. Cholinergic drugs induce the formation of copious amounts of secretions when these drugs are administered systemically. Secretions from the bulbourethral glands also contribute to the ejaculate. The bulbourethral glands do not store secretions but produce them during coitus. The secretory activity of the bulbourethral glands also appears to be under cholinergic control inasmuch as administration of atropine causes marked inhibition of secretion from these glands. Control of the motor activity of the ducts and the secretory activity of the accessory glands is complex and involves both the sympathetic and the parasympathetic divisions of the ANS. The central nervous system initiates and coordinates all these activities (Box 54-4).

Ejaculation is under the control of a spinal reflex As discussed, seminal emission transports semen to the proximal (posterior) part of the urethra. Ejaculation is the forceful expulsion of this semen from the urethra. Ejaculation is normally a reflex reaction triggered by the entry of semen from the prostatic urethra into the bulbous urethra.

1107

BOX 54-4  Retrograde Ejaculation

A

s noted in the text, emission is normally accompanied by constriction of the internal urethral sphincter. Retrograde ejaculation occurs when this sphincter fails to constrict. As a result, the semen enters the urinary bladder rather than passing down the urethra. Retrograde ejaculation should be suspected in patients who report absent or smallvolume ejaculation after orgasm. The presence of >15 sperm per high-power field in urine specimens obtained after ejaculation confirms the occurrence of retrograde ejaculation. Lack of emission or retrograde ejaculation may result from any process that interferes with innervation of the vas deferens and bladder neck. Several medical illnesses, such as diabetes mellitus (which can cause peripheral neuropathy) and multiple sclerosis, or the use of pharmaceutical agents that interfere with sympathetic tone can lead to retrograde ejaculation. Retrograde ejaculation may also occur as a result of nerve damage associated with certain surgical procedures, including bladder neck surgery, transurethral resection of the prostate, colorectal surgery, and retroperitoneal lymph node dissection. Retrograde ejaculation from causes other than surgery involving the bladder neck may be treated with pharmacological therapy. Sympathomimetic drugs such as phentolamine (an α-adrenergic agonist), ephedrine (which enhances norepinephrine release), and imipramine (which inhibits norepinephrine reuptake by presynaptic terminals) may promote normal (i.e., anterograde) ejaculation by increasing the tone of the vas deferens (propelling the seminal fluid) and the internal sphincter (preventing retrograde movement).

Thus, emission sets the stage for ejaculation. The ejaculatory process is a spinal cord reflex, although it is also under considerable cerebral control. The afferent (i.e., sensory) impulses reach the sacral spinal cord (S2 through S4) and trigger efferent activity in the somatic motor neurons that travel via the pudendal nerve. The resulting rhythmic contractions of the striated muscles of the perineal area—including the muscles of the pelvic floor, as well as the ischiocavernosus and bulbospongiosus muscles—forcefully propel the semen via the urethra through the external meatus. In addition, spasmodic contractions of the muscles of the hips and the anal sphincter generally accompany ejaculation. Orgasm is a term best restricted to the culmination of sexual excitation, as generally applied to both men and women. Orgasm is the cognitive correlation of ejaculation in the human male. Although orgasm, the pleasurable sensation that accompanies ejaculation, is not well understood, clearly it is as much a central phenomenon as it is a peripheral one.  N54-11

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 54  •  The Male Reproductive System

1107.e1

N54-11  Neuronal Lesions Affecting Erection and Ejaculation Contributed by Ervin Jones Erectile dysfunction is often associated with disorders of the central and peripheral nervous systems. Spinal cord disease and peripheral neuropathies are of particular interest, and effects of spinal cord injuries have been studied in some detail. Erectile capacity is usually preserved in men with lesions of the premotor neurons (neurons that project from the brain to the spinal cord; eTable 54-1). In these men, reflexogenic erections occur in 90% to 100% of cases, whereas psychogenic erections do not occur because the pathways from the brain are blocked. Ejaculation is more significantly impaired with upper than with lower motor neuron lesions, presumably because of loss of the psychogenic component. A clinically important feature of the spinal segmentation of nerve roots (i.e., thoracolumbar and lumbosacral) for generation of erection is that spinal or peripheral nerve damage may affect

only one of the effector systems. Because the lumbosacral system also carries most of the penile afferents, erection in response to penile stimulation (reflexogenic) is most affected by damage to the lower spinal cord or the nerves that project there. Evidence from men with spinal injuries in the T10 through T12 region has implicated the sympathetic thoracolumbar pathway in mediation of erections resulting from sexual stimuli received via the cranial nerves or generated within the brain as memories, fantasies, or dreams. In men with lower motor neuron lesions, reflexogenic erections are absent. However, psychogenic erections still occur in most men with incomplete lesions and in about one fourth of men with complete lesions. It remains uncertain whether this sympathetic pathway is normally the principal route for psychogenic erections or whether it just assumes the role when lumbosacral parasympathetic pathways are damaged.

eTABLE 54-1  Effects of Neural Lesions on Erection and Ejaculation LESION

REFLEXOGENIC ERECTION

PSYCHOGENIC ERECTION

EFFECT ON EJACULATION

Upper motor neuron

Present

Absent

Significantly impaired

Lower motor neuron

Absent

Present

Less impaired

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SECTION IX  •  The Reproductive System

REFERENCES Books and Reviews Ackland JF, Schwartz NB, Mayo KE, Dodson RE: Nonsteroidal signals originating in the gonads. Physiol Rev 72:731–787, 1992. Akingbemi BT: Estrogen regulation of testicular function. Reprod Biol Endocrinol 27:51–64, 2005. Andersson K-E, Wagner G: Physiology of penile erection. Physiol Rev 75:191–236, 1995. de Kretser D: Molecular Biology of Reproductive Systems: Molecular Biology of the Male Reproductive System. Academic Press, London, 1993. Giuliano F: Neurophysiology of erection and ejaculation. J Sex Med 4(Suppl):310–315, 2011. Hecht NB: Molecular mechanisms of male germ cell differentiation. Bioessays 20:555–561, 1998. Mather JP, Moore A, Li RH: Activins, inhibins, and follistatins: Further thoughts on a growing family of regulators. Proc Soc Exp Biol Med 215:209–222, 1997. Melmed S, Polonsky KS, Larsen PR, Kronenberg HM (eds): Williams Textbook of Endocrinology, 12th ed. Philadelphia, Saunders, 2011. Skinner MK: Cell-cell interaction in the testis. Endocrinol Rev 12:45–77, 1991.

Walters KA, Simanainen U, Handelsman DJ: Molecular insights into androgen actions in male and female reproductive function from androgen receptor knockout models. Hum Reprod Update 16(5):543–558, 2010. Journal Articles Carter AJ, Ballard SA, Naylor AM: Effect of the selective phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog. J Urol 160:242–246, 1998. Jones TM, Fang VS, Landau RL, Rosenfield R: Direct inhibition of Leydig cell function by estradiol. J Clin Endocrinol Metab 47:1368–1373, 1978. Koraitim M, Schafer W, Melchior H, Lutzeyer W: Dynamic activity of bladder neck and external sphincter in ejaculation. Urology 10:130–132, 1977. Ludwig DG: The effect of androgen on spermatogenesis. Endocrinology 46:453–481, 1950. Ricci G, Perticarari S, Fragonas E, et al. Apoptosis in human sperm: Its correlation with semen quality and the presence of leukocytes. Hum Reprod 17:2665–2672, 2002. Sofikitis N, Giotitsas N, Tsounapi P, et al: Hormonal regulation of spermatogenesis and spermiogenesis. J Steroid Biochem Mol Biol 109(3–5):323–330, 2008.

C H A P T E R 55  THE FEMALE REPRODUCTIVE SYSTEM Sam Mesiano and Ervin E. Jones

The female reproductive system functions to (1) pro­ duce haploid gametes—ova, (2) facilitate syngamy—or fertilization—between an ovum and a spermatozoon, (3) supply a site for implantation of the embryo (if syngamy occurs) and the establishment of pregnancy, (4) provide for the physical environment and nutritional needs of the developing fetus and its timely birth, and (5) nurture the neonate. The system consists of the gonads (the ovaries), the fal­ lopian tubes, the uterus and cervix, the vagina (Fig. 55-1A), the external genitalia, and the mammary glands, and is con­ trolled by hormones produced in the hypothalamus, pitu­ itary, and ovaries. The principal female sex hormones are estrogens (mainly estradiol) and progesterone, which are produced by the ovaries in a cyclic manner and regulate the growth and function of the female sex accessory structures and the development of secondary sexual characteristics. Function of the female reproductive system is ultimately regulated by hormones produced by the hypothalamicpituitary-gonadal axis under the control of higher brain centers. The system involves finely tuned neuroendocrine feedback interactions between hormones produced by the hypothalamus and anterior pituitary and hormones pro­ duced by the ovaries. The result is the cyclic production of gametes and the preparation of the sex accessory organs for the establishment of pregnancy.

Female reproductive organs include the ovaries and accessory sex organs The ovaries lie on the sides of the pelvic cavity (see Fig. 55-1A). Covered by a layer of mesothelial cells, each ovary consists of an inner medulla and an outer cortex. The cortex of the ovary in a mature female contains developing follicles and corpora lutea in various stages of development (see Fig. 55-1B). These elements are interspersed throughout the stroma, which includes connective tissue, interstitial cells, and blood vessels. The medulla comprises large blood vessels and other stromal elements. The female sex accessory organs include the fallopian tubes, the uterus, the vagina, and the external genitalia. The fallopian tubes provide a pathway for the transport of ova from the ovary to the uterus. The distal end of the fallopian tube expands as the infundibulum, which ends in multiple fimbriae. The fimbriae and the rest of the fallopian tubes are lined with epithelial cells, most of which have cilia that 1108

beat toward the uterus. The activity of these cilia and the contractions of the wall of the fallopian tube, particularly around the time of ovulation, facilitate transport of the ovum. Interspersed with ciliated cells are peg cells that secrete fluid and nutrients supporting the ovum and sper­ matozoa as well as the zygote that may result as fertilization occurs in the fallopian tubes. The uterus is a complex, pear-shaped, muscular organ that is suspended by a series of supporting ligaments. It is composed of a fundus, a corpus, and a narrow caudal portion called the cervix. The external surface of the uterus is covered by serosa, whereas the interior, or endometrium, of the uterus consists of complex glandular tissue and stroma. The bulk of the uterine wall consists of specialized smooth muscle, myometrium, that lies between the endometrium and the uterine serosa. The uterus is continuous with the vagina via the cervical canal. The cervix is composed of dense fibrous connective tissue and smooth-muscle cells. Glands lining the cervical canal produce a sugar-rich secre­ tion, the viscosity of which is conditioned by estrogen and progesterone. The human vagina is ~10 cm in length and is a single, expandable tube. The vagina is lined by stratified epithelium and is surrounded by a thin muscular layer. During develop­ ment, the lower end of the vagina is covered by the membra­ nous hymen, which is partially perforated during fetal life. In some instances, the hymen remains continuous. The external genitalia include the clitoris, the labia majora, and the labia minora, as well as the accessory secretory glands (including the glands of Bartholin), which open into the vestibule. The clitoris is an erectile organ that is homologous to the penis (see p. 1091) and mirrors the cavernous ends of the glans penis. The breasts can also be considered as part of the female reproductive system. Breast development (thelarche) begins at puberty in response to ovarian steroid hormones. The ductal epithelium of the breast is sensitive to ovarian steroids and especially during pregnancy becomes activated to produce milk (lactation) that will sustain the newborn infant.

Reproductive function in the human female is cyclic In some species (e.g., rabbits), female reproductive function, and specifically ovulation (the liberation of fertilizable oocytes), is triggered by mating. However, in most species,

Chapter 55  •  The Female Reproductive System

A

OBLIQUE VIEW OF THE INTERNAL FEMALE SEX ORGANS

Fallopian tube

Fundus of uterus

Intramural portion of fallopian tube

Ovary

1109

Ampulla of fallopian tube Isthmus of fallopian tube Ovary

Endometrium Myometrium Round ligament Infundibulum of fallopian tube

Broad ligament

Fimbriae Hilus Corpus (body) of uterus

Mesovarium Proper ovarian ligament Bladder Cervix of uterus Vagina

B

CROSS SECTION THROUGH AN OVARY

Primary follicles

Primordial follicle

Corpus albicans

Secondary follicle Tertiary follicle

Corpus luteum Figure 55-1  Anatomy of the female internal genitalia and accessory sex

Hilus

organs.

Blood vessels

Ruptured follicle

Graafian follicle

Discharged oocyte

Corona radiata

1110

SECTION IX  •  The Reproductive System

the female reproductive system functions in a cyclic manner. In some of these species with cyclic function (e.g., sheep, cattle, horses), females are receptive to males only around the time of ovulation, which maximizes the chances of fer­ tilization and pregnancy. This receptive behavior is known as estrus, and the animals are said to have seasonal estrus cycles, whereby the ovaries are active only at a certain time of the year. Such cyclic reproductive function in females enhances reproductive efficiency by coordinating gamete production with environmental (in seasonal species) and physiological changes that attract males and prepare the reproductive tract for sperm and ovum transport, fertiliza­ tion, implantation, and pregnancy. In a small subset of species (e.g., humans, baboons, apes), ovulation occurs in monthly cycles—known as menstrual cycles—that are asso­ ciated with regular episodes of uterine bleeding termed menstruation.

HYPOTHALAMIC-PITUITARY-GONADAL AXIS AND CONTROL OF THE MENSTRUAL CYCLE The human menstrual cycle coordinates changes in both the ovary and endometrium The human menstrual cycle involves rhythmic changes in two organs: the ovary and the uterus (Fig. 55-2). Although menstrual cycles are generally regular during the reproduc­ tive years, the length of the menstrual cycle may be highly variable because of disturbances in neuroendocrine func­ tion. Starting with the first day of the menses on day 0, the average menstrual cycle lasts 28 days. However, considerable variation occurs during both the early reproductive years and the premenopausal period, primarily because of the increased frequency of anovulatory cycles (Box 55-1). The ovarian cycle includes four key events: (1) folliculo­ genesis, (2) ovulation, (3) formation of the corpus luteum, and (4) death (atresia) of the corpus luteum. Temporally, the ovarian cycle includes two major phases: the follicular and luteal phases. The follicular phase begins soon after the corpus luteum degenerates, lasts 12 to 14 days, and ends at ovulation. The luteal phase begins at ovulation, lasts 12 to 14 days, and ends when the corpus luteum degenerates. Steroid hormones produced by the ovaries during the follicular and luteal phases induce changes in the endome­ trial lining of the uterus that constitute the endometrial cycle. The endometrial cycle consists of three key events: (1) menstruation, (2) endometrial growth and proliferation,

Ovarian cycle

and (3) differentiation of the endometrial epithelium into a glandular secretory phenotype. The endometrial cycle is divided into menses, the proliferative phase and the secretory phase (see Fig. 55-2). Ovarian and endometrial events are integrated into a single sequence as follows. Follicular/Proliferative Phase  The follicular/proliferative phase begins with the initiation of menstruation and aver­ ages ~14 days. The follicular phase of the ovarian cycle varies more in duration than any other phase of the cycle. During this time, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) stimulate the growth of a cohort of follicles, all of which (even those destined for atresia) produce estradiol. Consequently, circulating estradiol levels gradu­ ally increase during the follicular phase. Because estradiol stimulates rapid growth of the endometrium, this period is the proliferative phase of the endometrial cycle. Eventually, a single large, dominant preovulatory follicle develops in one

BOX 55-1  Effect of Energy Stores on Female Fertility

A

ctivity of the hypothalamic GnRH neurons in females is very sensitive to environmental and physiological conditions, as is particularly obvious in species with a seasonal estrus. The evolutionary rationale for this environmental sensitivity is that reproduction is most efficient when resources are available to sustain a pregnancy and nurture the newborn. In addition, pregnancy confers a survival risk to females. Leptin is produced by adipocytes and its levels in the circulation reflect the amount of energy stores (see pp. 1001– 1002). Because leptin promotes the production and release of GnRH by hypothalamic neurons, leptin signals the brain that fat stores are sufficient to support human female reproductive function. Indeed, increased leptin levels are associated with the onset of puberty in both sexes, and normal levels of leptin are needed to maintain menstrual cycles and normal female reproductive function. Low levels of leptin—due to starvation, anorexia, or strenuous exercise—are associated with amenorrhea (cessation of menstrual cycles). Thus, signals to the neuroendocrine reproductive axis are permissive for reproduction if fuel reserves are adequate, but inhibit the system if reserves are low. The practical consequence is to help ensure that reproduction occurs when the female has sufficient energy reserves to sustain a pregnancy and nurture an infant.

Follicular phase

Endometrial cycle

Menses 0

2

Luteal phase Secretory phase

Proliferative phase 4

6

8

10

12

14 Days

16

18

20

22

24

26

28–0

Figure 55-2  Ovarian and endometrial cycles. The menstrual cycle comprises parallel ovarian and endometrial cycles. The follicular phase of the ovarian cycle and the menses start on day 0. In this idealized example, ovulation occurs on day 14, and the entire cycle lasts 28 days.

Chapter 55  •  The Female Reproductive System

of the ovaries. This follicle becomes the principal source of estradiol as the follicular phase progresses. Ovulation  As we will see below, for most of the follicular phase, estradiol exerts negative feedback on gonadotropin secretion at the level of the hypothalamus and pituitary. However, toward the end of the follicular phase (day 12 to 13), when estradiol levels are maximal, the effect of estradiol on the hypothalamus and pituitary switches from negative to positive feedback. The result is a large transient surge in LH and a small increase in FSH secretion by the gonado­ trophs. The LH surge causes the dominant follicle to rupture and releases its oocyte—ovulation. Luteal/Secretory Phase  After release of the ovum, the remnants of the dominant follicle transform into a corpus luteum, which is why the second half of the ovarian cycle is called the luteal phase. Luteal cells produce progesterone and small amounts of estradiol, which together stimu­ late the endometrium to develop secretory glands—hence the term secretory phase of the endometrial cycle. If embryo implantation does not occur by day 20 to 22 of the cycle (i.e., midway through the luteal phase), the corpus luteum begins to degenerate and its production of progesterone and estra­ diol rapidly declines. The mechanisms that control the life span of the corpus luteum during a nonfertile cycle are not fully understood. If pregnancy is established, human chorionic gonadotropin (hCG) produced by the placenta main­ tains the corpus luteum. As a result, the corpus luteum maintains support for the endometrium, and menstruation does not occur. Menses  In the absence of pregnancy, withdrawal of pro­ gesterone (and estrogen) due to the demise of the corpus luteum leads to degeneration and shedding of the superficial part of the endometrium known as the functional layer. Degeneration of the functional layer is due to necrosis caused by the constriction of blood vessels that supply the endome­ trium. The necrotic tissue then sloughs away from the uterus and, in conjunction with blood from the underlying vessels and other uterine fluids, is shed as menstrual discharge (i.e., the period). Menstruation usually last 4 to 6 days. The first day of the menses (i.e., the first day of the endometrial cycle) is also the first day of the ovarian cycle. Rebuilding of the functional layer resumes when estrogen levels rise as a result of follicle growth during the new follicular phase.

The hypothalamic-pituitary-ovarian axis drives the menstrual cycle Neurons in the hypothalamus synthesize, store, and release gonadotropin-releasing hormone (GnRH). Long portal vessels carry the GnRH to the anterior pituitary, where the hormone binds to receptors on the surface of gonadotrophs. The results are the synthesis and release of both FSH and LH from the gonadotrophs (Fig. 55-3). These trophic hormones, LH and FSH, stimulate the ovary to synthesize and secrete the sex steroids estrogens and progestins as well as to produce mature gametes. The ovaries also produce peptides called inhibins and activins. Together, these ovarian steroids and peptides exert both negative and positive feedback on both

1111

the hypothalamus and the anterior pituitary. This complex interaction is unique among the endocrine systems of the body in that it generates a monthly pattern of hor­mone fluctuations. Because the cyclic secretion of estrogens and progestins primarily controls endometrial maturation, men­ struation reflects these cyclic changes in hormone secretion.

Neurons in the hypothalamus release GnRH in a pulsatile fashion A finely tuned neuroendocrine feedback between hormones produced by the brain and ovaries controls the menstrual cycle. As noted on pages 1092–1094, the process begins in the arcuate nucleus and the preoptic area of the hypothala­ mus, where neurons synthesize GnRH and transport it to their nerve terminals in the median eminence for storage and subsequent release. Higher centers in the brain trigger the release of GnRH near portal vessels, which carry GnRH to the gonadotrophs in the anterior pituitary. Before puberty, the GnRH neurons are quiescent and thus the reproductive system is inactive.  N55-1  After puberty, activity of the neurons increases, triggering release of GnRH in rhythmic pulses, about once per hour. Because the half-life of GnRH in blood is only 2 to 4 minutes, these hourly bursts of GnRH cause clearly discernible oscillations in GnRH levels in portal blood, leading to hourly surges in release of the gonadotro­ pins LH and FSH. Early in the follicular phase of the cycle, when the gonadotrophs are not very GnRH sensitive, each burst of GnRH elicits only a small rise in LH (Fig. 55-4A). Later in the follicular phase, when the gonadotrophs in the anterior pituitary become much more sensitive to the GnRH in the portal blood, each burst of GnRH triggers a much larger release of LH (see Fig. 55-4B).  N55-2 The frequency of GnRH release,  N55-3  and thus LH release, determines the specific response of the gonad. Pulses spaced 60 to 90 minutes apart upregulate the GnRH recep­ tors on the gonadotrophs, thus stimulating release of gonad­ otropins and activating the ovaries. However, continuous administration of GnRH (or an analog) causes downregula­ tion of the GnRH receptors, which suppresses gonadotropin release and gonadal function (Box 55-2). In addition to the hourly rhythm of GnRH secretion, a monthly rhythm of GnRH secretion also occurs in females of reproductive age. A massive increase in GnRH secretion by neurons in the preoptic area at midcycle is, in part, responsible for the LH surge, which, as we will see below (see p. 1116), leads to ovulation.

GnRH stimulates gonadotrophs in the anterior pituitary to secrete FSH and LH GnRH enters the anterior pituitary through the portal system and binds to GnRH receptors on the surface of the gonado­ troph, thus initiating a series of cellular events that result in the synthesis and secretion of gonadotropins. As discussed for the male on pages 1094–1095, occupation of the G protein–coupled GnRH receptor (GnRHR)  N55-4  leads to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG; see p. 58). The IP3 causes an increase in [Ca2+]i, triggering exocytosis and gonadotropin release (Fig. 55-5). In addition, the DAG stimulates protein kinase

Chapter 55  •  The Female Reproductive System

N55-1  Fetal Gonadotropin-Releasing Hormone

1111.e1

N55-2  Control of Pulsing of Gonadotropin-Releasing Hormone Neurons

Contributed by Ervin Jones GnRH is present in the hypothalamus at 14 to 16 weeks’ gestation, and its target, the gonadotropin-containing cells (gonadotrophs), are present in the anterior pituitary gland as early as 10 weeks’ gestation. The hypothalamic-pituitary system is functionally competent by ~23 weeks’ gestation, at which time fetal tissues release GnRH.

N55-3  Frequency versus Amplitude of Hypothalamic Releasing Hormones Contributed by Eugene Barrett For GnRH release, it appears that the important factor for signaling is the frequency of the GnRH pulses. On the other hand, in the case of corticotropin-releasing hormone (CRH; see pp. 1023–1025), it appears that amplitude is the primary factor in controlling adrenocorticotropic hormone (ACTH) release. Thus, depending on the target of the releasing hormone, either frequency or amplitude can be dominant.

N55-4  Gonadotropin-Releasing Hormone Receptor Contributed by Ervin Jones The GnRH receptor (GnRHR) is internalized and partially degraded in the lysosomes. However, a portion of the GnRHR is shuttled back to the cell surface. Return of the GnRHR to the cell membrane is referred to as receptor replenishment and is related to the upregulation of receptor activity discussed above in the text. The mechanism through which GnRH receptor replenishment occurs remains unclear.

Contributed by Sam Mesiano Although the mechanisms controlling the hourly pulses of GnRH remain unclear, the pulse generator for GnRH is thought to be located in the arcuate nucleus of the medial basal hypothalamus, where one group of GnRH neurons resides. GnRH neurons isolated from the rodent hypothalamus secrete GnRH in vitro in a rhythmic manner, with a frequency of approximately one pulse per hour. Those studies show that GnRH neurons have intrinsic pulsatile GnRH secretory activity and that the GnRH pulse generator resides within the GnRH neurons. In vivo studies show that bursts of nerve impulses from neurons in the arcuate nucleus correspond in time with the pulsatile release of GnRH from the hypothalamus and with the episodic release of LH from the anterior pituitary. These data suggest that a built-in system within the hypothalamus, and specifically the arcuate nucleus, controls the pulsatile discharge of GnRH from nerve terminals. Although the pulse generator is thought to be intrinsic to cells in the arcuate nucleus, it is significantly influenced by neurons from higher brain centers, predominantly in the cortex, that impact on the GnRH-secreting cells. Inhibitory and excitatory signals affect the pulse frequency of GnRH neurons. In general, kisspeptin neurons and glutamate neurons increase GnRH secretion frequency, whereas GABA neurons inhibit GnRH secretion and repress kisspeptin neurons. This GABA pathway is the main mechanism that keeps GnRH secretion relatively low during the juvenile prepubertal period. At puberty, GnRH secretion and pulse frequency increase, mainly due to increased activity of kisspeptin neurons and reduction in tonic GABA inhibition. Decreased GABA activity also is thought to enhance the stimulation of kisspeptin neurons by glutamatergic signaling through N-methyl-Daspartate (NMDA) receptors. Thus, the GnRH pulse-generating mechanism is intrinsic to the hypothalamic GnRH neurons, whose rhythmic activity is modulated by GABAergic, kisspeptinergic, and glutamatergic neurons from higher centers in the cortex. The modulation of GnRH pulse frequency via specific neurotransmitters in response to integration by higher brain centers is key to the control of puberty onset and cyclic reproductive function.

REFERENCE Terasawa E, Kurian JR, Guerriero KA, et al: Recent discoveries on the control of GnRH neurons in nonhuman primates. J Neuroendocrinol 22:630–638, 2010.

1112

SECTION IX  •  The Reproductive System

CNS

( / )

Hypothalamus

( / )

GnRH

( / ) Anterior pituitary

( / ) LH

FSH

Theca cell

Androgens

Granulosa cell

Inhibins Activins

Progestins

Estrogens

Negative feedback Positive feedback

Reproductive tract

Figure 55-3  Hypothalamic-pituitary-ovarian axis. Small-bodied neurons in the arcuate nucleus and the preoptic area of the hypothalamus secrete GnRH, which reaches the gonadotrophs in the anterior pituitary via the long portal veins. GnRH causes the gonadotrophs to synthesize and release two gonadotropins—FSH and LH. LH binds to receptors on theca cells to increase the biosynthesis of progestins and androgens. The androgens enter granulosa cells, which convert the androgens to estrogens. The dashed arrow indicates that the granulosa cells also have LH receptors. FSH binds to receptors on granulosa cells to increase the production of steroidogenic enzymes as well as activins and inhibins. The activins and inhibins act only on the anterior pituitary. The estrogens and progestins act on both the anterior pituitary and the hypothalamic neurons, exerting both positive- and negative-feedback controls. CNS, central nervous system.

C, which indirectly leads to increases in gene transcription. The net effect is an increase in synthesis of the gonadotropins FSH and LH, which are in the same family as thyroidstimulating hormone (TSH or thyrotropin; see pp. 1014– 1016) and hCG (see p. 1139).  N55-5 Before ovulation, the LH and FSH act on cells of the developing follicles. The theca cells (see p. 1117) of the fol­ licle have LH receptors, whereas the granulosa cells (see p. 1117) have both LH and FSH receptors. After ovulation, LH acts on the cells of the corpus luteum. Both the LH and the FSH receptors are coupled through Gαs to adenylyl cyclase (see p. 53), which catalyzes the conversion of ATP to cAMP. cAMP stimulates protein kinase A, which increases the expression of genes whose products enhance cell division or the production of peptide and steroid hormones.

The ovarian steroids (estrogens and progestins) feed back on the hypothalamic-pituitary axis As summarized in Figure 55-3, the ovarian steroids— primarily estradiol and progesterone—exert both negative and positive feedback on the hypothalamic-pituitary axis. Whether the feedback is negative or positive depends on both the concentration of the gonadal steroids and the duration of the exposure to these steroids (i.e., the time in the menstrual cycle). In addition, the ovarian peptides— the inhibins and activins—also feed back on the anterior pituitary. Negative Feedback by Ovarian Steroids  Throughout most of the menstrual cycle, the estradiol and progesterone that

Chapter 55  •  The Female Reproductive System

N55-5  The TSH-FSH-LH-hCG Family of Glycoprotein Hormones Contributed by Ervin Jones FSH and LH are in the same family as TSH (see p. 1014) and hCG (see p. 1139). All four are glycoprotein hormones with α and β chains. The α chains of all four of these hormones are identical; in humans, they have 92 amino acids and a molecular weight of ~20 kDa. The β chains are unique and confer the specificity of the hormones. In the female, the rhythm of GnRH secretion influences the relative rates of expression of genes encoding the synthesis of the α, βFSH, and βLH subunits of FSH and LH. GnRH pulsatility also determines the dimerization of the α and βFSH subunits, or α and βLH, as well as their glycosylation. Differential secretion of FSH and LH is also affected by several other hormonal mediators, including ovarian steroids, inhibins, and activins. The role of these agents is discussed in the section on feedback control of the hypothalamic-pituitary-ovarian axis. Thus, depending on the specific hormonal milieu produced by different physiological circumstances, the gonadotroph produces and secretes the α and β subunits of FSH and LH at different rates. The secretion of LH and FSH is further modulated by neuropeptides, amino acids such as aspartate, neuropeptide Y, corticotropin-releasing hormone (CRH), and endogenous opioids.

1112.e1

Chapter 55  •  The Female Reproductive System

A

120

Plasma 80 LH (mU/mL)

Plasma 80 LH (mU/mL)

0

LH

0

1

2 3 4 Time (hr)

C

pulses

120

40

BOX 55-2  Therapeutic Uses of GnRH

B LATE FOLLICULAR PHASE GnRH

EARLY FOLLICULAR PHASE GnRH pulses

LH

40

5

0

0

1

2 3 4 Time (hr)

1113

5

High levels of estradiol, typical of the late follicular phase, enhance the sensitivity of the gonadotrophs to GnRH. Figure 55-4  Pulsatile release of GnRH and pulsatile secretion of LH. (Data from Wang CF, Lasley BL, Lein A, Yen SS: The functional changes of the pituitary gonadotrophs during the menstrual cycle. J Clin Endocrinol Metab 42:718–728, 1976.)

are produced by the ovary feed back negatively on both the hypothalamus and the gonadotrophs of the anterior pitu­ itary. The net effect is reduced release of both LH and FSH. Estradiol exerts negative feedback at both low and high con­ centrations, whereas progesterone is effective only at high concentrations. Although estradiol inhibits the GnRH neurons in the arcuate nucleus and preoptic area of the hypothalamus, this inhibition is not direct. Rather, estradiol stimulates interneu­ rons that inhibit the GnRH neurons. In the arcuate nucleus, these inhibitory neurons exert their inhibition via opiates. However, in the preoptic area, the inhibitory neurons exert their inhibitory effect via gamma-aminobutyric acid (GABA), a classic inhibitory neurotransmitter (see p. 309). Positive Feedback by Ovarian Steroids  Although ovarian steroids feed back negatively on the hypothalamic-pituitary axis during most of the menstrual cycle, they have the oppo­ site effect at the end of the follicular phase. Levels of estradiol rise gradually during the first half the follicular phase of the ovarian cycle and then increase steeply during the second half (Fig. 55-6). After the estradiol levels reach a certain threshold for a minimum of 2 days—and perhaps because of the accelerated rate of estradiol secretion—the hypothalamicpituitary axis reverses its sensitivity to estrogens. That is, estradiol now exerts positive feedback on the axis. One mani­ festation of this positive feedback is that estradiol increases the sensitivity of the gonadotrophs in the anterior pituitary gland to GnRH. As discussed in the next section, this switch to positive feedback promotes the LH surge. Indeed, pitu­ itary cells that are cultured in the absence of estradiol have suboptimal responses to GnRH. Once high levels of estradiol have properly conditioned the gonadotrophs, rising levels of progesterone during the late follicular phase also produce a positive-feedback response and thus facilitate the LH surge.

ontinuous administration of GnRH leads to downregulation (suppression) of gonadotropin secretion, whereas pulsatile release of GnRH causes upregulation (stimulation) of FSH and LH secretion. Clinical problems requiring upregulation of gonadotropin secretion, which leads to stimulation of the gonads, are therefore best treated by a pulsatile mode of GnRH administration. In contrast, when the patient requires gonadal inhibition, a continuous mode of administration is necessary. An example of a disease requiring pulsatile GnRH administration is Kallmann syndrome. Disordered migration of GnRH cells during embryologic development causes Kallmann syndrome, which in adults results in hypogonadotropic hypogonadism and anosmia (loss of sense of smell). Normally, primordial GnRH cells originate in the nasal placode during embryologic development. These primitive cells then migrate through the forebrain to the diencephalon, where they become specific neuronal groups within the medial basal hypothalamus and preoptic area. In certain individuals, both male and female, proper migration of GnRH cells fails to occur. Females with Kallmann syndrome generally have amenorrhea (no menstrual cycles). However, the pituitary and gonads of these individuals can function properly when appropriately stimulated. Thus, females treated with exogenous gonadotropins or GnRH analogs—pulsatile administration with a programmed infusion pump—can have normal folliculogenesis, ovulation, and pregnancy. An example of a disease requiring continuous GnRH administration to downregulate gonadal function is endometriosis. Endometriosis is a common condition caused by the aberrant presence of endometrial tissue outside the uterine cavity. This tissue responds to estrogens during the menstrual cycle and is a source of pain and other problems, including infertility. In patients with endometriosis, continuous administration of GnRH analogs inhibits replenishment of the receptor for GnRH in the gonadotrophs in the anterior pituitary. As a result, insufficient numbers of GnRH receptors are available for optimum GnRH action; this deficiency diminishes gonadotropin secretion and produces relative hypoestrogenism. Because estrogen stimulates the endometrium, continuous administration of GnRH or GnRH analogs causes involution and diminution of endometriotic tissue. Leiomyomas (smooth-muscle tumors) of the uterus (also called uterine fibroids) are also estrogen dependent. When estrogen levels are decreased, the proliferation of these lesions is decreased. Therefore, leiomyomas of the uterus can also be effectively treated by continuous administration of GnRH analogs.

Ovaries produce peptide hormones—inhibins, activins, and follistatins—that modulate FSH secretion Inhibins, activins, and follistatins are gonadal peptide hormones, originally identified in follicular fluid, that selec­ tively affect the production and secretion of FSH but do not affect LH. Inhibins inhibit FSH production by gonadotrophs, activins activate FSH production, and follistatins inhibit FSH production by binding to and thereby inhibiting activins. The inhibins and the activins are glycoproteins that are members of the transforming growth factor-β (TGF-β) superfamily, which also includes antimüllerian hormone

1114

SECTION IX  •  The Reproductive System

8 2+ The [Ca ]i triggers exocytosis and release of gonadotropin.

1 Binding of GnRH to G-protein– linked receptor activates the PLC pathway and the release of 2+ Ca from internal stores. Ca

2+

From hypothalamus GnRH

Gq

2 The activated PLC also leads to the formation of DAG…

PLC 7 Ca2+ released from internal stores activates Ca2+ channels leading to 2+ sustained [Ca ]i.

Ca2+

PKC

3 …which stimulates PKC. PKC

Gonadotroph cell Follicular phase: estrogen

6 Gonadotropins are synthesized, dimerized, and glycosylated in the secretory pathway, regulated by the rhythm of GnRH.

α β-FSH β-LH

Luteal phase: estrogen and progesterone Inhibin (from ovary)

Activin (from ovary) 5 LH and FSH are αβ dimers. The α subunits are identical. The β subunit determines specificity.

4 PKC phosphorylates targets that indirectly stimulate gene transcription.

Figure 55-5  Gonadotropin secretion. PKC, protein kinase C; PLC, phospholipase C.

(AMH; see p. 1080). The inhibins and activins are dimers constructed from a related set of building blocks: a glycosyl­ ated 20-kDa α subunit and two nonglycosylated 12-kDa β subunits, one called βA and the other called βB (Fig. 55-7). The inhibins are always composed of one α subunit and either a βA or a βB subunit; the α and β subunits are linked by disulfide bridges. The α-βA dimer is called inhibin A, whereas the α-βB dimer is called inhibin B. The activins, however, are composed of two β-type subunits. Thus, three kinds of activins are recognized: βA-βA, βB-βB, and the het­ erodimer βA-βB. Follistatin is an unrelated monomeric poly­ peptide that binds to activin with high affinity.

The inhibins inhibit FSH secretion by the gonadotrophs of the anterior pituitary (hence the name inhibin) in a classic negative-feedback arrangement. The initial action of inhibin appears to be beyond the Ca2+-mobilization step in FSH secretion. In cultured pituitary cells, even very small amounts of inhibin markedly reduce mRNA levels for both the αLH/ FSH and the βFSH subunits. As a result, inhibins suppress FSH secretion. In contrast, inhibins have no effect on the mRNA levels of βLH. In addition to their actions on the anterior pituitary, the inhibins also have the intraovarian effect of decreasing androgen production, which can have secondary effects on intrafollicular estrogen production.

Negative Feedback by the Inhibins  FSH specifically stim­ ulates the granulosa cells to produce inhibins. Estradiol also stimulates inhibin production through an intraovarian mechanism. Just before ovulation, after the granulosa cells acquire LH receptors, LH also stimulates the produc­ tion of inhibin by granulosa cells. Inhibins are also produced by other tissues—including the pituitary, the brain, the adrenal gland, the kidney, the bone marrow, the corpus luteum, and the placenta. Nevertheless, the biological action of the inhibins is primarily confined to the reproductive system.

Positive Feedback by the Activins  The same tissues that produce the inhibins also produce the activins, which promote marked increases in βFSH mRNA and FSH release with no change in βLH formation. Activins augment GnRH production and release by hypothalamic neurons. However, the physiological role of activins in the female reproductive system is more complex than that of inhibins because multiple extragonadal tissues produce activin (and fol­ listatin), which may affect the hypothalamic-pituitarygonadal axis at many levels. Within the ovary, activins modulate folliculogenesis and steroid hormone production

1115

Chapter 55  •  The Female Reproductive System

Ovarian cycle

Follicular phase

Luteal phase

Events in the ovary Developing follicle

Corpus luteum Ovulation

80

LH FSH (mU/mL)

LH

Estradiol

FSH Inhibin

Progesterone

60 Estradiol (pg/mL) 1000

40

Progesterone (ng/mL) 10

800

8

600

6

1600

400

4

800

200

2

0

0

Inhibin (ng/mL)

0

20

0

0 Endometrial cycle

2 Menses

4

6

8

10

12

14 Days

Proliferative phase

16

18

20

22

24

26

28–0

Secretory phase

Figure 55-6  Hormonal changes during the menstrual cycle. The menstrual cycle is a cycle of the hypothalamicpituitary-ovarian axis, as well as a cycle of the target of the ovarian hormones: the endometrium of the uterus. Therefore, the menstrual cycle includes both the ovarian cycle—which includes the follicular phase, ovulation, and the luteal phase—and the endometrial cycle—which includes the menstrual, the proliferative, and the secretory phases.

by the corpus luteum. Each of these effects is inhibited by inhibin and follistatin. Activins bind to two types of cell-surface receptors (types I and II) that are serine/threonine kinases (see pp. 67–68). Upon ligand activation, the receptors couple to the SMAD second-messenger kinase cascade, which results in the modulation of transcription factors that affect the expression of a large variety of genes. Two cell-surface mol­ ecules that bind inhibin with high affinity antagonize the action of activin.

Modulation of gonadotropin secretion by positive and negative ovarian feedback produces the normal menstrual rhythm In premenopausal women, the pulsatile release of GnRH from the hypothalamus, generally occurring every 60 to 90 minutes (see p. 1111), triggers a corresponding pulsatile release of LH and FSH from the gonadotrophs of the anterior pituitary. The gonadotropins induce the production and

release of ovarian steroids, which in turn feed back on the hypothalamic-pituitary axis. This feedback loop is unusual because it elicits negative feedback on the hypothalamicpituitary axis throughout most of the menstrual cycle but positive feedback immediately before ovulation. Figure 55-6 illustrates the cyclic hormonal changes during the menstrual cycle. The time-averaged records of LH and FSH levels mask their hour-by-hour pulsatility. The follicular phase is characterized by a relatively high frequency of GnRH—and thus LH—pulses. Early in the follicular phase, when levels of estradiol are low but rising, the frequency of LH pulses remains unchanged, but their amplitude gradually increases with time. Figure 55-4 shows this increase in ampli­ tude between the early and late follicular phases. Later in the follicular phase, the higher estradiol levels cause both the frequency and the amplitude of the LH pulses to increase gradually. During this time of high estradiol levels, the ovarian steroids are beginning to feed back positively on the hypothalamic-pituitary axis. Late in the follicular phase, the net effect of this increased frequency and amplitude of

1116

SECTION IX  •  The Reproductive System

THE INHIBIN/ACTIVIN SUBUNITS α βA βB

INHIBINS α

ACTIVINS βA

S S βA

S S βA

α S S βB Inhibins are always composed of one α subunit and either a βA or a βΒ subunit. The α and β subunits are linked by disulfide bridges.

βB S S βB βA S S βB Activins are also dimers, but are composed of two β-type subunits: βA-βA, βB-βB, and the heterodimer βA-βB.

Figure 55-7  Inhibins and activins. The inhibins and activins are peptide hormones that are made up of a common set of building blocks. For both the inhibins and the activins, disulfide bonds link the two subunits.

LH and FSH pulses is an increase in their time-averaged circulating levels (see Fig. 55-6). The LH surge is an abrupt and dramatic rise in the LH level that occurs around the 13th to 14th day of the follicular phase in the average woman. The LH surge peaks ~12 hours after its initiation and lasts for ~48 hours. The peak concen­ tration of LH during the surge is ~3-fold greater than the concentration before the surge (see Fig. 55-6). The LH surge is superimposed on the smaller FSH surge. Positive feedback of estrogens, progestins, and activins on the hypothalamicpituitary axis is involved in the induction of this LH surge. The primary trigger of the gonadotropin surge is a rise in estradiol to very high threshold levels just before the LH surge. The rise in estrogen levels has two effects. First, the accelerated rate of increase in estradiol levels in the preovula­ tory phase sensitizes the gonadotrophs in the anterior pitu­ itary to GnRH pulses (see Fig. 55-4). Second, the increasing estrogen levels also modulate hypothalamic neuronal activ­ ity and induce a GnRH surge, presumably through GnRH neurons in the preoptic area of the hypothalamus. Thus, the powerful positive-feedback action of estradiol induces the midcycle surge of LH and, to a lesser extent, FSH. Gradually rising levels of the activins—secreted by granulosa cells— also act in a positive-feedback manner to contribute to the FSH surge. In addition, gradually increasing levels of LH trigger the preovulatory follicle to increase its secretion of

progesterone. These increasing—but still “low”—levels of progesterone also have a positive-feedback effect on the hypothalamic-pituitary axis that is synergistic with the positive-feedback effect of the estrogens. Thus, although pro­ gesterone is not the primary trigger for the LH surge, it augments the effects of estradiol. The gonadotropin surge causes ovulation and luteiniza­ tion. The ovarian follicle ruptures, probably because of weak­ ening of the follicular wall, and expels the oocyte and with it the surrounding cumulus and corona cells. This process is known as ovulation, and it is discussed in more detail in Chapter 56. As discussed below, a physiological change— luteinization—in the granulosa cells of the follicle causes these cells to secrete progesterone rather than estradiol. The granulosa and theca cells undergo structural changes that transform them into luteal cells, a process known as luteinization. The pulsatile rhythm of GnRH release and gonado­ tropin secretion is maintained throughout the gonadotropin surge. As the luteal phase of the menstrual cycle begins, circu­ lating levels of LH and FSH rapidly decrease (see Fig. 55-6). This fall-off in gonadotropin levels reflects negative feedback by three ovarian hormones—estradiol, progesterone, and inhibin. Moreover, as gonadotropin levels fall, the levels of ovarian steroids also fall. Thus, immediately after ovulation we see more or less concurrent decreases in the levels of both gonadotropins and ovarian hormones. Later, during the luteal phase, the luteal cells of the cor­ pus luteum gradually increase their synthesis of estradiol, progesterone, and inhibin (see Fig. 55-6). The rise in the concentration of these hormones causes—in typical negativefeedback fashion—the continued decrease of gonadotropin levels midway through the luteal phase. One of the mecha­ nisms of this negative feedback is the effect of progesterone on the hypothalamic-pituitary axis. Recall that at the peak of the LH surge, both the frequency and the amplitude of LH pulses are high. Progesterone levels rise, and high levels stimulate inhibitory opioidergic interneurons in the hypo­ thalamus, which inhibits the GnRH neurons. This inhibition decreases the frequency of LH pulses, although the ampli­ tude remains rather high. By ~48 hours before onset of the menses, the pulsatile rhythm of LH secretion has decreased to one pulse every 3 to 4 hours. As a result, circulating levels of LH slowly fall during the luteal phase. During the late luteal phase, the gradual demise of the corpus luteum leads to decreases in the levels of progesterone, estradiol, and inhibin (see Fig. 55-6). After the onset of menstruation, the hypothalamicpituitary axis returns to a follicular-phase pattern of LH secretion (i.e., a gradual increase in the frequency of GnRH pulses).

OVARIAN STEROIDS Starting from cholesterol, the ovary synthesizes estradiol, the major estrogen, and progesterone, the major progestin Estrogens in female humans are derived from the ovary and the adrenal gland and from peripheral conversion of androgens in adipose tissue. In a nonpregnant woman,

Chapter 55  •  The Female Reproductive System

estradiol, the primary circulating estrogen, is secreted principally by the ovary. The precursor for the biosynthesis of the ovarian steroids, as it is for all other steroid hor­ mones produced elsewhere in the body, is cholesterol. Cholesterol is a 27-carbon sterol that is both ingested in the diet and synthesized in the liver from acetate (see p. 968). Ovarian cells can synthesize their own cholesterol de novo. Alternatively, cholesterol can enter cells in the form of low-density lipoprotein (LDL) cholesterol and bind to LDL receptors. As shown in Figure 55-8, a P-450 enzyme  N54-4  (see Table 50-2) known as the side-chain-cleavage enzyme (or 20,22-desmolase) catalyzes the conversion of cholesterol to pregnenolone. This reaction is the rate-limiting step in estrogen production. Ovarian cells then convert preg­ nenolone to progestins and estrogens. The initial steps of estrogen biosynthesis from pregnenolone follow the same steps as synthesis of the two so-called adrenal androgens dehydroepiandrosterone (DHEA) and androstenedione, both of which have 19 carbon atoms. We discuss these steps in connection with both substances (see Figs. 50-2 and 54-6). The Leydig cells in the testis can use either of two pathways to convert these weak androgens to testosterone. Cells in the ovaries are different because, as shown in Figure 55-9, they have a P-450 aromatase (P-450arom) that can convert andro­ stenedione to estrone and testosterone to estradiol. This aro­ matization also results in loss of the 19-methyl group (thus, the estrogens have only 18 carbons), as well as conversion of the ketone at position 3 to a hydroxyl in the A ring of the androgen precursor. Once formed, estrone can be converted into the more powerful estrogen estradiol, and vice versa, by 17β-hydroxysteroid dehydrogenase (17β-HSD). Finally, the liver can convert both estradiol and estrone into the weak estrogen estriol. The two major progestins, progesterone and 17αhydroxyprogesterone, are formed even earlier in the biosyn­ thetic pathway than the adrenal androgens. Functionally, progesterone is the more important progestin, and it has higher circulating levels (Box 55-3).

Estrogen biosynthesis requires two ovarian cells and two gonadotropins, whereas progestin synthesis requires only a single cell A unique aspect of estradiol synthesis in the ovary is that it requires the contribution of two distinct cell types: the theca and granulosa cells within the follicle and the thecalutein and granulosa-lutein cells within the corpus luteum (see Fig. 55-9). The superficial theca cells and theca-lutein cells can take up cholesterol and produce DHEA and androstenedione (see Fig. 55-8), but they do not have the aromatase necessary for estrogen production. The deeper granulosa cells and granulosa-lutein cells have the aromatase, but they lack the 17α-hydroxylase and 17,20-desmolase (which are the same protein) necessary for making DHEA and androstenedione. Another difference between the two cell types is that, in the follicle, the superficial theca cell is near blood vessels, which supply LDL cholesterol. The granulosa cell, conversely, is far from blood vessels and, instead, is surrounded by LDLdeficient follicular fluid. Thus, in the follicular stage, the

1117

granulosa cells obtain most of their cholesterol by de novo synthesis. However, after formation of the corpus luteum, the accompanying vascularization makes it possible for the granulosa-lutein cell to take up LDL cholesterol from the blood and to thus synthesize large amounts of progesterone. A final difference between the two cell types is that theca cells have LH receptors, whereas granulosa cells have both LH and FSH receptors. Because of their unique physiological properties, neither the theca/theca-lutein cells nor the granulosa/granulosalutein cells can make estrogens by themselves. According to the two-cell, two-gonadotropin hypothesis, estrogen syn­ thesis occurs in the following steps: Step 1: LH stimulates the theca cell, through the adenylyl cyclase pathway, to increase its synthesis of LDL receptors and the side-chain-cleavage enzyme. Step 2: Thus stimulated, the theca cell increases its synthesis of androstenedione. Step 3: The androstenedione synthesized in the theca cells freely diffuses to the granulosa cells. Step 4: FSH, also acting through the adenylyl cyclase pathway, stimulates the granulosa cell to produce aromatase. Step 5: The aromatase converts androstenedione to estrone (see Fig. 55-8). 17β-HSD then converts the estrone to estradiol. Alternatively, 17β-HSD can first convert the same androstenedione to testosterone, and then the aro­ matase can convert this product to estradiol. By these pathways, theca-derived androgens are converted to estrogens in the granulosa cell. Step 6: The estradiol diffuses into the blood vessels. At low concentrations, the weak androgens produced by the theca cells are substrates for estrogen synthesis by the granulosa cells, in addition to enhancing the aromatase activity of granulosa cells. However, at high concentrations, conversion of androgens to estrogens is diminished. Instead, the weak androgens are preferentially converted by 5αreductase (see Fig. 54-6) to more potent androgens, such as dihydrotestosterone, a substance that cannot be converted to estrogen. Furthermore, these 5α-reduced androgens inhibit aromatase activity. Thus, the net effect of a highandrogen environment in the follicle is to decrease estrogen production. These androgens also inhibit LH receptor for­ mation on follicular cells. In the luteal phase of the cycle, luteinization of the follicle substantially changes the biochemistry of the theca and granulosa cells. As part of the formation of the corpus luteum, blood vessels invade deep toward the granulosalutein cells. Recall that in the follicle, the granulosa cells had been surrounded by follicular fluid, which is poor in LDL cholesterol. The increased vascularity facilitates the de­ livery of LDL cholesterol to the granulosa-lutein cells. In addition, LH stimulates the granulosa-lutein cells to take up and process cholesterol—as it does the theca cells. The net effect is the increased progesterone biosynthesis that is characteristic of the midluteal phase. Indeed, the major products of the corpus luteum are progesterone and 17αhydroxyprogesterone, although the corpus luteum also produces estradiol. As indicated in Figure 55-9, the granulosalutein cells cannot make either 17α-hydroxyprogesterone

1118

SECTION IX  •  The Reproductive System

Acetate

Cholesterol (27-carbon compound) 21 20 22 24 25 26 17 23 12 18 16 27 13

11 1

19 9

A

10

2

HO

4

8

B

5

D

C

14

15

7

6

Figure 55-8  Biosynthesis of the ovarian steroids. This scheme summarizes the synthesis of the progestins and estrogens from cholesterol. The individual enzymes are shown in the horizontal and vertical boxes; they are located in either the smooth endoplasmic reticulum (SER) or the mitochondria. The side-chaincleavage enzyme that produces pregnenolone is also known as 20,22-desmolase. The chemical groups modified by each enzyme are highlighted in the reaction product. The ovary differs from the testis in having aromatase, which converts androgens to estrogens. Certain of these pathways are shared in the biosynthesis of the glucocorticoids and mineralocorticoids (see Fig. 50-2) as well as androgens (see Fig. 54-6).

MITOCHONDRIA Side-chain–cleavage enzyme Pregnenolone

Progesterone

C

O

SER

CH3

CH3

D

C

CH3 A

B

A

PROGESTINS (21 carbons)

D

C

CH3

B

O

HO

SER 17 α-Hydroxylase

SER 17 α-Hydroxylase

17 α-Hydroxypregnenolone

17 α-Hydroxyprogesterone

CH3 C

B

HO

SER 17,20-Desmolase

CH3

A

D

C

CH3

O

CH3

A

B

O

Androstenedione

C

CH3 A

B

A

SER 17β-Hydroxysteroid Dehydrogenase

HO

A

A

OH

D

B

HO

SER 17β-Hydroxysteroid Dehydrogenase

OH CH3 CH3

C

B

er

Liv

Estradiol (E2)

D

B

OH CH3

Liver

HO

Testosterone C

O D

C

D A

OH CH3 CH3

Estriol (E3)

CH3

Aromatase

Androstenediol

Estrone (E1)

O CH3

O

SER 17β-Hydroxysteroid Dehydrogenase

ESTROGENS (18 carbons)

SER 17,20-Desmolase

B

HO

OH

D

C

CH3

SER

Dehydroepiandrosterone (DHEA)

3β-Hydroxysteroid Dehydrogenase

A

O

C OH

D

C

CH3

Same protein

CH3

O

CH3

ANDROGENS (19 carbons)

O

C CH3

CH3

C

CH3 C

D A

B

O

or estradiol directly because these cells lack the protein that has dual activity for 17α-hydroxylase and 17,20desmolase (see Fig. 55-8). Thus, 17α-hydroxyprogesterone synthesis necessitates that progesterone first moves to the theca-lutein cell (see Fig. 55-9), which can convert

OH D

B

HO

progesterone to 17α-hydroxyprogesterone, as well as an­ drostenedione. Furthermore, estradiol synthesis necessitates that androstenedione from the theca-lutein cell move to the granulosa-lutein cell for aromatization and formation of estradiol.

Chapter 55  •  The Female Reproductive System

1119

BOX 55-3  The Birth Control Pill

H

ormonal contraception is the most commonly used method of contraception in the United States; ~30% of sexually active women take the oral contraceptive pill (OCP).

Types of Oral Contraceptives Numerous combination (i.e., estrogen and progestin) oral contraceptives and progestin-only pills are available. The estrogens and progestins used in OCPs have varying potencies. In the United States, two estrogen compounds are approved for oral contraceptive use: ethinyl estradiol and mestranol. The progestins used in OCPs are modified steroids in which the methyl at position 19 (see Fig. 55-8) is removed; these progestins include norethindrone, norgestrel, norethynodrel, norethindrone acetate, and ethynodiol diacetate. A new generation of progestins— including gestodene and norgestimate—have reduced androgenic effects. The woman takes the OCPs daily for 21 days out of the 28-day cycle; she takes no pill, a placebo, or an iron pill during days 22 to 28. No medication is usually given during this fourth week to allow withdrawal bleeding to occur. Three regimens of contraceptive steroid administration are used: 1. Monophasic or fixed-combination OCPs. The pills taken for the first 21 days of the cycle are identical. 2. Multiphasic or varying-dose OCPs. The pills contain two or three different amounts of the same estrogen and progestin, the dosages of which vary at specific intervals during the 21-day medication period. Multiphasic OCPs generally maintain a low dose of estrogen throughout the cycle, combined with varying amounts of progestin. The rationale for this type of formulation is that the woman takes a lower total dose of steroid but is not at increased risk of breakthrough endometrial bleeding. 3. Progestin-only OCPs (“minipill”). These estrogen-free pills are taken daily for 3 weeks of a 4-week cycle. This regimen may be associated with irregular, low-grade breakthrough endometrial bleeding. The progestin-only OCP is a good option for nursing mothers as well as women for whom estrogens are contraindicated (e.g., those with thromboembolic disease, a history of cerebrovascular incidents, or hypertension).

TABLE 55-1  Benefits and Risks of Oral Contraceptives Oral Contraceptives Decrease the Risk of Ovarian cancer Endometrial cancer Ovarian retention cysts Ectopic pregnancy Pelvic inflammatory disease Benign breast disease

Oral Contraceptives Increase the Risk of Benign liver tumors Cholelithiasis (gallstones) Hypertension Heart attack Stroke Deep vein thrombosis Pulmonary embolus

Biological Action of Oral Contraceptives The contraceptive effectiveness of OCPs accrues from several actions. Like natural ovarian steroids, contraceptive steroids feed back both directly at the level of the hypothalamus (decreasing secretion of GnRH) and at the level of the gonadotrophs in the anterior pituitary (see Fig. 55-3). The net effect is suppressed secretion of the gonadotropins FSH and LH. The low FSH levels are insufficient to stimulate normal folliculogenesis; the low LH levels obviate the LH surge and therefore inhibit ovulation. However, in the commonly used doses, contraceptive steroids do not completely abolish either gonadotropin secretion or ovarian function. The progestin component of the OCP causes the cervical mucus to thicken and become viscid and scant. These actions inhibit sperm penetration into the uterus. The progestins also impair the motility of the uterus and oviducts and therefore decrease transport of both ova and sperm to the normal site of fertilization in the distal fallopian tube (see p. 1129). Progestins also produce changes in the endometrium that are not conducive for implantation of the embryo. These changes include decreased glandular production of glycogen and thus diminished energy for the blastocyst to survive in the uterus. Progestin-only OCPs do not effectively inhibit ovulation, as do the combination pills. However, they do produce other actions, as noted above: mucus thickening, reduced motility, and impaired implantation. Because they are inconsistent inhibitors of ovulation, the progestin-only OCPs have a substantially higher failure rate than does the combined type of OCPs. Side effects of the compounds in OCPs are those associated with estrogens and progestins and include nausea, edema, headaches, and weight gain. Specific side effects of progestins include depression, mastodynia, acne, and hirsutism. Many of the side effects associated with the progestin component of the pill, particularly acne and hirsutism, are the result of the androgenic actions of the progestins used. The potential benefits of the newer progestins include decreased androgenic effects, such as increased sex hormone–binding globulin, improved glucose tolerance (see p. 1038), and increased high-density and decreased low-density lipoprotein cholesterol (see Table 46-4). The clinical impact of these changes remains to be determined. Table 55-1 lists the major benefits and risks of OCPs.

Estrogens stimulate cellular proliferation and growth of sex organs and other tissues related to reproduction Most estrogens in blood plasma are bound to carrier pro­ teins, as are testosterone and other steroid hormones. In the case of estradiol, 60% is bound to albumin and 38% to sex hormone–binding globulin (SHBG; see p. 1099)—also known as testosterone-binding globulin (TeBG). TeBG is doubly a misnomer because this protein binds estradiol and, moreover, its levels are twice as high in women as in men. At least one reason for the higher levels in women is that estrogens (including birth control pills) stimulate the synthe­ sis of SHBG. Only 2% of total plasma estradiol circulates as the free hormone, which readily crosses cell membranes. The nuclear estrogen receptors ERα and ERβ function as dimers (αα, αβ, or ββ; see Table 3-6). When bound

1120

SECTION IX  •  The Reproductive System

LDL Theca cell

LDL

LH

Granulosa cell Cholesterol

Cholesterol cAMP

AC

Pregnenolone LH

Gs

cAMP

Pregnenolone

AC

These steps only become important during the luteal phase.

Gs

Progesterone 17α-OH Progesterone

Progesterone

Androstenedione 17β-HSD

Androstenedione

Testosterone Aromatase

Testosterone

17α-Hydroxylase and 17,20-desmolase activities are absent.

Aromatase Estrone

Gs

cAMP

FSH

AC

17β-HSD

Estradiol Capillary

Aromatase activity is absent. Figure 55-9  Two-cell, two-gonadotropin model. During the follicular phase, the major product of the follicle is estradiol, whereas during the luteal phase, the major products of the corpus luteum are the progestins, although estradiol synthesis is still substantial. In the follicular phase, LH primes the theca cell to convert cholesterol to androstenedione. Because the theca cell lacks aromatase, it cannot generate estradiol from this androstenedione. Instead, the androstenedione diffuses to the granulosa cell, whose aromatase activity has been stimulated by FSH. The aromatase converts the androstenedione to estradiol. In the luteal phase, the vascularization of the corpus luteum makes LDL available to the granulosa-lutein cells. Thus, both the theca-lutein and the granulosa-lutein cells can produce progesterone, the major product of the corpus luteum. For production of 17α-hydroxyprogesterone (17α-OH progesterone), some of the progesterone diffuses into the theca-lutein cell, which has the 17α-hydroxylase activity needed for converting the progesterone to 17α-hydroxyprogesterone. The theca-lutein cell can also generate androstenedione, which diffuses into the granulosa-lutein cell for estradiol synthesis. AC, adenylyl cyclase.

to estrogen, the ER dimer interacts with steroid response elements on chromatin and induces the transcription of spe­ cific genes. Over the next several hours, DNA synthesis increases, and the mitogenic action of estrogens becomes apparent. Estrogens almost exclusively affect particular target sex organs—including uterus and breasts—that have ERs. In addition to acting through nuclear receptors, estrogens can also exert nongenomic actions (see p. 989) by binding to the G protein–coupled receptor GPR30. The progestins, particularly progesterone, stimulate glandular secretion in reproductive tissue and promote the maturation of certain estrogen-stimulated tissue. One of the most prominent actions of progesterone, which binds to the dimeric progesterone receptor (PR; see Table 3-6), is the induction of secretory changes in the endometrium. In part because estrogens induce PR expression in endometrial cells, estrogens must condition the endometrium for progesterone to act effectively, as during the luteal phase. During the latter half of the menstrual cycle, progesterone induces final matu­ ration of the uterine endometrium for reception and implan­ tation of the fertilized ovum.

THE OVARIAN CYCLE: FOLLICULOGENESIS, OVULATION, AND FORMATION OF THE CORPUS LUTEUM Female reproductive life span is determined by the number of primordial follicles established during fetal life Unlike the male—which produces large numbers of mature gametes (sperm) continuously beginning at puberty and for the remainder of the man’s life—the female has a limited total number of gametes, determined by the number of oocytes formed during fetal life (see p. 1078). Oocyte maturation— the production of a haploid female gamete capable of fertil­ ization by a sperm—begins in the fetal ovary. Beginning at around the fourth week of gestation, primordial germ cells migrate from the endoderm of the yolk sac to the gonadal ridge (see Fig. 53-4B and C), where they develop into oogonia—immature germ cells that proliferate by mitosis. Primary Oocytes  By ~8 weeks’ gestation, ~300,000 oogonia are present in each ovary. At around this time, some oogonia enter prophase of meiosis I and become primary

Chapter 55  •  The Female Reproductive System

oocytes (Fig. 55-10A). From this point onward, the number of germ cells is determined by three ongoing processes: mitosis, meiosis, and death by apoptosis (see p. 1241). By 20 weeks, all the mitotic divisions of the female germ cells have been completed, and the total number of germ cells peaks at 6 to 7 million. All oogonia that have not already entered prophase of meiosis I by the 28th to 30th week of gestation die by apoptosis. The oocytes then arrest in the diplotene stage of prophase I. This prolonged state of meiotic arrest is known as the dictyotene state, which lasts until just before ovulation many years later, when the meiosis resumes and the first polar body is extruded. The second meiotic division occurs at syngamy (see p. 1072), at which stage maturation of the haploid oocyte is complete. Primordial Follicles  In the fetal ovary, dictyotene oocytes are surrounded by a single layer of flat, spindle-shaped pre­ granulosa cells to form a primordial follicle (see Fig. 55-10B). Each primordial follicle is 30 to 60 µm in diameter and enclosed by a basement membrane. By the 30th week of gestation, the ovaries contain around 5 to 6 million primor­ dial follicles. Unlike male gametes, new oocytes cannot form after this time because all gametogenic stem cells, in this case oogonia, have either died or entered meiosis. Therefore, by midgestation, the female gamete endowment is established. For the remainder of the female’s life, the number of primor­ dial follicles gradually decreases. One reason for the decline is that primordial follicles undergo a relentless process of apoptosis that begins at midgestation and ends at menopause when the endowment of primordial follicles is virtually exhausted. This progressive exhaustion is independent of gonadotropic hormones and is unaffected by pregnancy or the use of oral contraceptives. In addition, after puberty, each month a cohort of 10 to 30 primordial follicles is recruited to enter the irreversible process of folliculogenesis, which culminates in either ovulation (rupture of the follicle and expulsion of the ova) or atresia (a coordinated process in which the oocyte and other follicle cells undergo apoptosis, degeneration, and resorption). The mechanism by which some primordial follicles initiate folliculogenesis whereas others remain dormant is not known. Thus, even though the ovaries are invested with ~7 million oogonia at midgestation, the pool of primordial follicles is continually depleted, so that ~1 million exist at birth, ~300,000 remain at puberty, and there are virtually none at menopause. Of the ~300,000 primordial follicles present at puberty, only 400 to 500 are destined for ovulation between puberty and menopause (e.g., 12 per year for 40 years). Another 5000 to 15,000 are part of the monthly cohorts that undergo atresia. However, the vast majority of primordial and primary ovarian follicles are lost as a result of the rapid, continuous process of atresia during the reproductive life of the individual. The female gametes are stored in the ovarian follicles— the primary functional units of the ovary. Over the course of a female’s life, 90% to 95% of all primordial follicles never progress into folliculogenesis. Primordial follicles are dormant for most of their life. At any given time, a small proportion of primordial follicles begins a series of changes in size, morphology, and function referred to as folliculogenesis—the central event in the human female re­ productive system. Folliculogenesis—controlled by intrinsic

1121

A PRIMARY OOCYTE (4N DNA) Pregranulosa cell Primary oocyte (4N DNA) Basement membrane

B PRIMORDIAL FOLLICLE

Granulosa cell Zona pellucida Primary oocyte (4N DNA) Basement membrane

C PRIMARY FOLLICLE

D SECONDARY FOLLICLE

Granulosa cell Zona pellucida Theca cell Primary oocyte (4N DNA) Basement membrane Theca externa Theca interna Basement membrane Granulosa cells Antrum Zona pellucida

E EARLY TERTIARY FOLLICLE

Primary oocyte (4N DNA) First meiotic division completed

Antrum

F GRAAFIAN FOLLICLE

Theca externa Theca interna Basement membrane Granulosa cells

Secondary oocyte (2N DNA)

Zona pellucida

First polar body (2N DNA)

Corona radiata Cumulus oophorus

Beginning of second meiotic division, fertilization, and completion of second meiotic division Corona radiata

G OVULATED OVUM

Second polar body

Fertilized oocyte Figure 55-10  Maturation of the ovarian follicle.

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SECTION IX  •  The Reproductive System

factors within the ovary and by the gonadotropins (FSH and LH)—occurs by three processes: (1) enlargement and matu­ ration of the oocyte, (2) differentiation and proliferation of granulosa and theca cells, and (3) formation and accumula­ tion of a fluid. Primary Follicles  The first step in folliculogenesis is the emergence of a primordial follicle from its quiescent state to become a primary follicle (see Fig. 55-10C). This process involves proliferation of granulosa cells and their differentia­ tion from flattened pregranulosa cells to cuboidal cells. In addition, the oocyte increases in size and forms the zona pellucida—a glycoprotein shell surrounding the plasma membrane of the oocyte. Secondary Follicles  The further proliferation of granu­ losa cells and the appearance of the theca-cell layer converts the primary follicle into a secondary follicle (see Fig. 55-10D). Secondary follicles contain a primary oocyte surrounded by several layers of cuboidal granulosa cells. In addition, cells in the ovarian stroma surrounding the follicle are induced to differentiate into theca cells that populate the outside of the follicle’s basement membrane. The oocyte increases in size to a mean diameter of ~80 µm and the follicular diameter grows to 110 to 120 µm. As the developing follicle increases in size—becoming a late-stage secondary follicle—the number of granulosa cells increases to ~600 and the theca cells show increasing differentiation to form the theca interna layer closest to the granulosa and the theca externa that compresses the surrounding ovarian stroma. Progres­ sion to secondary follicles also involves the formation of a blood supply from arterioles that terminate in a wreath-like network of capillaries adjacent to the basement membrane surrounding the granulosa-cell layer, which remains avascu­ lar. The theca cells proliferate and acquire LH receptors, as well as the ability to synthesize steroids. Gap junctions also form between the oocyte and the adjacent layer of granulosa cells and between granulosa cells. The oocyte-granulosa junctions may function as thoroughfares to transport nutri­ ents and information from the granulosa cells to the oocyte and vice versa. The granulosa cells in this context are analo­ gous to the Sertoli cells (see pp. 1101–1102) in that they nurse the gamete and act as the barrier between the oocyte and the blood supply. Tertiary Follicles  The next stage of follicular growth is the maturation of secondary follicles into tertiary follicles (see Fig. 55-10E) as the increasingly abundant granulosa cells secrete fluid into the center of the follicle to form a fluidfilled space called the antrum. Tertiary follicles represent the first of two antral stages (the second being the graafian fol­ licle, below). FSH induces the transition of preantral second­ ary follicles to antral tertiary follicles. Graafian Follicles  As the antrum enlarges, it nearly encir­ cles the oocyte, except for a small mound or cumulus that attaches the oocyte to the rest of the follicle. At this second antral stage, the diameter of the follicle increases to 20 to 33 mm and it is called a preovulatory or graafian follicle (see Fig. 55-10F). The granulosa cells of the tertiary and graafian follicles are of three types: (1) Mural granulosa cells, which are the

farthest from the center of the follicle, are the most metaboli­ cally active and contain large quantities of LH receptors and enzymes that are necessary for the synthesis of steroids. (2) Cumulus granulosa cells are shed with the oocyte at the time of ovulation. (3) Antral granulosa cells, which face the antrum, are left behind within the follicle to become the large luteal cells of the corpus luteum. The capacity of the three types of granulosa cells to generate steroids differs. Cumulus cells contain neither the side-chain-cleavage enzyme (P-450SCC) nor aromatase (P-450arom) and therefore cannot generate estrogens. Moreover, cumulus cells respond less to LH and have a low overall LH receptor content. The exact role of the cumulus layer has not been definitively established, although investigators have postulated that the cumulus layer may function as a feeder layer and may provide stem cells that differentiate into other granulosa-cell types. The antral fluid provides a unique environment for oocyte growth and development. It facilitates the release of the oocyte-cumulus at the time of ovulation and serves as a medium for nutrient exchange and waste removal in the avascular compartment. The accumulation of antral fluid is a major factor in the formation of the dominant follicle. Between 5 and 6 days before ovulation the dominant follicle undergoes accelerated expansion, forming a cystic bulge on the surface of the ovary. After this final phase of growth, the follicle—now a graafian follicle—is prepared for ovulation (see Fig. 55-10G).

The oocyte grows and matures during folliculogenesis The principal role of folliculogenesis is to produce a mature oocyte that is capable of fertilization and formation of an embryo. The oocyte contributes the majority of the cytoplas­ mic and nuclear factors needed for embryo development, and these factors are not completely established until after the secondary follicle stage (see Fig. 55-10D). In addition, oocyte growth and maturation benefits from the gap junctions that connect cumulus granulosa cells to the oocyte, permitting the bidirectional exchange of nutrients, growth factors, and other molecules. Oocyte growth and maturation includes formation of the zona pellucida, formation of increased numbers of mitochondria, acquisition of competence to complete meiosis I. During maturation, the oocyte also re-establishes genomic imprints. Genomic imprinting (see p. 94) is the process by which certain genes—about 1% of the genome—are silenced; particular genes are silenced only in female gametes and others, only in male gametes.  N55-6

FSH and LH stimulate the growth of a cohort of follicles As described above, the development of primordial follicles to secondary follicles occurs continually from fetal life until menopause. However, almost all of these follicles undergo atresia (death of the ovum, followed by collapse of the fol­ licle and scarring) at some stage in their development. This gonadotropin-independent folliculogenesis and atresia is thought to be controlled by factors within the ovary, and especially between somatic cells and the oocyte, acting in a paracrine manner. Some key factors in this process are activin A, the forkhead transcription factor FOXO3, basic fibroblast growth factor, and kit ligand.

Chapter 55  •  The Female Reproductive System

N55-6  Genomic Imprinting Contributed by Emile Boulpaep, Walter Boron, and Sam Mesiano Genomic imprinting is the process by which certain genes— about 1% of the genome—are silenced; particular genes are silenced only in female gametes and others, only in male gametes. Thus, these genes are expressed in a manner specific to the parent of origin. Note that a female diploid oogonium has some paternal genes imprinted or silenced (i.e., only the maternal gene is active) and some maternal genes silenced (i.e., only the paternal gene is active). When the 2N genome splits into two 1N genomes, it is important that all genes in the oocyte have the female pattern of imprinting, which occurs during oocyte maturation. Failure of proper genomic imprinting causes aberrant gene expression and is associated with several human diseases, including Beckwith-Wiedemann, Prader-Willi, and Angelman syndromes. For example, the IGF2 gene is normally maternally imprinted (i.e., silenced). In Beckwith-Wiedemann syndrome, the material IGF2 gene becomes reactivated (by removal of methyl tags) during oocyte formation in the mother or early embryonic development. The result is that the offspring has two (rather than one) active copies of IGF2 and thus excess IGF2 protein. The most obvious sign is macrosomia (large body size) in the newborn.

REFERENCES Wikipedia. s.v. Genomic imprinting. http://en.wikipedia.org/ wiki/Genomic_imprinting. Accessed March 20, 2015.

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Chapter 55  •  The Female Reproductive System

At the time of puberty, increased levels of FSH and LH stimulate cohorts of secondary follicles to progress to the tertiary and preovulatory stages. Along the course of this development, most follicles undergo atresia until one domi­ nant graafian follicle remains at the time of ovulation. Con­ troversy exists about the length of this developmental process. Some believe that the entire developmental process occurs over three to four monthly cycles, so that the graafian follicle of the present ovulatory cycle was part of a cohort of secondary follicles recruited three to four cycles earlier. An alternative view is that FSH and LH induce the recruit­ ment of a cohort of follicles during the end of one cycle, and one of these follicles develops into the dominant graafian follicle in the next cycle. In any case, FSH is necessary for continued development of follicles beyond the secondary stage, and only a portion of the cohort of follicles continues to develop in response to FSH and LH. The other follicles undergo atresia.

Each month, one follicle achieves dominance Although the mechanism of selection of the dominant fol­ licle is not completely understood, it is thought to be caused by estrogen-induced events within the follicles. As estrogen levels rise during the follicular phase of the cycle, the pitu­ itary gradually lowers its secretion of FSH (see Fig. 55-6). Rising inhibin levels also feed back on the anterior pituitary to decrease FSH secretion. Peak inhibin levels correlate with the number of follicles present and rise in parallel with cir­ culating estradiol levels. Decreased levels of FSH cause a decline in FSH-dependent aromatase activity in granulosa cells (see p. 1117), which results in a decrease in estradiol production in the lessmature follicles (see Fig. 55-8). Conversely, estrogen increases the effectiveness of FSH in the more mature follicles by increasing the number of FSH receptors. The dominant fol­ licle therefore has more FSH receptors, a greater rate of granulosa-cell proliferation, more FSH-dependent aroma­ tase activity, and more estrogen production than the less dominant follicles. Because the less dominant follicles have less aromatase activity, they convert less androstenedione to estradiol. Thus, the weak androgen androstenedione either builds up or is converted to other androgens. As a result, the less dominant follicles have a lower estrogen/androgen ratio than the dominant follicle, and they undergo atresia under the influence of androgens in their local environment. In contrast, the production of estradiol and inhibins allows the dominant follicle to become prominent and to gain an even greater edge over its competitors. The vascular supply to the theca of the dominant follicle also increases rapidly, which may allow greater FSH delivery to the dominant follicle and thus help to maintain dominance of the follicle selected for ovulation.

Estradiol secretion by the dominant follicle triggers the LH surge and thus ovulation Ovulation occurs at the midpoint of every normal menstrual cycle, triggered by the LH surge, which in turn is stimulated by rapidly rising levels of estradiol. Estradiol secretion by the dominant follicle increases rapidly near the end of the late follicular phase (see Fig. 55-6). This dramatic rise in

1123

circulating estradiol switches the negative-feedback response of estradiol on the hypothalamus and anterior pituitary to a positive-feedback response and also sensitizes the anterior pituitary to GnRH. The result is the LH surge, which gener­ ally begins 24 to 36 hours after peak estradiol secretion. Ovulation usually occurs ~36 hours after onset of the LH surge, and ~12 hours after its peak. Thus, it appears that the developing follicle, through its increased estradiol secretion, signals the hypothalamic-pituitary system that follicular maturation is complete and that the hypothalamic-pituitary axis can now release a bolus of gonadotropin to induce ovu­ lation. The LH surge appears to terminate in part as a result of rising levels of progesterone, through negative feedback, and in part as a result of loss of the positive feedback that is derived from estradiol. Depletion of gonadotropin stores in the anterior pituitary gland may also contribute to termina­ tion of the LH surge. At the time of the LH surge, the primary oocyte (4N DNA), which had been arrested in the prophase of its first meiotic division since fetal life (see Fig. 53-2C), now resumes meiosis and completes its first meiotic division several hours before ovulation. The result of this first meiotic division is a small first polar body, which degenerates or divides to form nonfunctional cells, and a much larger secondary oocyte. Both the first polar body and the secondary oocyte, like secondary spermatocytes (see p. 1100), have a haploid number of duplicated chromosomes (2N DNA): 22 dupli­ cated somatic chromosomes and 1 duplicated X chromo­ some. This secondary oocyte begins its second meiotic division, but it becomes arrested in metaphase until the time of fertilization (see pp. 1131–1132). The secondary oocyte is surrounded by the zona pellucida and one or more layers of follicular cells, the corona radiata. Before ovulation, the cumulus oophorus expands under the influence of LH, and eventually the oocyte and its surrounding cells break free from the inner follicular-cell layer and, with their “stalk,” float inside the antrum, surrounded by follicular fluid. Breaking away of the oocyte-cumulus complex is probably facilitated by increased hyaluronidase synthesis that is stim­ ulated by FSH. Release of the oocyte from the follicle—ovulation— follows thinning and weakening of the follicular wall, probably under the influence of LH, progesterone, and pros­ taglandins (particularly those in the E and F series). These agents enhance the activity of proteolytic enzymes (e.g., collagenase) within the follicle, which leads to the diges­ tion of connective tissue in the follicular wall. Ultimately, a stigma—or spot—forms on the surface of the dominant follicle, in an area devoid of blood vessels. As this stigma bal­ loons out under the influence of increased follicular pressure and forms a vesicle, it ruptures and the oocyte is expelled. The expelled oocyte, with its investment of follicular cells, is guided toward the fallopian tube by the fimbriae that cover the surface of the nearby ovary (see Fig. 55-1). The oocyte is then transported through the infundibulum into the ampulla by ciliary movement of the tubal epithelium, as well as by muscular contractions of the tube. Fertilization, if it occurs, takes place in the ampullary portion of the fallopian tube. The resulting zygote resides in the ampulla for ~72 hours, followed by rapid transport through the isthmus to the uterine cavity, where it floats free for an additional 2 to 3 days before attaching to the endometrium.

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SECTION IX  •  The Reproductive System

After ovulation, theca and granulosa cells of the follicle differentiate into theca-lutein and granulosa-lutein cells of the corpus luteum After expulsion of the oocyte, the remaining follicular granu­ losa and theca cells coalesce into folds that occupy the fol­ licular cavity and, under the influence of LH, undergo a phenotypic transformation to form the corpus luteum—a temporary endocrine organ whose major product is proges­ terone. The mature corpus luteum is composed of two cell types, granulosa-lutein cells (also known as large luteal cells) derived from the granulosa cells, and theca-lutein cells (also known as small luteal cells) derived from the theca cell. The corpus luteum is highly vascularized, consistent with its primary function as an endocrine organ. During the early luteal phase, progesterone and estradiol produced by the corpus luteum exert negative feedback on the hypothalamicpituitary axis to suppress gonadotropin secretion and thus inhibit folliculogenesis. If pregnancy is not established, the corpus luteum regresses ~11 days after ovulation. One pos­ sible mechanism for this regression—or luteolysis—is that withdrawal of trophic support results in demise of the corpus luteum. A second possibility is that local factors, such as prostaglandin F2α produced by the endometrium, inhibit luteal function and terminate the life of the corpus luteum.

Growth and involution of the corpus luteum produce the rise and fall in estradiol and progesterone during the luteal phase Although the corpus luteum produces both estradiol and progesterone, the luteal phase is dominated by progesterone secretion. Estradiol production by the corpus luteum is largely a function of the theca-lutein cells because they produce the androgens that move to the granulosa-lutein cells (see Fig. 55-9) for conversion to estradiol. Progesterone production in the corpus luteum is primarily a function of the granulosa-lutein cells (see Fig. 55-9). Progesterone production rises modestly before follicular rupture but increases sharply after ovulation, peaking in ~7 days. Progesterone acts locally to inhibit follicular growth during the luteal phase. In addition, progesterone may act centrally by inhibiting gonadotropin secretion. Proges­ terone is also an antiestrogen in that it inhibits expression of ERs, thereby reducing estrogen responsiveness. The net effect is that increasing progesterone production suppresses folliculogenesis. Estradiol levels also rise during the luteal phase (see Fig. 55-6), which reflects production by the corpus luteum. Because estrogens induce expression of PRs in target cells, the estradiol produced during the luteal phase is necessary for progesterone-induced changes in the endometrium. Unless rescued by hCG—produced by the syncytial tro­ phoblasts of the blastocyst (see p. 1136)—luteal production of progesterone ceases toward the end of the menstrual cycle. hCG produced by the developing conceptus maintains ste­ roidogenic function of the corpus luteum until approxi­ mately the ninth week of gestation, at which time placental function is well established. If not rescued by pregnancy, the hormone-producing cells of the corpus luteum degenerate and leave behind a fibrotic corpus albicans.

THE ENDOMETRIAL CYCLE In the human female fetus, the uterine mucosa is capable of responding to steroid hormones by the 20th week of gesta­ tion. Indeed, some of the uterine glands begin secreting material by the 22nd week of gestation. Endometrial devel­ opment in utero apparently occurs in response to estrogens derived from the maternal placenta. By the 32nd week of gestation, glycogen deposition and stromal edema are present in the endometrium. As estrogenic stimulation is withdrawn after delivery, the endometrium regresses, and at ~4 weeks after birth, the glands are atrophic and lack vascularization. The endometrium remains in this state until puberty.

The ovarian hormones drive the morphological and functional changes of the endometrium during the monthly cycle The ovarian steroids—primarily estradiol and progesterone— control the cyclic monthly growth and breakdown of the endometrium. The endometrial cycle has three major phases: the menstrual, proliferative, and secretory phases. The Menstrual Phase  If the oocyte was not fertilized and pregnancy did not occur in the previous cycle, a sudden diminution in estradiol and progesterone secretion will signal the demise of the corpus luteum. As hormonal support of the endometrium is withdrawn, the vascular and glandu­ lar integrity of the endometrium degenerates, the tissue breaks down, and menstrual bleeding ensues; this moment is defined as the start of day 1 of the menstrual cycle (Fig. 55-11). After menstruation, all that remains on the inner surface of most of the uterus is a thin layer of nonepithelial stromal cells and some remnant glands. However, epithelial cells remain in the lower uterine segments as well as regions close to the fallopian tubes. The Proliferative Phase  After menstruation, the endome­ trium is restored by about the fifth day of the cycle (see Fig. 55-11) as a result of proliferation of the basal stromal cells on the denuded surface of the uterus (the zona basalis) as well as the proliferation of epithelial cells from other parts of the uterus. The stroma gives rise to the connective tissue components of the endometrium. Increased mitotic activity of the stromal and glandular epithelium continues through­ out the follicular phase of the cycle and beyond, until ~3 days after ovulation. Cellular hyperplasia and increased extracel­ lular matrix result in thickening of the endometrium during the late proliferative phase. The thickness of the endome­ trium increases from ~0.5 mm to as much as 5 mm during the proliferative phase. Proliferation and differentiation of the endometrium are stimulated by estrogen that is secreted by the developing follicles. Levels of estrogen rise early in the follicular phase and peak just before ovulation (see Fig. 55-6). ER levels in the endometrium also increase during the follicular phase of the menstrual cycle. Levels of endometrial ER are highest during the proliferative phase and decline after ovulation in response to changing levels of progesterone. Estradiol is believed to act on the endometrium in part through its effect on the expression of proto-oncogenes (see

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Chapter 55  •  The Female Reproductive System

Follicular phase

Ovarian cycle

Luteal phase

Events in the ovary Developing follicle Menstrual phase

Endometrial cycle

Corpus luteum

Proliferative phase Early

Secretory phase

Advanced

Early

Late (premenstrual)

Middle

Menstrual phase Zona compacta

Approximately 14 days Endometrial changes

Zona spongiosa

Functional layer

Zona basalis 0

4

8–9

14

Days

15–18

25

28–0

4

99 Basal body 98 temperature (°F) 97 Ovulation occurs on day 14. Figure 55-11  Endometrial cycle. The ovarian cycle includes the follicular phase (in which the follicle develops) and the luteal phase (in which the remaining follicular cells develop into the corpus luteum). The endometrial cycle has three parts: the menstrual, the proliferative, and the secretory phases.

p. 70). Estradiol also stimulates the synthesis of growth factors such as insulin-like growth factors (IGFs; see p. 996), transforming growth factors (TGFs), and epidermal growth factor (EGF) by endometrial cells that then act in an auto­ crine and paracrine manner to induce maturation and growth of the endometrium. Estradiol also induces the syn­ thesis of PRs in endometrial tissue. Levels of PRs peak at ovulation, when estradiol levels are highest, to prepare the cells for the high progesterone levels of the luteal phase of the cycle. Progesterone, in contrast, opposes the action of estradiol on the epithelial cells of the endometrium by inhibiting ER expression and stimulating expression of 17β-HSD and sulfotransferase. 17β-HSD converts estradiol to estrone (see Fig. 55-8), which is a weaker estrogen. Sulfotransferase conjugates estrogens to sulfate, making them biologically inactive. The Secretory Phase  During the early luteal phase of the ovarian cycle, progesterone further stimulates the 17βHSD and sulfation reactions (see above) and decreases ER levels in endometrial cells. These three antiestrogenic effects halt the proliferative phase of the endometrial cycle. Progesterone also stimulates the glandular components of the endometrium and thus induces secretory changes in the endometrium. The epithelial cells exhibit a marked increase in secretory activity, as indicated by increased amounts of endoplasmic reticulum and mitochondria. These increases

in synthetic activity occur in anticipation of the arrival and implantation of the blastocyst. The early secretory phase of the menstrual cycle (see Fig. 55-11) is characterized by the development of a network of interdigitating tubes within the nucleolus—the nucleolar channel system—of the endome­ trial epithelial cells. During the middle to late secretory phase, the secretory capacity of the endometrial glands increases. Vascularization of the endometrium increases, the glycogen content increases, and the thickness of the endometrium increases to 5 to 6 mm. The endometrial glands become engorged with secre­ tions. They are no longer straight; instead, they become tor­ tuous and achieve maximal secretory activity at approximately day 20 or 21 of the menstrual cycle. The changes in the endometrium are not limited to the glands; they also occur in the stromal cells between the glands. Beginning 9 to 10 days after ovulation, stromal cells that surround the spiral arteries of the uterus enlarge and develop eosinophilic cytoplasm, with a prominent Golgi complex and endoplasmic reticulum. This process is referred to as predecidualization. Under the influence of progester­ one, spindle-shaped stromal cells become rounded decidual cells and form an extracellular matrix consisting of laminin, fibronectin, heparin sulfate, and type IV collagen. Multiple foci of decidual cells spread throughout the upper layer of the endometrium and form a dense layer called the zona compacta (see Fig. 55-11). This spreading is so exten­ sive that the glandular structures of the zona compacta

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SECTION IX  •  The Reproductive System

become inconspicuous. Inflammatory cells accumulate around glands and blood vessels. Edema of the midzone of the endometrium distinguishes the compact area from the underlying zona spongiosa, where the endometrial glands become more prominent. Together, the superficial zona compacta and the midlevel zona spongiosa make up the so-called functional layer of the endometrium. This functional layer is the region that prolif­ erates early in the monthly endometrial cycle, that later interacts with the embryo during pregnancy, that is shed after pregnancy, and that is also shed each month during menstruation. The deepest layer of the endometrium—the zona basalis—is the layer left behind after parturition or menstruation. The cells of the zona basalis give rise to the proliferation at the beginning of the next endometrial cycle. During the late luteal phase of the menstrual cycle, just before the next menstruation, levels of both estrogens and progestins diminish, and these decreased ovarian steroid levels lead to eventual demise of the upper two thirds of the endometrium. During this period, the spiral arteries rhyth­ mically go into spasm and then relax. This period of the cycle is sometimes referred to as the ischemic phase. As cells begin to die, hydrolases are released from lysosomes and cause further breakdown of the endometrium. Prostaglandin production increases as a result of the action of phospholi­ pases liberated from lysosomes. Necrosis of vascular cells leads to microhemorrhage. The average loss of blood, tissues, and serous fluid amounts to ~30 mL. Menstrual blood does not clot because of the presence of fibrolysins released from necrotic endometrial tissue.

The effective implantation window is 3 to 4 days Based on studies of embryo transfer to recipient mothers in oocyte donation programs (see Box 56-1) when both the age of the donated embryo and the time of the endometrial cycle of the recipient are known, the period of endometrial receptivity for implantation of the embryo is estimated to extend from as early as day 16 to as late as day 19 of the menstrual cycle. Of course, because implantation must nor­ mally follow the ovulation that occurs on day 14 and because fertilization normally occurs within 1 day of ovulation, the effective window is 18 g/dL, and hematocrit increases from 40% to 45% to >55%. Normally, the body regulates RBC mass within fairly tight limits. However, renal hypoxia and norepinephrine stimulate the production and release of erythropoietin (EPO) from fibroblast-like cells in the kidney (see pp. 431–433). EPO is a growth factor that stimulates production of proerythroblasts in bone marrow and also promotes accelerated development of RBCs from their progenitor cells.  N18-2

Chapter 61  •  Environmental Physiology

N61-22  Capillary-Tissue PO2 Gradients Contributed by Arthur DuBois The oxygen tension or (partial pressure, PO2 ) in mixed-venous blood—a measure of average end-capillary PO2—is normally 40 mm Hg, sufficient to allow O2 to diffuse into the tissue cells. Although mitochondria can use O2 down to very low levels, the radial O2 diffusion gradient between capillary blood and cell surfaces becomes barely sufficient when the mixedvenous PO2 is ≤20 mm Hg. A sign of tissue hypoxia would be lactic acidosis. Oxygen electrodes measure PO2 in arterial or venous blood samples. The difference between arterial and mixed-venous PO2 is a measure of the average axial O2 gradient along systemic capillaries. Note that finger pulse oximeters measure the oxyhemoglobin saturation in the pulsating (i.e., arterial) blood that rushes into the tissue of the finger (see Box 29-2). At high altitude, the fall in arterial PO2 causes a reflex hyperventilation that lowers arterial and thus tissue PCO2 and thereby increases the affinity of Hb for oxygen. By itself, this Bohr effect (see p. 652) would further lower tissue PO2. Conversely, raising tissue PCO2 raises tissue PO2 by displacing O2 from Hb.

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Pulmonary Diffusing Capacity  Acclimatization to high altitude also causes a 2- to 3-fold increase in pulmonary diffusing capacity (see p. 668). Much of this increase appears to result from a rise in the blood volume of pulmonary capillaries (see p. 664) and from the associated increase in capillary surface area available for diffusion (see p. 661). This surface area expands even further because hypoxia stimulates an increase in the depth of inspiration. Finally, right ventricular hypertrophy raises pulmonary arterial pressure, thereby increasing perfusion to the upper regions of the lungs (see Fig. 31-9). Capillary Density  Hypoxia causes a dramatic increase in tissue vascularity. Tissue angiogenesis (see pp. 481–482) occurs within days of exposure to hypoxia, triggered by growth factors released by hypoxic tissues. Among these angiogenic factors are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiogenin. Oxidative Enzymes  Hypoxia promotes expression of oxidative enzymes in the mitochondria, thereby enhancing the tissues’ ability to extract O2 from the blood (see pp. 1220– 1222). Thus, acclimatization to high altitude increases not only O2 delivery to the periphery but also O2 uptake by the tissues.

High altitude causes mild symptoms in most people and acute or chronic mountain sickness in susceptible individuals Symptoms of Hypoxia  The first documented evidence of the ill effects of high altitude was in 35 BC, when Chinese travelers called the Himalayas the “Headache Mountains.” Recreational mountain climbing became popular in the mid19th century, and with modern transportation, many people can now travel rapidly to mountain resorts. In fact, it is possible to ascend passively from sea level to high altitude in a matter of minutes (e.g., in a balloon) to hours. A rapid ascent may precipitate a constellation of relatively mild symptoms: drowsiness, fatigue, headache, nausea, and a gradual decline in cognition. These uncomfortable effects of acute hypoxia are progressive with increasing altitude. They occur in some people at altitudes as low as 2100 m and occur in most people at altitudes higher than 3500 m. Initially, these symptoms reflect an inadequate response (i.e., compensatory hyperventilation) to hypoxemia, which results in insufficient O2 delivery to the brain. In the longer term, symptoms may stem from mild cerebral edema, which probably results from dilation of the cerebral arterioles leading to increased capillary filtration pressure and enhanced transudation (see p. 468). Acute Mountain Sickness  Some people who ascend rapidly to altitudes as seemingly moderate as 3000 to 3500 m develop acute mountain sickness (AMS). The constellation of symptoms is more severe than those described in the previous paragraph and includes headache, fatigue, dizziness, dyspnea, sleep disturbance, peripheral edema, nausea, and vomiting. The symptoms usually develop within the first day and last for 3 to 5 days. The primary problem in AMS is hypoxia, and the symptoms probably have two causes. The first is thought to be a progressive, more severe case

of cerebral edema. The second cause of the symptoms is pulmonary edema, which occurs as hypoxia leads to hypoxic pulmonary vasoconstriction (see p. 687), which in turn increases total pulmonary vascular resistance, pulmonary-capillary pressure, and transudation. Certain people have an exaggerated pulmonary vascular response to hypoxia, and they are especially susceptible to AMS. Cerebral or pulmonary edema can be fatal if the exposure to hypoxia is not rapidly reversed, first by providing supplemental O2 to breathe and then by removing the individual from the high altitude. Although being physically fit provides some protection against AMS, the most important factor is an undefined constitutional difference. Persons who are least likely to develop symptoms ventilate more in response to the hypoxia and therefore tend to have a higher PO2 and a lower PCO2. The higher PO2 and lower PCO2 lead to less cerebral vasodilation, and the higher PO2 minimizes pulmonary vasoconstriction.  N61-23 Chronic Mountain Sickness  After prolonged residence at high altitude, chronic mountain sickness may develop. The cause of this disorder is an overproduction of RBCs—an exaggerated response to hypoxia. In such conditions, the hematocrit can exceed 60%—polycythemia—which dramatically increases blood viscosity and vascular resistance, and increases the risk of intravascular thrombosis. The combination of pulmonary hypoxic vasoconstriction and increased blood viscosity is especially onerous for the right heart, which experiences a greatly increased load. These conditions eventually lead to congestive heart failure of the right ventricle.  N61-23

FLIGHT AND SPACE PHYSIOLOGY Acceleration in one direction shifts the blood volume in the opposite direction To accelerate a rocket from rest, we must apply enough force to overcome its inertial force (i.e., its weight, the product of its mass, and the acceleration caused by gravity), as well as the frictional forces of the environment. This requirement is merely a restatement of Newton’s second law of motion.  N61-10  With the rocket accelerating vertically, astronauts inside experience an inertial G force (see p. 1225), as required by Newton’s third law, a force that presses the astronauts into their seats in the direction opposite that of the rocket’s acceleration. Before liftoff, an astronaut experiences only the force of gravity, +1G. As a rocket blasts off from earth, the astronaut experiences higher G forces. In early rockets, astronauts sometimes experienced G forces as high as +10G. Maximal G forces in the space shuttle were only approximately +4G (Fig. 61-7). Similarly, pilots of high-performance aircraft experience positive G forces as they pull out of a dive, and we all experience negative G forces when an aircraft hits turbulence, suddenly loses altitude, and lifts us out of our seats. Although G forces can frequently have potentially large effects on aircraft pilots, they affect astronauts only during the liftoff and re-entry phases of space flight. To ensure that acceleration effects have a minimal influence on body

Chapter 61  •  Environmental Physiology

N61-23  High-Altitude Diseases Contributed by Emile Boulpaep and Walter Boron For a discussion of acute mountain sickness (AMS), highaltitude cerebral edema (HACE), and high-altitude pulmonary edema (HAPE), visit http://www.everestnews.com/ stories2005/illness01112005.htm (accessed February 2015). For a discussion of chronic mountain sickness (CMS), see the papers by León-Velarde and colleagues (the consensus statement) and by Zubieta-Castillo and colleagues (an alternative view).

REFERENCES León-Velarde F, Maggiorini M, Reeves JT, et al: Consensus statement on chronic and subacute high altitude diseases. High Alt Med Biol 6:147–157, 2005. Zubieta-Castillo G Sr, Zubieta-Calleja G Jr, Zubieta-Calleja L: Chronic mountain sickness: The reaction of physical disorders to chronic hypoxia. J Physiol Pharmacol 57(Suppl 4):431–442, 2006.

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4 3 Acceleration 2 (G) 1 0

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Figure 61-7  G forces during ascent into space on the space shuttle.

Before liftoff, astronauts experience +1G, the acceleration that is due to earth’s gravity. After liftoff, the solid rockets burn for ~2 minutes, during which time the G force ramps up to slightly more than +3G. After the solid-rocket burn, the G force falls back to +1G. Thereafter, the main engine gradually builds up the G force to about +4G before engine cutoff. These G-force data were generated in a human centrifuge to simulate the profile of a shuttle launch. (Data from Buckey JC, Goble RL, Blomqvist CG: A new device for continuous ambulatory central venous pressure measurement. Med Instrum 21:238–243, 1987.)

function, astronauts sit with their backs perpendicular to the direction of the accelerating force, so the G force acts across the chest from front to back.  N61-24 G forces propel the body’s tissues in the direction opposite that of acceleration; these forces compress soft tissues against underlying structural elements (e.g., bone) or pull these tissues away from overlying structural elements. In addition, G forces tend to shift the blood volume away from the direction of acceleration, thereby adding to the other component forces that determine blood pressure (see p. 414). In high-performance aircraft, the rapid motions associated with changes in flight direction or altitude produce G forces that can be considerable for several minutes, exceeding 8G. Even in relatively primitive aircraft, aerobatic maneuvers can shift blood volume away from the head, resulting in transient reductions in cerebral blood flow and O2 delivery. If these reductions are sufficiently large, they can result in loss of consciousness. The early warnings of such an event are narrowing of the visual field (i.e., loss of peripheral vision) and loss of color perception as the retina is deprived of O2, a phenomenon called gray-out. The term blackout describes a total loss of consciousness that occurs during acceleration that lasts for tens of seconds or minutes. Pilots experiencing gray-out or blackout are at extreme risk. As early as World War II, fighter pilots used G suits that provided counterpressure to the lower extremities during repeated tight maneuvers during dogfights. The counterpressure opposed the pooling of blood in the extremities and maintained sufficient cardiac filling, cardiac output, and blood flow to the brain, thereby eliminating the tendency toward gray-out.

“Weightlessness” causes a cephalad shift of the blood volume and an increase in urine output An astronaut in an orbiting spacecraft experiences “weightlessness,” a state of near-zero G force, also called a microgravity environment. Although an astronaut at an altitude of 200 km still experiences ~94% of the force of the earth’s

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gravity at sea level (i.e., the astronaut truly has weight), the centrifugal force of the spacecraft’s orbital trajectory balances the earth’s gravitational force, and the astronaut experiences no net acceleration forces and thus has the sensation of weightlessness. This weightlessness, however, differs from the true near-zero-gravity environment in “outer space.” We are adapted to life at +1G, and arteriolar tone in the lower extremities prevents pooling of blood in the capacitance vessels, thereby ensuring adequate venous return to the right heart (see p. 576). The acute effects of microgravity on the circulatory system are exactly what one would expect for a system designed to oppose the effect of gravity in a standing person: blood volume redistributes toward the head. This cephalad shift of blood volume—away from the capacitance vessels of the legs—expands the central blood volume, increasing the cardiac preload and increasing the filtration of plasma water into the interstitium of the facial region. The resulting edema explains the dramatically bloated facial appearance of astronauts in microgravity within 24 hours of the launch. From this discussion, one would think that the central venous pressure (CVP) is higher in space. However, such an increase in CVP has been difficult to confirm. In laboratory studies involving prolonged head-down tilt (i.e., a model intended to simulate microgravity exposure), the cephalad shift of blood volume produces the expected increase in CVP and rapid reflex responses to the apparent volume overload. First, the increased stretch on the right atrium causes release of atrial natriuretic peptide (ANP; see p. 547). Second, stimulation of the low-pressure baroreceptors inhibits secretion of arginine vasopressin, or antidiuretic hormone (see p. 547), from the posterior pituitary. These two events increase excretion of salt and water by the kidneys (see pp. 838–840), which tends to correct the perceived volume overload and explains the tendency for astronauts to remain relatively underhydrated during space flight. In orbiting spacecraft, the cephalad shift of blood volume, even without an increase in CVP, causes a small increase in cerebral arterial pressure and thus in blood flow to the brain. Such regional alterations in blood volume and flow do not substantially impact total peripheral resistance in space. Thus, mean arterial pressure and cardiac output are not significantly different from their values on the earth’s surface.

Space flight leads to motion sickness and to decreases in muscle and bone mass Despite training (e.g., in three-dimensional motion simulators), more than half of all astronauts experience motion sickness during the initial days of microgravity. Motion sickness (i.e., nausea and vomiting) results from conflicting sensory input to the brain regarding the position of the body. In space flight, motion sickness is the consequence of altered inertial stimulation of the vestibular system in the absence of normal gravitational forces. Nearly all cases of motion sickness resolve within the first 96 hours of microgravity exposure as the vestibular system or the CNS accommodates to the novel input. The increased cerebral blood flow and blood volume in microgravity, accompanied by increased capillary filtration of fluid from the intravascular space, contribute to the

Chapter 61  •  Environmental Physiology

N61-24  Effects of Acceleration on Astronauts Contributed by Arthur DuBois If we sit in a human centrifuge with acceleration directed from head to foot, and if the seat pushes headward by the same force, blood drains from our heads and we nearly pass out at +4G. Fighter pilots wear tight leggings with air bladders inside inflated to support the circulation, which extends tolerance an extra +2G. Some pilots in an aircraft undergoing a tight turn can withstand up to +8G or +9G by straining to provoke vasoconstriction. Astronauts in takeoff or landing mode lie transversely to the acceleration or deceleration to be able to tolerate up to 9G. But above 10G, they can hardly move the chest wall to breathe.

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increased incidence of headache, nausea, and motion sickness, at least during the period of transition to microgravity. These symptoms reduce performance. Astronauts attempt to minimize these effects by restricting water intake before launch. Numerous other changes occur during prolonged residence in microgravity, many of which are related to the markedly diminished aerobic power output in space, where the force of gravity does not oppose muscle contraction. The major physiological alterations include reductions in body water content, plasma and RBC volume, total-body nitrogen stores, muscle mass, and total-body calcium and phosphate (associated with a loss in bone mass). The bone loss appears to be continuous during time in a weightless environment, whereas the other changes occur only during the first weeks in space. The reductions in plasma and RBC volumes result in a marked decrease in maximal cardiac output (see pp. 1214–1215), a determinant of maximal aerobic power. The reduction of muscle mass decreases the maximal force developed by muscle. The reduction in bone mass similarly decreases bone strength. Although these changes are appropriate adaptations to a microgravity environment, in which great strength and high aerobic capacity have little inherent value, they are decidedly disadvantageous on return to the earth’s surface. Prolonged bed rest simulates weightlessness by causing loss of calcium from the bones and protein from the muscles.

Exercise partially overcomes the deconditioning of muscles during space flight The intermittent loading of muscles, bone, and the cardiovascular system prevents—to some extent—the deconditioning effects of space flight on muscle mass and performance. Astronauts have used bungee (i.e., elastic) cords and ergometric (i.e., work-measuring) stationary bicycles to provide resistance against which to exert force. The most effective exercise regimen appears to be walking on a motor-driven treadmill with the lower body encased in a negative-pressure chamber. Reducing the chamber pressure to 100 mm Hg lower than ambient pressure creates transmural pressure differences across the blood vessels in the feet that are similar to pressure differences when standing upright on the earth’s surface. However, this arrangement greatly exaggerates transmural pressure differences near the waist. For this reason, the astronauts also wear positive-pressure pants that

compress the tissues by 70 mm Hg at the level of the waist and decrease the compression decrementally to 0 mm Hg at the feet. The net effect of the negative-pressure chamber and the graded positive-pressure pants is to create a physiological toe-to-waist gradient of transmural pressures across the blood vessels of the lower body. The aerobic activity, the impact of the feet on the treadmill, and the generation of physiological transmural pressure gradients appear to be sufficient to simulate exercise at +1G. This regimen can reduce or even eliminate the deconditioning effects of space flight.

Return to earth requires special measures to maintain arterial blood pressure The problems associated with re-entry reflect a return to full gravity on earth’s surface. The most dramatic effects result from reduced blood volume and decreased tone of the leg vessels. Both factors contribute to reductions in cardiac preload, orthostatic tolerance, and exercise capacity. It has been common practice to shield astronauts from public view immediately after return to the earth’s surface, until they have regained a good orthostatic response. In recent years, astronauts have employed various strategies just before re-entry to counter the adaptations to microgravity. The countermeasure to orthostatic intolerance is restoration of blood volume before re-entry. One means of attenuating the reduction of blood volume in space flight is an exercise program. Even a brief period (e.g., 30 minutes) of intense exercise expands plasma albumin content (see p. 1220), increasing plasma oncotic pressure and plasma volume by 10% within 24 hours. The problems with exercise programs are difficulties in logistics and the astronauts’ lack of motivation. A second means of minimizing the reduced blood volume is increasing salt and fluid intake. However, this practice has proven difficult to implement because of the consequent increase in urine flow. Currently, astronauts are educated about the effects of prolonged space flight and then are maintained under continuous observation after re-entry until they have regained a normal orthostatic response. This usually occurs within hours, and certainly within 1 day, of re-entry.

REFERENCES The reference list is available at www.StudentConsult.com.

Chapter 61  •  Environmental Physiology

REFERENCES Books and Reviews Bunn HF, Poyton RO: Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 76:839–885, 1996. Crystal RG, West JB: The Lung. New York, Raven Press, 1991. Duffner GJ: Medical problems involved in underwater compression and decompression. Ciba Clin Symp 10:99–117, 1958. Krakauer J: Into Thin Air. New York, Anchor Books/Doubleday, 1997. Monge C: Chronic mountain sickness. Physiol Rev 23:166–184, 1943.

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Vann RD, Butler FK, Mitchell SJ, Moon RE: Decompression illness. Lancet 377:153–164, 2011. West JB: Man in space. News Physiol Sci 1:189–192, 1986. Journal Articles Cain SM, Dunn JE II: Low doses of acetazolamide to aid the accommodation of men to altitude. J Appl Physiol 21:1195–1200, 1966. Schoene RB, Lahiri S, Hackett PH, et al: Relationship of hypoxic ventilatory response to exercise performance on Mount Everest. J Appl Physiol 56:1478–1483, 1984. West JB: Human physiology at extreme altitudes on Mount Everest. Science 223:784–788, 1984.

C H A P T E R 62  THE PHYSIOLOGY OF AGING Edward J. Masoro

Biomedical science paid surprisingly little attention to a remarkable change in human biology during the 20th century—the marked increase of human life expectancy  N62-1  in developed nations. Life expectancy is the projected mean length of life of those born in a given calendar year (e.g., 1984)—or those of a particular age (e.g., 30 years)—computed from the mortality characteristics of the entire population in a particular year (e.g., 2016). In the United States, life expectancy for men progressively increased from 47.9 years in 1900 to 76.4 years in 2012, and for women, from 50.7 years in 1900 to 81.2 years in 2012.  N62-2

CONCEPTS IN AGING During the 20th century, the age structure of populations in developed nations shifted toward older individuals The fraction of the U.S. population ≥65 years of age was only 4% in 1900 but 12.4% in 2000. This trend in age structure is projected to continue (Fig. 62-1). Moreover, because women have a greater life expectancy, they comprised 70.5% of the population >80 years of age in 1990 in developed nations. The shift in the age structure of the U.S. population during the 20th century depended only modestly on an increase in life expectancy from birth. More important was the progressive decrease in birth rates. As a result, the elderly have become an ever-increasing fraction of the population, particularly in developed nations. Indeed, the effect of the post– World War II “baby boom” generation on population age structure is clearly apparent in Figure 62-1. If birth rates do not fall much further, future changes in the age structure of the U.S. population will depend mainly on further increases in life expectancy.  N62-3

The definition, occurrence, and measurement of aging are fundamental but controversial issues The age of an organism usually refers to the length of time the individual has existed. Biogerontologists and members of the general public alike usually use aging to mean the process of senescence. For example, we may say that a person is young for her age, an expression meaning that the processes of senescence appear to be occurring slowly in that person. Aging—the synonym for senescence that we use throughout this chapter—is the progressive deteriorative

changes during the adult period of life that underlie an increasing vulnerability to challenges and thereby decrease the ability of the organism to survive. Biogerontologists distinguish biological age from chronological age. Although we easily recognize the biological aging of family members, friends, and pets, it would be helpful to have a quantitative measure of the rate of aging of an individual. Biomarkers of aging—morphological and functional changes that occur with time in the adult organism—could in principle serve as a measure of senescent deterioration. Alas, a generally agreed-on panel of biomarkers of aging has yet to emerge, so it is currently impossible to quantitate the aging of individuals. Although measuring the aging of individuals is difficult, it has long been possible to measure the rate of aging of populations. In 1825, Benjamin Gompertz, a British actuary, published a report on the human age-specific death rate— the fraction of the population entering an age interval (e.g., 60 to 61 years of age) that dies during the age interval. For the British population, Gompertz found that, after early adulthood, the age-specific death rate increases exponentially with increasing adult age. The same is true for other human populations (Fig. 62-2) and for many animal populations. Based on the assumption that the death rate reflects the vulnerability caused by senescence, it has generally been accepted that the slope of the curve in Figure 62-2 reflects the rate of population aging. Although gompertzian and related analyses had long been viewed as the “gold standard” for measuring population aging, some biogerontologists have challenged this approach.

Aging is an evolved trait Most evolutionary biologists no longer accept the once popular belief that aging is an evolutionary adaptation with a genetic program similar to that for development. The current view is that aging evolved by default as the result of the absence of forces of natural selection that might otherwise eliminate mutations that promote senescence. For example, consider a cohort of a species that reaches reproductive maturity at age X. At that age, all members of the cohort will be involved in generating progeny. Furthermore, assume that this species is evolving in a hostile environment— the case for most species. As the age of this cohort increases past X, fewer and fewer members survive so that all members of the cohort die before exhibiting senescence. In this cohort, 1235

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N62-1  Life Expectancy

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N62-2  National Institute on Aging

Contributed by Emile Boulpaep and Walter Boron

Contributed by Edward Masoro

Human life-expectancy data—such as those cited in the first paragraphs of this chapter—are computed from mortality data for a particular year (e.g., 2002)—that is, the ages of all people who happen to have died in a particular year. Note that some of these individuals who died in 2002 were born in 2002, and some were born in 1900. The first step in computing life expectancy is to use the mortality data of 2002, for example, to compute age-specific death rates, from which we can derive a variety of other statistics. For example, we can compute the life expectancy at a particular age. The life expectancy at birth in the United States in 2002 was 74.5 years for men and 79.9 years for women. However, based on 2012 data, the life expectancy at birth in the United States had already risen to 76.4 for men and 81.2 for women. Clearly, these life expectancies are not predictions about how long someone alive today will live. Rather, they are death rates that are frozen in time. Another way of approaching the question is to analyze an extinct cohort, such as all those born in the year 1800. Based on the age at death of each member of this cohort, we could compute the true life expectancy of those born in the year 1800. Note that it is impossible—today—to predict the true life expectancy of those born in the year 2000 because that cohort is not extinct.

Responding to the increase in life expectancy, the United States in 1974 established the National Institute on Aging (NIA) in the National Institutes of Health. The NIA has had a major impact in the United States and throughout the world in the promotion of research on aging and in the development of geriatric medicine.

REFERENCES Arias E: United States life tables, 2004. Natl Vital Stat Rep 56(9):1–40, 2007. http://www.cdc.gov/nchs/data/nvsr/ nvsr56/nvsr56_09.pdf. Accessed July 16, 2015. Heron MP, Hoyert DL, Murphy SL, et al: Deaths: Final data for 2006. Natl Vital Stat Rep 57(14):1–135, 2009. http:// www.cdc.gov/nchs/data/nvsr/nvsr57/nvsr57_14.pdf. Accessed July 16, 2015. Wikipedia. s.v. Life expectancy. Last modified July 15, 2015. http://en.wikipedia.org/wiki/Life_expectancy. Accessed July 16, 2015.

N62-3  Socioeconomic Impact of a Graying Population Contributed by Edward Masoro There is concern that the increasing fraction of the population ≥65 years will have a negative socioeconomic impact. Part of the potential problem is cultural in that individuals expect to exit the work force at or around 65 years of age. Also, with advancing age, there is progressive deterioration of physiological capacity and an increasing prevalence of age-associated diseases. Thus, with advancing age, individuals need greater assistance in living and more medical care. In the United States, hospital admissions in 1993 for those >65 years of age were more than twice the admissions for those 45 to 64 years of age.

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1955 Females

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Figure 62-1  Age structure of the 1955 U.S. population and the projected age structure of the 2010 U.S. population. (From Tauber C: Sixty-five Plus in America. Washington, DC, US Bureau of the Census, 1992; revised 1993.)

genes with detrimental actions expressed only at advanced ages would not be subjected to natural selection. If we now move the progeny of our cohort to a highly protective environment, many may well live to ages at which the deleterious genes can express their effects, thereby giving rise to the aging phenotype. This general concept led biologists to put forward three genetic mechanisms that we discuss in the following three paragraphs. These are not mutually exclusive, and each has experimental support. In 1952, Peter Medawar  N62-4  proposed a variant of the foregoing model, now referred to as the mutationaccumulation mechanism. He proposed that most deleterious mutations in gametes will result in progeny that are defective during most of life, and natural selection removes

such genes from the population. However, a very few of mutated genes will not have deleterious effects until advanced ages, and natural selection would fail to eliminate such genes. George Williams proposed another variant in 1957. He postulated that the genes that have deleterious actions in late life actually increase evolutionary fitness in early adulthood. Natural selection will strongly favor such alleles because they promote the ability of the young adult to generate progeny and because they have a negative impact only after reproduction—antagonistic pleiotropy. In this scenario, aging is a byproduct of natural selection. In 1977, Tom Kirkwood proposed the disposable soma theory, according to which the fundamental life role of organisms is to generate progeny. Natural selection would

Chapter 62  •  The Physiology of Aging

N62-4  Peter Medawar For more information about Peter Medawar and his work on acquired immunological tolerance, for which he shared a Nobel Prize, visit http://nobelprize.org/nobel_prizes/medicine/ laureates/1960/index.html (accessed February 2015).

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Cross-Sectional Design  The usual approach to the foregoing difficulty is a cross-sectional design in which investigators study cohorts with several different age ranges (e.g., 20- to 29-year-olds, 30- to 39-year-olds) over a brief period (e.g., a calendar year). However, this design suffers from two serious potential confounders. One is the cohort effect; that is, different cohorts have had different environmental experiences. For example, in studies of the effects of aging on cognition, a confounding factor could be that younger cohorts have had the benefit of a relatively higher level of education. If aware of a potential confounder, the investigator may be able to modify the study’s design to avoid the confounder. The second potential confounder is selective mortality— individuals with risk factors for diseases that cause death at a relatively young age are underrepresented in older age groups. For example, in a study on the effect of age on plasma lipoproteins, mortality at a young age from cardiovascular disease would preferentially eliminate individuals with the highest low-density lipoprotein levels.

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Longitudinal Design  To circumvent the confounders encountered in cross-sectional designs, investigators can repeatedly study a subject over a significant portion of his or her lifetime. However, this longitudinal design has other problems. Long-term longitudinal studies require a special organizational structure that can outlive an individual investigator and ensure completion of the study. Even shorter longitudinal studies are very costly. Some problems are inherent in the time course of longitudinal studies, including the effect of repeated measurements on the function being assessed, changes in subjects’ lifestyle (e.g., diet), dropout of subjects from the study, and changes in professional personnel and technology.

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apportion the use of available energy between reproduction and body (i.e., somatic) maintenance to maximize the individual’s lifetime yield of progeny. As a consequence, less energy is available for somatic maintenance than needed for indefinite survival. This theory further proposes that a hostile environment increases the fraction of energy expended in reproduction, so that a smaller fraction is left for somatic maintenance.

Human aging studies can be cross-sectional or longitudinal Measuring the effects of aging on the human physiology presents investigators with a difficulty—the subjects’ life span is longer than the investigator’s scientific life span.

Whether age-associated diseases are an integral part of aging remains controversial Age-associated diseases are those that do not cause mor­ bidity or mortality until advanced ages. Examples are coronary artery disease, stroke, many cancers, type 2 diabetes, osteoarthritis, osteoporosis, cataracts, Alzheimer disease, and Parkinson disease. These are either chronic diseases or acute diseases that result from long-term processes (e.g., atherogenesis). Most gerontologists have held the view that age-associated diseases are not an integral part of aging. These gerontologists developed the concept of primary and secondary aging to explain why age-associated diseases occur in almost all elderly people. Primary aging refers to intrinsic changes occurring with age, unrelated to disease or environmental influences. Secondary aging refers to changes caused by the interaction of primary aging with environmental influences or disease processes. In contrast, some gerontologists adhere to a view expressed by Robin Holliday: “The distinction between agerelated changes that are not pathological and those that are pathological is not at all fundamental.” Moreover, the genetic mechanisms proposed for the evolution of aging (see p. 1235) may apply equally to the processes underlying both primary and secondary aging.

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SECTION X  •  Physiology of Everyday Life

CELLULAR AND MOLECULAR MECHANISMS OF AGING In this subchapter, we consider three major classes of cellular and molecular processes that may be proximate causes of organismic aging: (1) damage caused by oxidative stress and other factors, (2) inadequate repair of damage, and (3) dysregulation of cell number. No one of these is the underlying mechanism of aging. The basic mechanism of aging is likely to be the long-term imbalance between damage and repair. During growth and development, the genetic program not only creates a complex structure, but also repairs damaged molecules that arise in the process. Following development is a brief adult period when damage and repair are in balance, and then begins a long-term imbalance in favor of damage. The factors underlying the imbalance vary among species and among individuals within species, as a result of both genetic and environmental variability. For example, oxidative stress is one of many damaging processes that underlie aging, but an individual’s genome and environment determine the extent to which it is an important causal factor.

Oxidative stress and related processes that damage macromolecules may have a causal role in aging Raymond Pearl in 1928 proposed that organisms have a finite amount of a “vital principle,” which they deplete at a rate proportional to the rate of energy expenditure. Although this once-dominant “rate of living” theory of aging has now been discarded, some of its concepts helped to spawn the oxidative stress theory of aging.  N62-5 Reactive Oxygen Species  As illustrated in Figure 62-3A, reactive oxygen species (ROS) include molecules such as hydrogen peroxide. (H2O2), neutral free radicals such as the hydroxyl radical ( OH), and anionic radicals such as the superoxide anion radical (O2i−). Free radicals have an

unpaired electron in the outer orbital, shown in red in Figure 62-3A. These free radicals are extremely unstable because they react with a target molecule to capture an electron, so that they become a stable molecule with only paired electrons in the outer shell. However, the target molecule left behind becomes a free radical, which initiates a chain reaction that continues until two free radicals meet to create . a product with a covalent bond. ROS—particularly OH, which is the most reactive of them all—have the potential to damage important biological molecules, such as proteins, lipids, and DNA. However, ROS also play important physiological roles in the oxidation of iodide anions by thyroid peroxidase in the formation of thyroid hormone (see pp. 1006–1010), as well as in the destruction of certain bacteria by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase in phagocytic cells.  N62-6  Finally, the highly reactive signaling molecule nitric oxide (see p. 66) is a free radical (see Fig. 62-3A).  N62-7 Quantitatively, the most important source of ROS is the mitochondrial electron transport chain (see p. 118). Complex I and complex III of the electron transport chain generate O2i− as byproducts (see Fig. 62-3B). The enzyme superoxide dismutase (SOD) converts O2i− to hydrogen peroxide, which . in turn can yield the highly reactive OH. Only a small fraction of the oxygen used in aerobic metabolism (