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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science Publishers,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

PHYSIOLOGY - LABORATORY AND CLINICAL RESEARCH

GHRELIN

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PRODUCTION, ACTION MECHANISMS AND PHYSIOLOGICAL EFFECTS

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Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

PHYSIOLOGY - LABORATORY AND CLINICAL RESEARCH

GHRELIN PRODUCTION, ACTION MECHANISMS AND PHYSIOLOGICAL EFFECTS

HIROMASA YAMADA Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

AND

KINTARO TAKAHASHI EDITORS

Nova Science Publishers, Inc. New York

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com

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Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

Contents Preface Chapter I

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Chapter II

Chapter III

Chapter IV

Chapter V

vii Nucleotide Polymorphisms, Transcriptional Analysis, Gene Expression of the Bovine Growth Hormone Secretagogue Receptor 1A (GHS-R1A) Gene and Its Genetic Association with Growth and Carcass Traits in Cattle Masanori Komatsu, Yuki Fujimori, Tomohito Itoh, Yoichi Sato, Hiroaki Okamura, Motohide Nishio, Osamu Sasaki, Aduli E. O. Malau-Aduli, Hideaki Takahashi, Hisato Takeda and Masahiro Satoh The Role of the Pro-Ghrelin Derived Peptides in the Iris Muscle Regulation: Implications in Glaucoma Pathophysiology Sara Azevedo-Pinto, Marta Tavares-Silva, Paulo Pereira-Silva, A. Leite-Moreira and A. Rocha- Sousa Ghrelin: Expression and Functions in the Central Nervous System Irina I. Stoyanova Role of Central Ghrelin in the Gastric Accommodation and Reflex Swallowing Motoi Kobashi, Satoshi Mizutani, and Yuichi Shimatani Physiological Relevances of Ghrelin Synthesis, Transport and Degradation Christine Delporte

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

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Contents

Chapter VI

Ghrelin, Obesity and Atherosclerosis Emina Sudar, Sanja Soskic, Bozidarka L. Zaric, Zorica Rasic-Milutinovic, Katarina Smiljanic, Djordje Radak, Dimitri P. Mikhailidis, Manfredi Rizzo and Esma R.Isenovic

Chapter VII

Rikkunshito, An Endogenous Signal Enhancer of Ghrelin, Improves Gastrointestinal Dysfunction Tomohisa Hattori, Naoki Fujitsuka, Haruka Amitani, Akihiro Asakawa and Akio Inui

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Index

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143

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Preface This book presents current research in the study of ghrelin, a gastric peptide hormone and neurotransmitter, which has been identified in the hypothalamus of the central nervous system, and has importance in food intake, body-weight regulation and glucose homeostasis. Topics discussed in this compilation include the pro-ghrelin derived peptides in iris muscle regulation and their implication in glaucoma pathophysiology; the expression, mechanisms of activity and role of ghrelin in the development of brain and neuronal networks; the role of central ghrelin in gastric accommodation and reflex swallowing and physiological relevances of ghrelin synthesis, transport and degradation. Chapter I - Ghrelin - Growth hormone secretagogue receptor 1a (GHS-R1a) is involved in many important functions including growth hormone (GH) secretion and food intake. In this chapter, we explore existing nucleotide polymorphisms, transcriptional analysis, gene expression of the bovine GHS-R1a gene and its genetic association with growth and carcass traits in cattle. Firstly, we evaluated haplotype variety and characterized the microsatellite ((TG)n, 5’-untransrated region (UTR)) and nucleotide polymorphisms of the GHS-R1a gene in cattle. Nucleotide sequencing of this gene (~6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)n, Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and divided into 3 major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of about 2.2 kb (nt667(C>T) ～ nt2884 (A>G)) were identified in Bos taurus breeds. Significant breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found. A DelR242 was found in the Japanese Shorthorn (frequency: ~ 0.44), Japanese Brown, 5 European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black nor the Mishima Island cattle. Secondly, 5'-rapid amplification of cDNA ends (5’-RACE) and reverse transcriptionpolymerase chain reaction (RT-PCR) analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5’-UTR (GHS-R1a); and non-spliced, with the microsatellite (GHS-R1b). Thirdly, we examined age-related changes in the expressions of GHS-R1a and GHS-R1b (the truncated-type receptor) in the arcuate nucleus, pituitary gland and other tissues by real-time RT-PCR in cattle. The GHS-R 1a mRNA expression in the arcuate nucleus of post-weaning calves was more than 10-fold higher than those of preweaning calves and cows, and its expression level was the highest in all tissues examined. The GHS-R1a mRNA expression in the pituitary gland of pre-weaning calves was higher than those of post-weaning calves and cows. The GHS-R1b mRNA expression was widespread in

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

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viii

Hiromasa Yamada and Kintaro Takahashi

all tissues examined and predominantly occurred in the pancreas, pituitary gland, spleen and arcuate nucleus in adult. Fourthly, we carried out a genetic association study between five nucleotide polymorphisms (5’UTR microsatellite ((TG)n), nt-7(C>A), L24V, DelR242 and Intron 1 microsatellite (GTTT)n) of the GHS-R1a gene and growth and carcass traits in 1,285 steers sired by 117 Japanese Black bulls in a progeny testing program. Statistical analysis revealed that the 5’UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG). One of the four major microsatellite alleles (19TG allele) with an allele frequency of 0.145, had a significantly desirable effect on CW and ADG. We proposed a translational hypothesis that the association is due to differences in the secondary structure of GHS-R1b mRNA (the non-spliced type with the 5’UTR microsatellite) among the GH-SR1a gene haplotypes. Finally, we predicted the potential increase in profitability due to increased CW in cow-calf fattening enterprises through planned matings based on DNA testing of the 5’UTR microsatellite. We concluded that the 19-TG allele could potentially be an economically useful nucleotide marker for growth and carcass traits in Japanese Black cattle. Chapter II - Ghrelin is a 28 aminoacid peptide first described in the rat’s stomach oxyntic musosa in 1999 by Kojima et al. This peptide derives from the pro-ghrelin and is the endogenous ligand of the growth hormone secretagogues receptor-type 1a (GHSR-1a), being ghrelin’s acylation in the serine 3 residue essential for this linkage. There is also a non acylated ghrelin form, des-octanoil-ghrelin, which does not bind GHSR-1a and represents 90% of the circulating hormone. Ghrelin exerts its actions through different subcellular pathways, being the GHSR-1a related to the IP3-DAG pathway. Besides promoting growth hormone release from the pituitary, ghrelin exerts its actions in several organ systems, namely the endocrine, cardiovascular, musculo-skeletal and the eye, among others. Nowadays, it is accepted that there are other receptors responsible for ghrelin’s action than GHSR-1a. Through an alternative splicing method, pro-ghrelin may also originate another peptide, called obestatin. Obestatin, a 23 aminoacid peptide, was first isolated through bioinformatic techniques, being subsequently described in both rat’s and human stomach. This peptide has been reported to exert actions opposite to those of ghrelin in several systems, such as the endocrine system. All these pro-ghrelin derived peptides have been proven to exert significant effects in the several components of the ocular tissue. The ghrelin-obestatin system has recently been described to exert an active role on the kinetics of the iris sphincter muscle. On the one hand, ghrelin promotes the decrease of the muscle’s tension, either the actively developed after carbachol pre-contraction, or the basal one. This relaxing effect is not species dependent, being independent from GHSR-1a and from nitric oxide and dependent on prostaglandins’ production. On the other hand, obestatin showed to potentiate the iris’ sphincter muscle cholinergic contraction, although when the muscle stimulation was achieved through electrical field stimulation it seemed to induce a tendency of the developed tension to decrease. The other ocular muscle studied was the iris dilator muscle, a smooth muscle also presenting several pathways of neurohumoral regulation. In this muscle, ghrelin was reported to decrease the norepinephrine induced muscular contraction, through a mechanism dependent on GHSR-1a. Concerning obestatin, it decreased the iris’ dilator muscle basal tension, but showed no effect on the epinephrine induced active tension. The anterior segment of the eye is also influenced by the pro-ghrelin derived peptides. Ghrelin’s mRNA has been observed in the iris posterior segment and in the non pigmented cilliary epithelium. The interaction between this system and the anterior segment has also been reported in glaucoma,

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Preface

ix

an optic nerve damage which presents as the main cause an increase in intra-ocular pressure. Both open angle and pseudoexfoliation glaucoma were associated with increased aqueous humor ghrelin levels, which may imply these peptides in the patophisiology of the disease. Concerning other effects in the eye, obestatin was shown to promote the proliferation of the retinal pigmented epithelium cells, through a pathway dependent on the activation of ERK ½, while ghrelin was implicated in the retina neovascular process. Chapter III - Ghrelin is a gastric peptide hormone and neurotransmitter, ligand for the growth hormone secretagogue receptor (GHS-R1). The hypothalamus was identified as the main source of ghrelin in the CNS, therefore the effects of the peptide have been mainly related to this part of the brain; numerous studies over the past decade demonstrate its importance in food intake, body-weight regulation and glucose homeostasis. Data about the existence of extrahypothalamic ghrelinergic neurons are still controversial, however the distribution of GHS-R1 outside the hypothalamus indicates that ghrelin also has an important role in the regulation of many other processes. The spectrum of functions and biological effects of ghrelin on neurons is remarkably wide and complex. It varies from modulation of the membrane excitability, to control of neurotransmitter release, neuronal gene expression, neuronal survival, proliferation and differentiation. Ghrelin and its effects have been described not only in situ in different parts of the brain, but also in vitro. It has a stimulating effect on the electrical network activity, and dissociated cortical neurons express ghrelin immunorectivity since the earliest stage of culturing. As ghrelin is present in the majority of cultured neurons during the first week in vitro, when the neuronal differentiation and network formation take place, it may also influence early synaptic formation and cell-to-cell interactions, which are both very important for network functions such as learning and memory. This chapter gives an overview on the expression, mechanisms of activity and role of ghrelin in development of brain and neuronal networks. Chapter IV - Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R), and was originally isolated from the stomach. In addition to GH-releasing activity, ghrelin has an orexigenic (appetite-enhancing) effect. This is rational, since the blood and stomach ghrelin levels are high during fasting in rats. Ghrelin has been shown to be present not only in the stomach but also in the hypothalamus, and participates in the regulation of food intake itself and feeding-related phenomena through the peripheral and central nervous system. Although the hypothalamic nuclei have an essential role in feeding, the nuclei in the caudal brainstem modify food intake in response to ghrelin. This region has a substantial role in swallowing, gastric motility and gastric secretion. Since swallowing and gastric accommodation are important for smooth digestion and are involved in the sequence of events, we examined the central effect of ghrelin on reflex swallowing and the reservoir function of the stomach in urethane-chloralose anaesthetized rats. Fourth ventricular administration of ghrelin but not a vehicle induced relaxation of the proximal stomach lasting for more than 30 min. Administration of ghrelin with a growth hormone secretagogue receptor antagonist ([D-Lys3] GHRP-6) into the fourth ventricle did not induce a significant change in intragastric pressure. Bilateral sectioning of the vagi at the cervical level abolished the relaxation induced by the administration of ghrelin into the fourth ventricle. Microinjections of ghrelin into the caudal part of the dorsal vagal complex (DVC) induced obvious relaxation of the proximal stomach. These results revealed that ghrelin induced relaxation in the proximal stomach via GHS-R situated in the caudal DVC. Fasting is usually followed by feeding; therefore, gastric relaxation achieved by the actions of orexigenic

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neuropeptides, which are released by hunger, to enhance the easy accommodation of food is rational. Since gastric relaxation is closely related to swallowing, we further examined the effect of ghrelin on reflex swallowing in anaesthetized rats. Fourth ventricular administration of ghrelin significantly decreased swallowing frequency during electrical stimulation of the central cut end of the superior laryngeal nerve (SLN stimulation). The administration of ghrelin with [D-Lys3] GHRP-6 did not change swallowing frequency during SLN stimulation. Since microinjection of ghrelin into the vicinity of the solitary tract inhibited swallowing frequency induced by SLN stimulation, ghrelin inhibited reflex swallowing by modifying neural activities of the dorsal medulla where the swallowing center is housed. In conclusion, central ghrelin regulates extensive autonomic phenomena associated with food intake by way of the caudal brainstem. Chapter V - Ghrelin, previously coined GHS (growth hormone secretagogue), is a 28 amino acid peptide possessing an unusual octanoyl moiety on the serine in position 3. Ghrelin is predominantly produced and secreted into the blood stream by the endocrine X/A like cells of the stomach mucosa in rodents (P/D1 cells in humans). The processing of the 117 amino acids prepro-ghrelin precursor yields ghrelin and another peptide called obestatin. The unusual acylation is catalyzed by ghrelin O-acyl transferase (GOAT), a member of the membrane-bound O-acyl transferase family, adding an octanoyl moiety before the translocation of ghrelin to the Golgi apparatus. Various modified ghrelin peptides have been identified and can be classified into two groups based on their length (28 or 27 amino acids) and/or into four groups based on the presence and nature of the acyl group on the serine in position 3 (non-acylated, octanoylated, decanoylated, decenoylated). Human plasma ghrelinimmunoreactivity consists of more than 90% of des-acyl ghrelin. The shorter half-life of ghrelin compared to des-acyl ghrelin and plasma ghrelin deacylation could account for this observation. Several enzymes have been shown to participate in ghrelin degradation in the stomach and systemically. In the circulation, des-acyl ghrelin is mostly present as a free peptide, while the vast majority of acyl ghrelin is bound to larger molecules such as lipoproteins. Phosphorylation of ghrelin on serine 18 has been reported to affect the amphipathic helix formed by about two third of the C-terminal part of the peptide. However, to date, it is yet unknown if such phosphorylation could occur in cells under specific conditions. The biological activity of ghrelin, via its binding to its growth hormone secretagogue receptor of type A (GHS-R1a), relies on the presence of the voluminous hydrophobic groups on serine 3. The biological activity of the other ghrelin peptides has been pharmacologically evaluated on the GHS-R1a. Biological activities of non-acylated ghrelin have been reported and suggested to occur through an as yet unidentified receptor. Ghrelin synthesis, post-translational modifications, transport, receptor(s) recognition and enzymatic degradation represent important mechanisms that are likely to collectively and co-ordinately modulate the biological activity of ghrelin. Chapter VI - Cardiovascular disease (CVD) is common cause of death in humans and its major underlying pathology is atherosclerosis. Atherosclerosis is a chronic inflammatory disease that predisposes to coronary artery disease (CAD), stroke and peripheral arterial disease, responsible for most of the cardiovascular morbidity and mortality. This inflammatory process, triggered by the presence of lipids in the vascular wall, and encompasses a complex interaction among inflammatory cells, vascular elements, and lipoproteins through the expression of several adhesion molecules and cytokines. Obesity is a risk factor for CVD but this association is not fully understood. Altered levels of obesity

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Preface

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related peptides such as ghrelin may play an important role in this pathophysiology. Recent evidence indicates that ghrelin features several cardiovascular activities, including increased myocardial contractility, vasodilatation and protection from myocardial infarction. Recent data demonstrate that ghrelin can influence important key events in atherogenesis and thus they may play a role in atherosclerosis. In this review we present the latest data from recent animal and clinical studies which focus on a novel approach to ghrelin as a potential therapeutic agent in the treatment of a complex disease like atherosclerosis. Thus, ghrelin may become a new therapeutic target for the treatment of CVD. Further studies are necessary to investigate the potential mechanisms involved in the effects of ghrelin on the cardiovascular system. Chapter VII - Herbal medicine has been practiced since centuries in Japan, China, and other countries for restoring the balance of energy in the body. Herbal medicines, containing biologically active compounds, are also widely used to alleviate various disorders. For example, recent studies have shown that rikkunshito, Japanese traditional medicine alleviates nausea, loss of appetite, and cachexia associated with cancer and chemotherapy—conditions that decrease life expectancy and quality of life (QOL). The mechanism of action of rikkunshito involves the enhancement of signaling by ghrelin, an appetite-stimulating peptide produced in the stomach that was discovered in 1999. We and other researchers have demonstrated that the regulation of peripheral ghrelin levels may be partly associated with the activation of the serotonin 5-HT2b/2c receptor pathway, resulting in a change from fasted motor activity to fed motor activity and the negative control of feeding behavior in animal models of upper gastrointestinal disorders. The unique effects of rikkunshito induce the stimulation of acylated ghrelin secretion from the stomach into peripheral circulation by the inhibition of the 5-HT2b receptor, located in the gastric fundus, and the 5-HT2c receptor, widespread in the central area. In addition, administration of rikkunshito may block leptin receptor signaling in the arcuate nucleus of the hypothalamus by inhibiting the activity of the phosphodiesterase type III receptor and increasing ghrelin receptor gene expression, leading to the activation of ghrelin signaling. These studies focused on rikkunshito and its active components, which can potentiate ghrelin signaling and can alleviate symptoms of gastrointestinal diseases, such as functional dyspepsia. This review provides novel scientific insights into herbal medicine.

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

In: Ghrelin Editors: H. Yamada and K. Takahashi

ISBN: 978-1-61942-400-5 © 2012 Nova Science Publishers, Inc.

Chapter I

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Nucleotide Polymorphisms, Transcriptional Analysis, Gene Expression of the Bovine Growth Hormone Secretagogue Receptor 1A (GHS-R1A) Gene and Its Genetic Association with Growth and Carcass Traits in Cattle Masanori Komatsu1*, Yuki Fujimori2, Tomohito Itoh3, Yoichi Sato4, Hiroaki Okamura5, Motohide Nishio1, Osamu Sasaki1, Aduli E. O. Malau-Aduli6, Hideaki Takahashi1, Hisato Takeda1 and Masahiro Satoh1 1

National Institute of Livestock and Grassland Science (NILGS), Tsukuba, Japan 2 Ibaraki Prefecture Livestock Research Centre, Hitachi-Ohmiya, Japan 3 Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc. (LIAJ), Maebashi, Japan 4 Iwate Prefecture Livestock Research Centre, Takizawa, Japan 5 National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan 6 Animal Production and Genetics, School of Agricultural Science/ Tasmanian Institute of Agricultural Research (TIAR), University of Tasmania, Hobart, Tasmania, Australia

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2

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

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Abstract Ghrelin - Growth hormone secretagogue receptor 1a (GHS-R1a) is involved in many important functions including growth hormone (GH) secretion and food intake. In this chapter, we explore existing nucleotide polymorphisms, transcriptional analysis, gene expression of the bovine GHS-R1a gene and its genetic association with growth and carcass traits in cattle. Firstly, we evaluated haplotype variety and characterized the microsatellite ((TG)n, 5’-untransrated region (UTR)) and nucleotide polymorphisms of the GHS-R1a gene in cattle. Nucleotide sequencing of this gene (~6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)n, Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and divided into 3 major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of about 2.2 kb (nt667(C>T) ～ nt2884 (A>G)) were identified in Bos taurus breeds. Significant breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found. A DelR242 was found in the Japanese Shorthorn (frequency: ~ 0.44), Japanese Brown, 5 European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black nor the Mishima Island cattle. Secondly, 5'-rapid amplification of cDNA ends (5’RACE) and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5’-UTR (GHS-R1a); and non-spliced, with the microsatellite (GHS-R1b). Thirdly, we examined age-related changes in the expressions of GHS-R1a and GHS-R1b (the truncated-type receptor) in the arcuate nucleus, pituitary gland and other tissues by realtime RT-PCR in cattle. The GHS-R 1a mRNA expression in the arcuate nucleus of postweaning calves was more than 10-fold higher than those of pre-weaning calves and cows, and its expression level was the highest in all tissues examined. The GHS-R1a mRNA expression in the pituitary gland of pre-weaning calves was higher than those of postweaning calves and cows. The GHS-R1b mRNA expression was widespread in all tissues examined and predominantly occurred in the pancreas, pituitary gland, spleen and arcuate nucleus in adult. Fourthly, we carried out a genetic association study between five nucleotide polymorphisms (5’UTR microsatellite ((TG)n), nt-7(C>A), L24V, DelR242 and Intron 1 microsatellite (GTTT)n) of the GHS-R1a gene and growth and carcass traits in 1,285 steers sired by 117 Japanese Black bulls in a progeny testing program. Statistical analysis revealed that the 5’UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG). One of the four major microsatellite alleles (19-TG allele) with an allele frequency of 0.145, had a significantly desirable effect on CW and ADG. We proposed a translational hypothesis that the association is due to differences in the secondary structure of GHS-R1b mRNA (the non-spliced type with the 5’UTR microsatellite) among the GH-SR1a gene haplotypes. Finally, we predicted the potential increase in profitability due to increased CW in cow-calf fattening enterprises through planned matings based on DNA testing of the 5’UTR microsatellite. We concluded that the 19-TG allele could potentially be an economically useful nucleotide marker for growth and carcass traits in Japanese Black cattle.

Keywords: Growth hormone secretagogue receptor (GHS-R), nucleotide polymorphism, mRNA expression, tissue distribution, mRNA secondary structure, growth and carcass traits, profitability, cattle

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Nucleotide Polymorphisms, Transcriptional Analysis …

3

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Introduction Pre- and post-weaning growth in the early stages of a calf’s life cycle is an important trait in calf production, because of the significant feed change from liquid (milk) to solids (grass, hay, concentrates, etc.) and production pressure on calves to grow faster than in the other periods of life. Growth is controlled by growth hormone (GH) action based on aging. The secretion of GH by pituitary somatotropes is primarily stimulated by the hypothalamic growth-hormone-releasing hormone (GHRH) through the activation of a specific G protein-coupled receptor (GPCR), GH-releasing hormone receptor (GHRH-R). Growth hormone is also released in response to ghrelin, a peptide produced in the stomach, hypothalamus and pituitary gland that activates somatotropes via a distinct GPCR, the growth hormone secretagogue receptor 1a (GHS-R1a)[1-3]. Somatostatin inhibits the action of pituitary somatotrope cells through distinct GPCRs, somatostatin receptors (ssts), to release GH [3]. It is likely that the GHRH/GHRH-R-arm of the GH pathway serves primarily in the production of de novo GH, and secondarily in the release of (pre-made) GH, while ghrelin/GHS-R1a may serve primarily in the release of stored GH and secondarily in the production of de novo GH [1]. Ghrelin stimulates GH secretion by primarily binding to GHS-R1a on GHRH neurons in the arcuate nucleus of the hypothalamus. The GHRH neurons have been found to be mostly located in the arcuate nucleus in cattle [4]. Moreover, ghrelin-containing neurons in the arcuate nucleus pre-synaptically induce neuropeptide Y (NPY) neurons to release NPY, thus stimulating appetite [3, 5]. GHS-R1a mRNA is detected in NPY and GHRH neurons in the arcuate nucleus, and GHS-R1a is involved in the up-regulation of GHRH and NPY expression in the arcuate nucleus in rats [6]. Since GHRH and NPY are hypothalamic orexigenic neuropeptides, the interactions between ghrelin/GHS-R1a and between GHRH/GHRH-R are involved in the regulatory mechanism of appetite as well as GH secretion. The function of the other type of GHS-R, type 1b (GHS-R1b, the truncated receptor polypeptide), has been suggested to regulate the GHS-R1a expression in the form of the GHS-R1a/GHS-R1b heterodimer [7]. It is well documented that plasma GH concentration decreases with age in a variety of species, and there are distinct differences in the decline patterns. In cattle, it is well known that the plasma GH concentration is elevated at birth and declines during sexual maturation [8-12]. Furthermore, age-dependent changes in GH increase the response to stimulation with GH-releasing factor (GRF) [10], GHRH and GH-releasing peptide 6 (GHRP-6) [13], GHRH [11], and ghrelin and GHRH [12, 14] have been reported in cattle. Interestingly, a synergistic effect of ghrelin and GHRH on GH secretion has been reported in pre-weaning calves [14]. Changes in the hormone responsiveness of tissues can occur via changes in the number and/or affinity of receptors and through modification of the signal transduction systems subsequent to the binding of the hormone to its receptor [15]. Age-related changes in the GHS-R1a mRNA expression have been reported in rats [16] and in mice [17]. Furthermore, comprehensive tissue distributions of the GHS-R1a and/or GHS-R1b mRNA expressions have been reported in humans [18] and in mice [17]. However, in cattle, no comprehensive tissue distributions of the GHS-R1a and/or GHS-R1b mRNA expressions in the arcuate nucleus, pituitary gland and other bovine tissues have been reported in cattle.

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4

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

Through the use of anti-sense GHS-R1a mRNA expression, transgenic rats with impaired GHS-R function in the arcuate nucleus have been found to be significantly smaller at birth, and have lower body weight and less adipose tissue compared to controls [19]. Furthermore, the genetic linkage of single nucleotide polymorphisms (SNPs) of the GHS-R1a gene and the association of a mis-sense mutation (A204E) within the gene with obesity and/or short stature have been reported in humans [20, 21]. In cattle, the GHS-R1a gene was reported as a potential candidate gene when we detected growth trait QTLs in Japanese Black cattle using microsatellite DNA markers and half-sib regression analysis [22, 23]. Furthermore, nucleotide polymorphisms of the coding regions of the GHS-R1a gene [24] and its association with growth traits have also been reported [25]. With respect to the bovine GHS-R1a gene, the genomic DNA sequences of this locus have been defined by the Bovine Genome Project (Accession number : NW_001493714; http://www.hgsc.bcm.tmc.edu/projects/bovine/). It is of great interest that a polymorphic microsatellite ((TG)n) was located within 5'-flanking region of this locus [26] , because no microsatellite had previously been found within the GHS-R1a locus in either humans [27], mice [28] or rats [29]. However, there had been no report on nucleotide polymorphisms from the 5’-flanking region to the 3’-UTR nor on the transcriptional analysis of the 5’-UTR of the GHS-R1a gene in cattle. However, recently, we reported nucleotide polymorphisms from the 5’-flanking region to the 3’-UTR (~6 kb) and two different kinds of spliced and non-spliced transcripts (spliced, without a microsatellite within 5’-UTR (GHS-R1a); and non-spliced, with the microsatellite (GHS-R1b)) of the bovine GHS-R1a gene) [30]. Furthermore, we reported a significant additive effect of the 5’-UTR microsatellite locus on cattle average daily gain (ADG) and carcass weight (CW) due to differences in the secondary structure of GHS-R1b mRNA among the GHS-R1a gene haplotypes [31]. Additionally, in order to develop a better understanding of the age-related functions of GHS-R 1a and GHS-R1b in the hypothalamus/pituitarymediated regulation of GH secretion and feeding/growth in cattle, we examined the agerelated changes in the GHS-R 1a and GHS-R1b mRNA expressions in several tissues including the arcuate nucleus and pituitary gland [32]. In this chapter, we structure and present our recent studies on the bovine GHS-R1a gene as follows: 1) Nucleotide polymorphisms from the 5’-flanking region to the 3’-UTR of the GHSR1a gene and its molecular evolution; 2) 5’ -UTR transcriptional analysis of the bovine GHS-R1a gene; 3) Age-related changes in the GHS-R1a and GHS-R1b mRNA expressions in several tissues including the arcuate nucleus and pituitary gland; 4) Genetic association between the 5’UTR microsatellite ((TG)n) of the GHS-R1a gene and growth and carcass traits in Japanese Black cattle. 5) The translational hypothesis that any significant genetic association with growth and carcass traits is attributable to differences in the secondary structure of GHS-R1b mRNA; 6) Prediction of the potential increase in profitability due to increased carcass weight through planned matings based on DNA testing of the 5’UTR microsatellite ((TG)n) of the GHS-R1a gene

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Nucleotide Polymorphisms, Transcriptional Analysis …

5

1. Nucleotide Polymorphisms from the 5’-Flanking Region to the 3’-UTR of the GHS-R1A Gene and its Molecular Evolution We performed fragment analysis of the microsatellite DNA ((TG)n) of a total of 356 individuals belonging to 11 breeds that included 3 Wagyu breeds (Japanese Black, Japanese Shorthorn and Japanese Brown), the Mishima Island cattle (the oldest breed of native Japanese cattle [33], 5 European cattle breeds, the Philippine native cattle breeds (Batangas, Ilocos and Iloilo; Bos indicus / Bos taurus mixture breeds [34]) and an American-Brahman cross. To compare microsatellite TG-repeats among the alleles, we sequenced the microsatellites from 10 individuals of 8 breeds (comprising 9 homozygotes and 1 heterozygote) as depicted on Table 1. For the nucleotide polymorphism analysis of the GHS-R1a gene, we sequenced the genomic DNA from the 5’-flanking region to the 3’-UTR (~6 kb) using 26 individuals belonging to 10 breeds and an American-Brahman cross based on the microsatellite genotypes (comprising 15 homozygotes and 11 heterozygotes), thus defining as large a variety of actual nucleotide polymorphisms as possible (Table 1).

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Microsatellite ((TG)n) Polymorphism and Molecular Evolution A total of 17 alleles (10-TG to 33-TG) was found in 11 cattle breeds (Table 1). There were breed differences in allele frequencies and major alleles. Specifically, the major alleles were 19-TG, 23-TG and 24-TG in Japanese Black, and alleles 20-TG, 23-TG, 25-TG and 28TG in Japanese Shorthorn, alleles 21-TG, 22-TG, 23-TG, and 24-TG in Japanese Brown, alleles 24-TG and 25-TG in Mishima Island cattle, alleles 19-TG, 21-TG, 22-TG and 24-TG in Holstein-Friesian, alleles 19-TG, 21-TG, 22-TG and23-TG in the 4 European cattle breeds, and allele10-TG and 22-TG in the Philippine native cattle breeds. The short repeat number alleles 10-TG, 15-TG, 16-TG and 18-TG were found in the Philippine native cattle. The microsatellite TG-repeat sequences included one cytosine(C) instead of guanine (G) at the 9th repeat position from the 3’-end of this locus in 7 alleles of the Bos taurus breeds and allele 22-TG of the American-Brahman cross (mtDNA type: Bos taurus type). Interestingly, alleles 10-TG and 19-TG of the Philippine native cattle (mtDNA type: Bos indicus type) did not reveal such nucleotide substitution (Table 1). The common Bos taurus breeds had a TC-repeat at the 9th repeat position in the microsatellite ((TG)n) locus. This position was constant and independent of the TG-repeat number. These results suggest that the TG-repeat number of the microsatellite increased with evolution from the 3’-end to the 5’-end direction of this sequence. Furthermore, and interestingly, the 2 alleles, TG-10 and TG-19 from the Philippine native cattle (Bos indicus) did not include the TC-repeat in the microsatellite locus. Thus, there may be a different lineage of molecular evolution in the microsatellite of the GHS-R1a gene between Bos taurus and Bos indicus breeds.

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Table 1. Allele frequencies of the microsatellite ((TG)n) of the GHS-R1a gene in several cattle breeds including Wagyu Allele ((TG) n) £

Breed

n

$,†

10 ,15, 16,18

19†

20

21†

22†

23†

24†

25†

26

27

28†

29

31

33

χ2#

(Bos taurus) Wagyu Japanese Black

93

-

0.22§

-

0.01

0.09

0.24

0.37

-

-

-

-

0.04

-

0.04

a

Japanese Shorthorn

77

-

0.02

0.20

0.01

0.11

0.22

0.06

0.14

0.03

-

0.20

-

0.01

-

b

31

-

0.03

0.08

0.18

0.18

0.16

0.23

-

0.13

-

-

-

-

0.02

c

58

-

-

-

0.01

-

0.14

0.35

0.50

-

-

-

-

-

-

d

Japanese Brown ¢

Mishima Island cattle Holstein-Friesian

34

-

0.29

0.03

0.13

0.16

0.07

0.16

0.02

0.08

-

-

-

-

0.06

ce

¶

30

-

0.17

0.02

0.25

0.17

0.18

-

0.05

0.10

0.02

0.05

-

-

-

e

Philippine native cattle

28

0.23

0.05

-

0.02

0.57

0.07

-

0.02

0.04

-

-

-

-

-

f

American-Brahman cross

5

0.10

-

-

0.10

0.60

0.10

-

-

0.10

-

-

-

-

-

-

Four European breeds (Bos indicus and /or

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Bos taurus mixture)

£

†

No of animals. Number of (TG) repeats; Sequenced. ¢Japanese native cattle. Jersey + Swiss Brown + Hereford + Aberdeen-Angus. # The different alphabets mean that allele frequencies are statistically different. §A bold font means a major allele frequency. Sequence of each allele (5' →3', breeds): allele 10, TG(10), Philippine native cattle; allele 19、TG(10)TC(1)TG(8), Japanese Black, Holstein-Friesian; allele 19,TG(19), Philippine native cattle; allele 21, TG(12)TC(1)TG(8), Aberdeen-Angus; allele 22, TG(13)TC(1)TG(8), Japanese Brown, American-Brahman cross; allele 23 、 TG(14)TC(1)TG(8), Japanese Black; allele 24 、 TG(15)TC(1)TG(8), Japanese Black; allele 25, TG(16)TC(1)TG(8), Mishima Island cattle; allele 28, TG(19)TC(1)TG(8), Japanese Shorthorn. $

¶

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Nucleotide Polymorphisms, Transcriptional Analysis …

7

Nucleotide Polymorphism We detected a total of 52 nucleotide polymorphisms, including 47 SNPs (the 5’-flanking region, 16; Exon 1, 4; Intron 1, 23; Exon 2, 1; the 3’-UTR, 3), 4 indels (the 5’-flanking region, a 1-bp indels; Exon 1, a 3-bp indels; Intron 1, a 1-bp indels and a 3-bp indels) and a microsatellite ((GTTT)n) were identified in 26 individuals from 11 breeds (Tables 2). Twenty-five of the 47 SNPs (the 5’-flanking region, 7; Exon 1, 4; Intron 1, 13; the 3’UTR, 1), two 1-bp indels (the 5’-flanking region and Intron 1), a 3-bp indel (Exon 1) and three Intron 1 microsatellite alleles ((GTTT)5, (GTTT)6 and (GTTT)8) were identified in Bos taurus breeds. Their approximate positions are indicated in Figure 1(A). The 19 haplotypes were constructed from all nucleotide variability patterns, and were divided into 3 major groups by the phylogenetic analysis (neighbor-joining (NJ) tree) using MEGA4 [35]. Two were found in Bos taurus breeds (the Group 1, Hap 01-09; the Group 2, Hap21-26) and one in Bos indicus (the Group 3, Hap 41-43) (Figure 2). Table 2. Summary of nucleotide polymorphisms of the bovine GHS-R1a gene Items

Classification

Region 5'-flanking + 5'-UTR

(Subtotal) SNP 1-bp indels†

Exon 1

SNP

#

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3-bp indels Intron 1

No. of polymorphism

§

SNP 1-bp indels$ 3-bp indels

¶

16

[5, 2, 9]£

1

[1, 0, 0]

4

[4, 0, 0]

1

[1, 0, 0]

23

[6, 7, 10]

1

[0, 1, 0]

1

[0, 0, 1]

Exon 2

SNP

1

[0,0,1]

3'-UTR

SNP

3

[0,1 , 2]

Total

51

[17, 11, 23]

Microsatellite

(17)††

[3, 8, 5]

(TG)n

(10-33)##

[27-33, 19-26, 10-18]

Microsatellite

(4)††

[1, 2, 1]

(GTTT)n

(4,5,6,8)¶¶

[ 8, (5,6), 4]

5'-UTR

Intron 1

£

[Bos taurus only, Bos taurus and Bos indicus, Bos indicus only]. †nt-1177(A>－). #L24V(nt70(C>G), nt456(G>A), D191N(nt580(G>A), nt667(C>T). §DelR242(nt724-726(AGG>－)). $nt2323(T>－). ¶ nt1449-1451(TTT>－). ††Number of alleles. ##Number of (TG) repeats. ¶¶Number of (GTTT) repeats.

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8

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

Figure 1. (A) Scheme of the bovine GHS-R1a gene showing the region sequenced and the polymorphisms detected. Vertical dashes (│) indicate SNPs; additional arrows (↓) indicate indels. Differences in the polymorphisms between Bos taurus and Bos indicus were observed:

●, both in Bos

□

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taurus and Bos indicus; 〇, Bos taurus; , Bos indicus. MS indicates a microsatellite locus. (B) Structure of the bovine GHS-R1a gene transcript. Two transcription start sites (1a and 1b) are shown with dotted arrows. Splicing of an intron including microsatellite (wave box) in the 5’UTR is demonstrated only for the GHS-R1a (1a type) but not for the GHS-R1b (1b type). The 3’UTR is shown as a dot line.

The 2 Wagyu breeds (Japanese Black and Japanese Brown) and Holstein-Friesian and Swiss Brown were found to belong to both Groups 1 and 2, while the Japanese Shorthorn and the Mishima Island cattle belonged to the Group 1. Hap04 and Hap05 were observed in the Mishima Island cattle, and the Japanese Black cattle shared the Hap04. The Hap07 in Group 1 was the most common haplotype in the Bos taurus breeds (Holstein-Friesian, AberdeenAngus, Swiss Brown, Japanese Black and Japanese Brown). The Mishima Island cattle, the oldest breed of native Japanese cattle [33], showed 2 unique haplotypes (Hap04 and Hap05) belonging to Group 1 and Japanese Black cattle shared the Hap05. The 2 major haplotype lineages of the GHS-R1a gene, Groups 1 and 2, were observed in 2 Wagyu breeds (Japanese Black and Japanese Brown) as well as in the European breeds. These results suggest that Hap04 and Hap05 may be the fundamental haplotypes unique to the Wagyu breeds and that the Group 2 haplotypes observed in the 2 Wagyu breeds may reflect the crossbreeding history of each Wagyu breed with several European cattle breeds (e.g. Swiss Brown) imported to Japan [36]. Four SNPs (L24V, nt456 (G>A), D191N, nt667(C>T) and DelR242 in Exon 1 were found in Bos taurus breeds. L24V and D191N were linked to each other and belonged to Hap21, Hap22 or Hap23 in the Group 2. DelR242 (a 3-bp indel) was linked to the nt456 (G>A) and the microsatellite 28-TG allele, and belonged to the Group 1. DelR242 was found in the Japanese Shorthorn (homozygote; Hap09), Japanese Brown and Jersey breeds (heterozygotes; Hap06/Hap10; Figure 2). A 3-bp indel (nt1449-1451) in Intron 1 was identified in the Philippine native cattle (Hap43) and allele (GTTT)6 of the Intron 1 microsatellite was identified in both the Bos taurus and Bos indicus breeds.

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Nucleotide Polymorphisms, Transcriptional Analysis …

9

(J. Black, Swiss Brown) (J. Shorthorn) (J. Brown, Jersey) (Hereford)

(J. Black, J. Brown, Holstein, Angus, Swiss Brown) (Hereford, J. Brown) (J. Brown, Jersey) (J. Black)

(J. Black, Mishima Island cattle) (Mishima Island cattle) (Swiss Brown) (J. Black) (J. Black, J. Brown) (J. Black)

(J. Black) (J. Brown, Holstein) (Philippine native cattle , American-Brahman cross) (Philippine native cattle) (Philippine native cattle)

: DelR242

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Figure 2. Neighbor-joining (NJ) tree for the GHS-R1a gene (19 haplotypes) and an estimated lineage of the DelR242. The numbers at selected branches are the bootstrap values after 500 re-sampling draws. Hap: Haplotype numbers. J. Black: Japanese Black; J. Brown: Japanese Brown; J. Shorthorn: Japanese Shorthorn. The square box shows the most common haplotypes in Bos taurus breeds.

In the Bos taurus breeds, Group 2 haplotypes were characterized by allele (GTTT)6, a 1bp indel (nt2323(T>-) and 10 SNPs (nt667(C>T), nt811(G>A), nt980(G>T), nt990(T>A), nt1150(T>C), nt1171(C>T), nt2543(C>T), nt2593(C>A), nt2883(G>T), nt2884(A>G)[30]. A haplotype block of about 2.2 kb (from nt667(C>T) to nt2884 (A>G)) was detected in Bos taurus breeds using 26 SNPs markers (Hap01～Hap26) (Figure 1 (A)). We found no significant changes in the 17 nucleotide polymorphisms in the 5’-flanking region (16 SNPs and a 1-bp deletion) as transcriptional factor recognition sites except the nt2022 (G>A) that contributes a W element consensus sequence: WGNAMCYG . This mutation (nt-2022(G>A)) is lost in this site.

Allele Frequency of nt-7(C>A), L24V, DelR242 and the Microsatellite ((GTTT)n) nt-7(C>A): There were clear breed differences in allele frequencies in the nt-7(C>A ) locus (Table 3). Allele A was predominant in Japanese Shorthorn, Holstein-Friesian, Aberdeen-Angus, Hereford and Jersey. Allele C was predominant in Japanese Black, the Mishima Island cattle and Swiss Brown, especially allele C was very high in the Mishima Island cattle (0.96).

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Table 3. Allele frequencies of the nt-7(C>A), L24V, DelR242 and the microsatellite ((GTTT)n) loci in several cattle breeds

Breed

No. of Animals

nt-7(C>A)

L24V (nt+70(C>G))

DelR242 (3-bp deletion)

A

C

C

G

AGG

DelR242

(GTTT)4

(GTTT)5

(GTTT)6

(GTTT)8

Microsatellite((GTTT)n)

Japanese Black

93

0.34

0.66

0.995

0.005

1.00

—

—

0.93

0.07

—

Japanese Shorthorn

77

0.71

0.29

1.00

—

0.57

0.43

—

1.00

—

—

Japanese Brown

31

0.47

0.53

0.93

0.07

0.79

0.21

—

0.82

0.18

—

Mishima Island cattle

58

0.04

0.96

1.00

—

1.00

—

—

0.99

—

0.01#

Holstein-Friesian

34

0.88

0.12

1.00

—

0.91

0.09

—

0.93

0.07

—

Four European breeds¶

30

0.78

0.22

0.87

0.13

0.77

0.23

—

0.83

0.13

0.03#

Philippine native cattle

28

0.55

0.45

1.00

—

0.98

0.02

0.09

0.86

0.05

—

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Jersey + Swiss Brown + Hereford + Aberdeen-Angus. #Jersey.

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Nucleotide Polymorphisms, Transcriptional Analysis …

11

Both allele frequencies were almost equal in Japanese Brown and the Philippine native cattle. L24V: Allele G was found in Swiss Brown with a high frequency (0.67) in contrast to the low frequencies in Japanese Brown (0.07) and Black (0.005) cattle, but none was detected in the Japanese Shorthorn, Mishima Island, Holstein-Friesian, Aberdeen-Angus, Hereford, Jersey and the Philippine native cattle. Allele G, found in Japanese Black and Brown breeds, which encodes valine residue instead of leucine, might have been derived from the Swiss Brown breed because of a decline in frequency of the unique haplotype shared between the Swiss Brown and the 2 Wagyu breeds. DelR242: DelR242 was found in high frequencies in the Japanese Shorthorn (0.43), Japanese Brown (0.21) and four European breeds (0.23), but with low frequencies in Holstein-Friesian (0.09) and the Philippine native cattle (0.02). DelR242 was never found in Japanese Black and Mishima Island cattle. Colinet et al. (2009)[24] reported the DelR242 in Holstein-Friesian, Belgian Blue and Limousin cattle breeds. We also identified DelR242 in Japanese Shorthorn, Japanese Brown, four European breeds (Holstein-Friesian, Aberdeen-Angus, Hereford, Jersey and Swiss Brown) and the Philippine native cattle [30]. This mutation causes a truncated 3 arginine residues (normal type: 4 arginine residues) in the third loop of the intercellular loop domain of the GHS-R1a protein. The DelR242 is a fundamental allele of the GHS-R1a gene in mice and rats. Taken together, these results show that DelR242 is widely distributed in many cattle breeds (Figure 2) and it may be an older allele than the non-DelR242. Although the frequency of this mutation has not yet been reported in the Shorthorn breed, a possible reason for the observed high frequency of this mutation in Japanese Shorthorn might have been derived from the native cattle population (mostly Nanbu-Gyu). The DelR242 allele may have some effects on growth and carcass traits. Intron 1 microsatellite ((GTTT)n): Allele (GTTT)6 was found in Brown Swiss with a high frequency (0.67), but with low frequencies in Japanese Brown (0.18), Holstein-Friesian (0.07), Japanese Black (0.07), and the Philippine native cattle (0.05), and was not detected in Japanese Shorthorn, Aberdeen-Angus and Hereford. Furthermore, the 2 additional alleles were found as follows: (1) allele (GTTT)8 in Mishima Island cattle (0.01) and Jersey (0.14) and (2) allele (GTTT)4 in the Philippine native cattle (0.09). A difference of four alleles of the microsatellite ((GTTT)n) locus is due to the difference in number of the GTTT-repeat as follows: (GTTT)4, (GTTT)5, (GTTT)6 and (GTTT)8. The GTTT-repeat number of the microsatellite ((GTTT)n) locus may be increased with evolution as well as the TG-repeat number of the microsatellite ((TG)n).

Haplotype frequency of the [microsatellite ((TG)n] – [nt-7(C>A) ] – [L24V] - [DelR242] – [microsatellite ((GTTT)n)] To further reveal nucleotide variety of the GHS-R1a gene in 3 Wagyu breeds and Mishima Island cattle, the haplotype among the 5’-UTR microsatellite ((TG)n), nt-7(C>A), L24V, DelR242 and the microsatellite ((GTTT)n) was constructed. A total of 42 haplotypes (Group 1, 28; Group 2, 8; Group 3, 6) was found in 11 cattle breeds [30]. There were breed differences in haplotype frequencies and major haplotypes. The microsatellite ((TG)n), nt-

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12

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

7(C>A), L24V, DelR242, and microsatellite ((GTTT)n) are recommended as nucleotide markers of choice for the GHS-R1a gene for association studies with growth and carcass traits in Wagyu breeds. The clear differences observed in the features of the nucleotide polymorphisms of the bovine GHS-R1a gene, strongly suggest that the gene may have accumulated several dramatic nucleotide mutations during the process of long-term selection and crossbreeding as an important farm animal.

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2. 5’ -UTR Transcriptional Analysis of the Bovine GHS-R1a Gene There are two different kinds of transcripts, GHS-R1a mRNA (spliced type) and GHSR1b mRNA (non-spliced type) in human, mice and rats. As mentioned previously, GHS-R1b (the truncated receptor polypeptide) has been suggested to regulate the expression of GHSR1a in the form of GHS-R1a/GHS-R1b heterodimer [7]. The polymorphic microsatellite ((TG)n) is located within the 5'-flanking region of the bovine GHS-R1a locus [26], however, no microsatellite has been detected within the GHS-R1a locus in either humans, mice or rats. Therefore, we sought to define the transcript sequences of the 5’-UTR of the gene in order to determine whether or not the microsatellite ((TG)n) locus is transcribed. A tissue block containing the arcuate nucleus was dissected from the brain of a 2.5month-old Holstein-Friesian bull calf. The block contained a large proportion of arcuate nucleus, a small proportion of ventromedial nucleus (VMH) and dorsomedial nucleus, and a large proportion of median eminence [32]. Total RNA (1 µg) was extracted and reversetranscribed to single-stranded cDNA (ss-cDNA) by reverse transcriptase. This was then followed by the 5’-rapid amplification of cDNA ends (RACE), TA-cloning and sequence analysis of the 5'-UTR of the GHS-R1a gene in order to determine whether or not the microsatellite locus within the 5’-flanking region was transcribed. Thirty independent positive clones containing the bovine 5’-UTR of the GHS-R1a cDNA were obtained. Sequencing of these clones revealed that the cDNA sequences (type 1, 26 clones) were identical to the genome sequence of the GHS-R1a gene but did not include the sequences between positions -390 and -135. The other sequences (type 2, 4 clones) were identical to the genome sequences including those between positions -390 and -135 (Figure 1). Therefore, the type 2 transcripts contained the splice-out sequence of the 5’-UTR and included the microsatellite TG-repeats between positions -277 and -232. These results show that the type 1 is the 1a type and the type 2 is the 1b type. The structure of the bovine GHSR1a gene transcripts is shown in Figure 1(B). The sequences of the clones showed multiple 5'-ends of the cDNA between positions -1,015 and -976 for all types 1 (types 1-1, 1-2, 1-3, 14) and those between positions -763 and -568 for types 2 (types 2-1, 2-2, 2-3). The intron region (from -390 to -135) contained the typical donor and acceptor sequence (GT/AG) and the pyrimidine-rich tract (py tract) (from -160 and to -138). Furthermore, this alternative splicing of the GHS-R1a gene was confirmed by RT-PCR analysis. Therefore, we found evidence for 2 different kinds of transcripts, spliced, without a microsatellite within 5’-UTR (the GHS-R1a type); and non-spliced, with the microsatellite (the GHS-R1b type)[30].

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Nucleotide Polymorphisms, Transcriptional Analysis …

13

To find out if there was a species-specific motif within the promoter region of the GHSR1a gene in cattle, we compared potential transcriptional regulatory sequences in approximately 2.6 kb of the 5'-flanking region the GHS-R1a gene among cattle, human and mouse [30]. Six bovine specific motifs were identified as follows: (1) S1 nucleasehypersensitive site (S1_HS), (2) Apolipoprotein E_B1 element (ApoE_B1), (3) A-activator binding site (AABS)_consensus sequence (CS), (4) Nuclear protein factors and erythroid specific 1_CS1 ((NF_E1_CS1), (5) Nuclear protein factors I_CS7((NFI_CS7), and (6) NF_E1_CS2. Apolipoprotein E (apoE) is a major constituent of very low density lipoprotein and can be found associated with all of the major classes of lipoprotein particles [37]. Furthermore, AABS motif is a binding site for C/EBP beta (CCAAT/enhancer-binding protein beta) [38]. C/EBP beta is a transcriptional regulator of the UCP1 (uncoupling protein1) gene, the specific marker gene of brown adipocytes that is responsible for their thermogenic capacity [39]. Expression of mRNA of the GHS-R1a gene in cattle may be more coordinated with lipoprotein metabolism than in humans and mice.

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3. Age-Related Changes in the GHS-R1a and GHS-R1b mRNA Expressions in Several Tissues Including the Arcuate Nucleus and Pituitary Gland Total RNA was extracted from 16 tissues (the arcuate nucleus, pituitary gland, liver, spleen, kidney, heart, skeletal muscle, rib eyes, adipose tissue, pancreas, adrenal gland, thyroid gland, jejunum, colon, abomasum, and mammary gland) collected from 11 HolsteinFriesian cattle that ranged in age from 19 days to 8.1 years. The tissue samples were divided into three age-classes for age-related analysis of mRNA expression as follows: (I) preweaning (19 - 26 d-old male calves); (II) post-weaning (2 - 6.5 mo-old steers); and (III) Adult (3.2 – 8.1 yr-old dams). The total RNA was reverse-transcribed to single-stranded complementary DNA (ss-cDNA). A portion of each cDNA for GHS-R1a (150 bp), GHS-R1b (170 bp) and GAPDH (147 bp, an internal control) was synthesized and cloned into the pUC57 vector. The plasmid DNA containing the correct insert for the respective cDNA was used to generate the standard curve in real-time RT-PCR. The amount of each mRNA was quantified by real-time RT-PCR using the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Polymerase chain reaction (PCR) was carried out with TaqMan Universal PCR MasterMix (Applied Biosystems), Custom TaqMan Assays-by-Design Primers and Probe set. The standard curve for each gene was generated using the plasmid-DNA-containing fragment of each gene, which was serially 10-fold diluted (from101-fold to 109-fold). Relative copy numbers of each mRNA were expressed as follows: (copy numbers of each mRNA / copy numbers of GAPDH mRNA) × 1000. Relative copy numbers were log-transformed [32]. Age-related changes in copy numbers (CNs) of GHS-R1a and GHS-R1b mRNAs in the arcuate nucleus and pituitary gland are shown in Table 4 and the Log10 expression levels of these mRNAs after normalization with GAPDH are described in Table 5. In the arcuate

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nucleus, there was an 18-fold CN increase in GHS-R1a mRNA from pre-weaning (2.3 x 105) to post-weaning (52.3 x 105). Table 4. Age-related changes in GHS-R1a and 1b mRNA copy numbers / μg total RNA in the arcuate nucleous and pituitary gland Tissue and Age#

GHS-R1a Mean (x105)

GHS-R1b Mean (x105)

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Arcuate nucleous Pre-weaning (2)¶ 2.3 1.3 Post-weaning (3) 52.3 0.9 Cow (6) 1.6 2.2 Pituitary gland Pre-weaning (2) 32.5 22.3 Post-weaning (1) 0.7 0.4 Cow (4) 8.8 8.7 # Pre-weaning, 19 -26days after birth (male calves); Post-weaning, 2-6.5 months after birth (steers); Cow, 3.2-8.1 years after birth (cows). ¶Number of animals.

In contrast, the CN of GHS-R1a mRNA in cow (1.6 x 105) declined to 3.1% CN of that observed post-weaning. The post-weaning expression level of GHS-R 1a mRNA in the arcuate nucleus was more than 10-fold higher (P < 0.01) than those in pre-weaning and adult stages. The CN of GHS-R1b mRNA at post-weaning tended to be lower than those in preweaning and adult stages. In the pituitary gland, the CNs of GHS-R1a mRNA in cow decreased compared with those in pre-weaning (16%) and GHS-R1b mRNA also decreased (39%). The CN of GHS-R1b mRNA at post-weaning tended to be lower than those in preweaning and adult (Table 4). An age-dependent decline in plasma GH concentration from birth to sexual maturity was observed in cattle [8-12]. However, the plasma level of active ghrelin did not show an age-dependent decline in cattle even though a decline was observed between pre-weaning and post-weaning phases of growth [12,14]. Furthermore, hypothalamic GHRH and somatostatin mRNAs had been reported to remain relatively constant throughout development in rats [40] and plasma somatostatin level remained unchanged after weaning in cattle [41]. Taken together, our results suggest that the age-dependent decline in plasma GH concentration is partly due to the age-dependent declines in GHS-R1a mRNA expression in the pituitary gland. Ghrelin / GHS-R stimulates appetite in the arcuate nucleus [3, 5]. In the arcuate nucleus, the ghrelin-containing neurons send efferent fibers onto neuropeptide Y (NPY)- and agouti-related protein (AgRP)-expressing neurons to stimulate the release of these orexigenic peptides and onto proopiomelanocortin (POMC) to suppress the release of this anorexigenic peptide in rodents [2]. In sheep, offering feed ad libitum (resulting in greater ME intake) decreased hypothalamic mRNA expression of NPY and AgRP and tended to increase that of POMC compared with the wethers under restricted-feeding [42]. GHS-R1a mRNA is detected in NPY and GHRH neurons in the arcuate nucleus, and GHS-R1a is involved in the up-regulation of NPY and GHRH expression in the arcuate nucleus [6]. In cattle, voluntary feed intake increases significantly with age and reaches or exceeds ‘adult’ levels within 6 weeks after weaning [43].

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Table 5. Age-related changes in the logarithmic 10 transformed (Log 10) relative expression levels of GHS-R1a and GHS-R1b mRNAs in various tissues Age# and Tissue

GHS-R1a mRNA Mean‡ (Log10)

(1) Pre-weaning

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Pituitary gland Liver Arcuate nucleus Spleen Kidney Adipose tissue Heart Rib Eye Skeletal muscle (2) Post-weaning Arcuate nucleus Liver Pituitary gland Thyroid Spleen Leucocyte

Age and Tissue

GHS-R1b mRNA Mean (Log10)

(1) Pre-weaning 1.97 1.11 0.85 0.02 -1.05 -1.27 -1.65 -2.38 -2.82

Pituitary gland Spleen Arcuate nucleus Heart Liver Kidney Adipose tissue Skeletal muscle Rib Eye (2) Post-weaning Spleen Pancreas Leucocyte Thyroid Jejunum Pituitary gland

1.81 0.88 0.64 0.45 0.05 -0.11 -0.35 -0.63 -0.72

Jejunum

2.14 1.13 1.01 0.24 0.09 0.06 -0.06

Abomasum

1.85 1.72 1.21 0.83 0.77 0.73 0.43

Pancreas

-0.40

Arcuate nucleus

0.31

Adipose tissue

Heart

0.22

Abomasum

-0.84 -0.95

Liver

0.21

Colon

-0.97

Colon

0.04

Adrenal

-1.00

Adrenal

-0.09

Heart (3) Cow

-1.24

Adipose tissue (3) Cow

-0.10

Pituitary gland

1.31

Pancreas

2.32

Arcuate nucleus

0.96

Spleen

1.68

Liver

0.76

Pituitary gland

1.31

Spleen

Arcuate nucleus

0.86

Pancreas

0.35 0.29

Adipose tissue

0.85

Mammary gland

-0.60

Leucocyte

0.84

Adipose tissue

-0.62

Kidney

0.46

Kidney

-1.05

Mammary gland

0.51

Heart Skeletal muscle Leucocyte Rib Eye

-1.72 -1.85 -2.01 -2.46

Liver Skeletal muscle Heart Rib Eye

0.04 -0.12 -0.51 -0.53

#

Age: (1) Pre-weaning, 19～26 days of age, ♂ ; (2) Post-weaning,, 2～6.5 months of age, steer; (3) Cow, 3.2～8.1 years of age, ♀. ‡ Data are expressed relative to GAPDH mRNA {Log10 ( [copy numbers of GHS-R1a mRNA or GHSR1b mRNA in 1 μg total RNA / copy number of GAPDH mRNA in 1 μg total RNA] × 1,000)}.

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Furthermore, voluntary feed intake per unit of metabolic weight (dry matter (g) / live weight (kg)/ day) from weaning to sexual maturity shows a steady decline with increasing weight [44]. The plasma levels of leptin, which serves as a satiety signal secreted by white adipose tissue, and those of active ghrelin, remain constant until 1 year of age in Holstein cattle (Leptin: [45]; ghrelin [12, 14]). Furthermore, in cattle, there are age-dependent declines of plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels, which are additional gastrointestinal peptides involved in satiety [46]. These findings show that plasma leptin, ghrelin, CCK and PP levels seem not to be responsible for the fast increase in voluntary feed intake during the post-weaning period. Since GHRH interacts with GHS-R1a, which modifies the ghrelin-associated intracellular signaling [47], the very high expression of GHS-R1a mRNA and the lower expression of GHS-R1b mRNA in the arcuate nucleus during the post-weaning period dramatically amplify ghrelin signaling that stimulates the release of orexigenic peptides (e.g., NPY, AgRP, GHRH). These conditions also suppress the release of anorexigenic peptides (e.g., α melanocyte stimulating hormone) as well as the secretion of GH in cattle. For this reason, post-weaning calves exhibit a very high voluntary feed intake. GHS-R1a mRNA was highly expressed in the liver, arcuate nucleus and pituitary gland, but expressed at much lower levels in the spleen (Table 5). The expression level of GHS-R1a mRNA in the liver was relatively high in all age groups (from the pre-weaning to the cow phases). The expression of the GHS-R1b mRNA was detected in all tissues examined. To compare with that in all tissues examined in each age group, the GHS-R1b mRNA was highly expressed in the pituitary gland, spleen and arcuate nucleus in pre-weaning and adult, and in the pancreas, adipose tissue and leucocyte in adult than that of the other tissues examined. Interestingly, a relatively large animal to animal variation was observed in the pancreas in post-weaning and adult (data not shown). Comprehensive analysis of the expression of GHS-R1a and GHS-R1b mRNA in tissues has been accomplished in humans and GHS-R1a mRNA expression described in the pituitary, thyroid, pancreas, spleen, myocardium and adrenal glands, whereas GHS-R1b mRNA was found in all tissues studied [18]. The tissue distribution of the GHS-R1a mRNA has also been studied in mice and GHS-R1a mRNA expression has been described in the pituitary gland, brain, heart, thymus, testis, lung, adrenal gland, small intestine, thyroid gland, spleen, pancreas, and kidney [17]. In peripheral tissues, there were 3 marked differences in mRNA expression between cattle and monogastric animals (i.e., humans and / or mice) as follows: (1) the GHS-R1a mRNA expression in the liver was high in cattle and very low in humans and mice; (2) the GHS-R1b mRNA expression in the liver was low in cattle and high in humans; (3) the GHSR1b mRNA expression in the pancreas was very high and showed dramatic animal to animal variation in cattle. Murata et al. [48] reported that the GHS-R1a mRNA is expressed in hepatoma cells and that ghrelin up-regulates the mRNA level of phosphoenolpyruvate carboxykinase (PEPCK), which is the rate-limiting enzyme of glyconeogenesis and modulates downstream molecules involved in insulin-signaling in humans. Furthermore, GHS-R1a and ghrelin mRNAs are expressed in human T lymphocytes and monocytes, where ghrelin acts via GHS-R1a to especially inhibit the expression of pro-inflammatory anorectic cytokines such as IL-6, IL-1 βand TNF-α [49]. IL-6 is well-known to inhibit the expression of the glyconeogenic genes

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(phosphoenolpyruvate carboxylase-1(PCK-1) and glucose 6-phosphatase (G6PC)) in the liver via the signal transducer and activator of the transcription-3 (STAT-3) signal cascade [50]. Since hepatic and renal gluconeogenesis is crucially important in the glucose metabolism in ruminants [51], the high expression of GHS-R1a mRNA and the low expression of GHS-R1b mRNA in the liver seem to be important for gluconeogenesis and represent the bovinespecific expression pattern for maintaining glucose homeostasis in cattle. The very high expression of splenic GHS-R1b mRNA described here may attenuate the inhibition of pro-inflammatory anorectic cytokines and leptin-induced anorectic cytokine expression in monocytes and T cells by the action of ghrelin via GHS-R1a for maintaining the immune system and appetite control. Ghrelin is expressed in pancreatic islets and there appears in vivo, to be a negative association between ghrelin and insulin secretion from the pancreas [52]. Ghrelin has been known to function as a potent inhibitor of pancreatic CCK-induced exocrine secretion in rats [52], and the CCKA and CCKB/gastrin receptors, which are GPCRs, are expressed in the pancreas in cattle [53]. Moreover, PP is expressed in the endocrine pancreas and is released in response to meals as an anorexigenic peptide. A receptor with a high affinity for PP, the Y4 receptor, which is also a GPCR, is expressed in the pancreas in humans [54]. A very high expression of GHS-R1b mRNA and a relatively large animal to animal variation in the GHS-R1b mRNA expression in the pancreas may support the hypothesis that the GHS-R1b alters the basal expression of GHS-R1a by the GHS-R1a – GHS-R1b heterodimer formation [7]. Furthermore, other GPCRs expressed in the pancreas, such as the CCKA and CCKB/gastrin receptors and the Y4 receptor, may interact with the GHS-R1b to alter their basal expression and to ensure a ready response to changes in nutritional / physiological body conditions.

4. Genetic Association between the 5’UTR Microsatellite ((TG)n) of the GHS-R1a Gene and Growth and Carcass Traits in Japanese Black Cattle 5 nucleotide polymorphic loci (5’UTR microsatellite (TG)n, Intron 1 microsatellite (GTTT)n, nt-7(C>A), L24V and DelR242) were recommended as nucleotide markers of choice for investigating genetic association between the GHS-R1a gene and growth and carcass traits in Wagyu breed [30]. Therefore, we investigated the genetic association between the 5 nucleotide polymorphisms of the GHS-R1a gene and growth and carcass traits in Japanese Black cattle [31]. We used a population of 1,285 Japanese Black steers sired by 117 bulls (ranging from 10 to 14 progeny per sire) in a progeny-testing program of the Livestock Improvement Association of Japan (LIAJ). They were allowed to suckle their dams in addition to being fed concentrates and corn silage until weaning. After weaning, they were moved to the grower’s barn and reared until the attainment of seven to eight months of age when the steer calves were divided into two groups and transported to two progeny-testing stations in Japan. They were fed with the conventional grower ration with an allowance of 20 days for adjustment

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and acclimatization followed by the standard 364 days (52 weeks) progeny testing duration prior to slaughter. Routine management of the animals involved the recording of body weight, body shape, concentrate intake and grass-silage intake every four weeks. Steers were weighed at the beginning and end (WT) of the testing period so that average daily gain (ADG) could be computed. Carcass data collected included weight at slaughter (WS), cold carcass weight (CW), average daily gain (ADG), rib eye area (REA), rib thickness (RT), carcass yield estimate (YE), subcutaneous fat thickness (SFT), inter-muscular fat thickness (IFT) and beef marbling score (BMS). Genomic DNA was extracted from the ear and semen. Fragment analysis for the two microsatellites and DelR242 were carried out. Modified Sequence-Specific Primer Cycle Elongation (SSPCE) for nt-7(C>A) and L24V (nt70(C>G)) was also carried out [31]. Genetic association analysis between DNA markers and growth and carcass traits was carried out using a univariate model within the framework of a derivative-free restricted maximum likelihood algorithm as applied in the MTDFREML [55]. The following linear mixed animal model was used:

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y=X1b+X2g+Zu+e, where y is a vector of phenotypic observations; b is a vector of fixed effects and includes the year and month of birth, place of birth, location of test-stations and the linear covariate of age at the beginning of the progeny test; g is a vector of the fixed additive effect of the GHS-R1a microsatellite or SNP alleles or haplotypes of the microsatellite and SNP; u is a vector of random additive genetic effect of the polygene, which is assumed to be distributed across N(0, Aσ a2), where A is the additive relationship matrix among animals andσ a2 is the additive genetic variance of polygene; e is a vector of random residual effects, which is assumed to be distributed across N(0, Iσ e2) ; and X1, X2 and Z are the corresponding incidence matrices. Although L24V and Intron 1 microsatellite loci were major alleles with frequencies over 0.94, we chose to rather focus on the 5’UTR microsatellite and nt-7(C>A) loci for the association study with growth and carcass traits because the effects of L24V and Intron 1 microsatellite loci on these traits did not reach statistical significance during the preliminary analysis. Furthermore, the allele DelR242 was not detected in this population. Preliminary statistical analysis revealed that the 5’UTR microsatellite locus had a highly significant additive effect on WT (pA)), Hap03 (the haplotype number); Right: A (nt7(C>A)), Hap02. The square box shows a translation start region. The square dotted arrow shows a Kozak sequence of the GHS-R1a mRNA (AGCAUGU) and the AUG is the translation start codon. The underlined bases in this sequence are bound in a secondary structure of the GHS-R1a mRNA. The solid arrow shows the 5’ end of the transcript. (B) Optimal secondary structure of the 5’UTR microsatellite region of the 4 haplotypes of the GHS-R1b mRNAs. The circle dotted arrow shows the 5’UTR microsatellite ((TG)n) region. A putative secondary structure type for the 5’UTR region: S type, [19TG]; B type, [non–19-TG] ( [24-TG], [29-TG], [33-TG]). (C) Optimal secondary structure of the GHSR1b mRNAs. Left: [19-TG] - A (the [5’UTR microsatellite] – [nt-7(C>A)] haplotypes), Hap07; Right: [24-TG] - C, Hap04. The circle dotted oval box shows the 5’UTR microsatellite ((TG)n) region. The square box, the square dotted arrow, the AUG, the underlined bases and the solid arrow mean as referred to Figure 3(A). A secondary structure of the 1b mRNAs: S-V type, [19-TG]-A; B-T type, [24TG]-C.

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Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

Fold algorithms and basic options used were as follows: (1) minimum free energy (MFE) and partition function, (2) no GU pairs at the end of helices, and (3) no isolated base pairs. As shown in Figure 3(A) and Table 7, the optimal RNA secondary structures of the 1a mRNAs appeared to be almost the same among haplotypes or between the A and C types (nt7(C>A)). Thus, the number of Kozak sequence bases (“AGCAUGU”) bound in secondary structure of these types was the same as 2 regardless of the haplotypes. In addition, there seemed to be no fundamental difference in the minimum free energy ( Δ G) among the haplotypes of the GHS-R1a gene (Table 7). Taken together, these results indicate that there seems to be no differences in the GHSR1a mRNA translation efficiency and the GHS-R1a protein level among these haplotypes (Table 8). On the other hand, the optimal RNA secondary structure of the 5’UTR regions of the 1b mRNAs appeared to be different between the [19-TG] and [non-19-TG] types or between the A and the C types (nt-7(C>A)) (Figure 3(B,C), Table 7). A secondary structure of 5’UTR microsatellite region for the [non-19-TG] 1b mRNAs seems to have a bending knot but not in the [19-TG] type (Figure 3(B)). It would seem that the secondary structure of the 5’UTR microsatellite region of the [19-TG]-1b mRNAs had a small dominant negative effect on the translation efficiency of the GHS-R1b mRNA due to its unique structure. Therefore, the GHS-R1b mRNA translation efficiency based on the secondary structure of the 5’UTR microsatellite region seems to be the following order: [non19-TG] > [19-TG]. There were 5 Kozak sequence bases bound in the secondary structures for the A type (nt7(C>A)) and 2 for the C type (Figure 1(C)). The A type of the 1b mRNAs seemed to have a lower effect on translation efficiency than that of the C type because of the different number of Kozak sequence bases bound in the secondary structure. Therefore, the GHS-R1b mRNA translation efficiency based on the nt-7(C>A) seems to be the following order: C > A. Taking these differences in GHS-R1b mRNA translation efficiency, the GHS-R1b mRNA translation efficiency based on the secondary structure of the 5’UTR microsatellite region and the nt-7(C>A) seems to be the following order: [non-19-TG] – A > [non-19-TG] – C > [19TG] – C > [19-TG] – A. In addition, the [19-TG] – A type mRNA is assumed to have a specific effective interaction to translation machinery. On the basis of these results, we proposed a translational hypothesis that the differences in the RNA secondary structure of the GHS-R1b mRNAs among the 5’UTR microsatellite the nt-7(C>A) haplotypes affect the functional Ghrelin receptor (GHS-R1a) level (Table 8). As already mentioned, the function of the GHS-R1b has been suggested to regulate the GHS-R1a expression in the form of the GHS-R1a/GHS-R1b heterodimer [7]. The estimated functional Ghrelin receptor levels of each homozygote are [the GHS-R1a protein level] minus [GHS-R1b protein level] and the following order: [19-TG] – A > [19-TG] – C > [non-19-TG] – C > [non-19-TG] – A. The differences in the RNA secondary structure around the 5’UTR region of the 1b mRNAs between the [19-TG] and [non-19-TG] or between the A and C (nt-7(C>A)) affect translational levels of the 1b mRNAs and the functional Ghrelin receptor level during growth, GH release from the pituitary gland, plasma GH concentration, appetite, glyconeogenesis and finally growth traits in cattle [31]. This hypothesis should be validated by a molecular biological study in the future.

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6. Prediction of the Potential Increase in Profitability Due to Increased Carcass Weight through Planned Matings Based on DNA testing of the 5’UTR Microsatellite ((TG)n) of the GHS-R1a gene

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We predicted the potential increase in profitability (ΔVind) due to increase in carcass weight (CW) of Japanese Black cattle cow-calf fattening farms using planned mating based on DNA testing of the [19-TG] allele for 5’UTR microsatellite. The BeefIncome Program (http://nilgs.naro.affrc.go.jp/prog/BeefIncome.html) [64] was used for the prediction of profitability. The assumptions and explored diverse scenarios: (1) Initial frequency of the19-TG allele in Japanese Black cattle (p) = 0.145. (2) The 19-TG (excellent) to non-19-TG (ordinary) allele additive substitution effect for CW (ΔCWQTL), 8.125 kg ( = 6.5 kg ×(440/352)). (3) Carcass weight (CW), 440 kg. (4) Per-kg unit price for CW of Japanese Black cattle (CWPU), ¥1,900/kg. (5) Charge for DNA typing (CTYP), ¥0 or ¥5,000. (6) Semen price difference between sires selected and unselected by DNA typing through reproduction cycles (SEM), ¥0 or ¥10,000. (7) The DNA typed sires (the [19-TG] homozygote) were used to breed dams (HardyWeinberg population). (8) Sire/dam ratio (R(s/d)), 1/30. (9) Number of dams (Number of cows in the population) (N), 30 or 1,200. (10) The age at first calving and the productive lifetime of dams (N: number of dams) were 3 and 8 yr, respectively. 1 5 (11) The replacement rate for cows was N. Calves not for replacement ( N) and cows 6 6 1 over 8 yr ( N) were moved for fattening and slaughter. 6 (12) The planned time horizon (T) was 1 or 6 yr. (13) Priority sequence of dams for slaughter: [non-19-TG] homozygote → [19-TG/ non19-TG] heterozygote → [19-TG] homozygote. (14) [Scenario 1]: Sires were known for DNA type (the 19-TG homozygote): CTYP= ¥0, SEM = ¥0 or ¥10,000. [Scenario 2]: Sires were DNA typed and selected (the 19-TG homozygote): CTYP = ¥5,000, SEM = ¥0 or ¥10,000. We predicted the potential increase in profitability in both small-scale cow-calf fattening farms utilizing Japanese Black cattle through the use of planned mating based on DNA testing of the 19-TG allele for 5’UTR microsatellite (usage of the19-TG homozygote sire) to increase CW (Table 9).

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Table 9. Increase in total profit in CW (￥/head) (ΔVind) predicted by DNA typing for the 19-TG allele through 3 or 6-year planned time horizon under the two scenarios† Case (Scenario) / Item

ΔVind ( ￥/head)

Number of dams (N)

30

30

Sire/dam ratio (R(s/d)) Planned time horizon (years) (T)

(1/30) 3

(1/30) 6

( 1-1) CTYP = ￥ 0; SEM = ￥ 0

10,608

11,193

( 1-2) CTYP =￥ 0; SEM = ￥10,000

-503

217

( 2-1) CTYP =￥ 5,000; SEM = ￥0 ( 2-2) CTYP =￥ 5,000; SEM = ￥10,000

10,423

11,010

-688

34

3,080

3,080

Scenario (1)

Scenario (2)

Scenario (0): Hardy-Weinberg Population †

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Assumptions, see below; (1) Initial frequency of the19-TG allele in Japanese Black cattle (p) = 0.145. (2) Carcass weight (CW) ,440kg/head. (3) The 19-TG allele additive effect on CW (ΔCWQTL) , 8.125 kg (4) Per-kg unit price for CW of Japanese Black cattle (CWPU), 1,900 ￥/kg. (5) Charge for DNA typing (CTYP), ¥0 or ¥5,000. (6) Semen price difference between sires selected and unselected by DNA typing through reproduction cycles (SEM), ¥0 or ¥10,000. (7) Sire/dam ratio (R(s/d)), 1/30. (8) Number of dams (Number of cows in the population) (N), 30. (9) Planned time horizon (years) (T), 3 or 6. The BeefIncome Program (http://nilgs.naro.affrc.go.jp/prog/BeefIncome.html) was used for the prediction of profitability.

It was demonstrated that it was possible to achieve about ¥10,000-worth of profitability increase (ΔVind) provided that the cost of DNA diagnosis was ¥5,000/head or less and that the semen price differential was ¥0/head in a small-scale cow-calf fattening farms utilizing Japanese Black cattle within a 3-year time frame. This result indicates that the 19-TG allele for 5’UTR microsatellite is potentially an economically useful DNA marker for Japanese Black cow-calf fattening enterprises.

Conclusion (1) Nucleotide sequencing of this gene (~6 kb) revealed 47 single nucleotide polymorphisms (SNPs), 4 indels and 2 microsatellites ((TG)n, 5’UTR and (GTTT)n, Intron 1). (2) The 19 haplotypes constructed from all nucleotide viability patterns belonged to 3 major groups. (3) A DelR242 was found in the Japanese Shorthorn (frequency: ~ 0.44), Japanese Brown, 5 European cattle breeds, the Philippine native cattle, but none detected in neither the Japanese Black nor the Mishima island cattle. (4) 5’-RACE and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5’UTR (GHS-R1a); and non-spliced, with the microsatellite (GHS-R1b). (5) The microsatellite ((TG)n), nt-7(C>A), L24V, DelR242, and microsatellite ((GTTT)n) are recommended as

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nucleotide markers of choice for the GHS-R1a gene in association study with growth and carcass traits in Japanese Black cattle. (6) GHS-R 1a mRNA expression in the arcuate nucleus of post-weaning calves was significantly more than 10-fold higher than those of pre-weaning calves and cows, and its expression level was the highest in all tissues examined. (7) GHSR1a mRNA expression in the pituitary gland of pre-weaning calves was higher than in postweaning calves and cows. (8) The GHS-R1b mRNA expression was widespread in all tissues examined and predominantly occurred in the pancreas, pituitary gland, spleen and arcuate nucleus. (9) The [5’UTR microsatellite] – [nt-7(C>A)] haplotype of the bovine GHS-R1a gene affects growth and carcass traits in Japanese Black cattle. (10) We proposed a translational hypothesis that the association is due to differences in the secondary structure of GHS-R1b mRNA (the non-spliced type with the 5’UTR microsatellite) among the GHS-R1a gene haplotypes. (11) The frequency of the 19-TG allele of 5’UTR microsatellite ((TG)n) is 0.145 and the 19-TG allele has a significantly desirable effect on CW and ADG in Japanese Black cattle. The 19-TG allele is potentially an economically useful DNA marker for Japanese Black cow-calf fattening farms.

References [1] [2]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[3] [4]

[5] [6]

[7]

[8] [9]

Lin-Su K, Wajnrajch MP. Growth hormone releasing hormone (GHRH) and the GHRH receptor. Rev Endocrine and Metabolic Disorders. 2002;3:313-323. Anderson LL, Jeftinija S, Scanes CG, Stromer MH, Lee J-S, Jeftinija K, GlavaskiJoksimovic A. Physiology of ghrelin and related peptides. Domestic Anim. Endocrinol. 2005;29:111-144. Kojima M, Kangawa K. Ghrelin: Structure and function. Physiol. Rev. 2005;85:495522. Leshin LS, Barb CR, Kiser TE, Rampacek GR, Kraeling RR. Growth hormonereleasing hormone and somatostatin neurons within the porcine and bovine hypothalamus. Neuroendocrinology. 1994; 59:251-264. Cruz CR, Smith RG. The growth hormone secretagogue receptor. Vitamins and Hormones. 2008;77:47-88. Mano-Otagiri A, Nemoto T, Sekino A, Yamauchi N, Shuto Y, Sugihara H, Oikawa S, Shibasaki T. Growth hormone-releasing hormone (GHRH) neurons in the arcuate nucleus (Arc) of the hypothalamus are decreased in transgenic rats whose expression of ghrelin receptor is attenuated: Evidence that ghrelin receptor is involved in the upregulation of GHRH expression in the Arc. Endocrinology. 2006;147:4093-4103. Leung P-K, Chow KBS, Lau P-N, Chu K-M, Chan C-B, Cheng CHK, Wise H. The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor. Cel. Signal. 2007;19:1011-1022. Purchas RW, Macmillan KL, Hafs HD. Pituitary and plasma growth hormone levels in bulls from birth to one year of age. J. Anim. Sci. 1970;31:358-363. McAndrews JM, Stroud CM, MacDonald RD, Hymer WC, Deaver DR. Age-related changes in the secretion of growth hormone in vivo and in vitro in infantile and prepubertal Holstein bull calves. J. Endocrinol. 1993;139:307-315.

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28

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al.

[10] Baumgard LH, Weber WJ, Kazmer GW, Zinn SA, Hansen LB, Chester-Jones H, Crooker BA. Effects of selection for milk yield on growth hormone response to growth hormone releasing factor in growing Holstein calves. J. Dairy Sci. 2002 ;85:2529-2540. [11] Weber JM, Baumgard LH, Kazmer GW, Zinn SA, Hansen LB, Chester-Jones H, Crooker BA. Growth hormone response to growth hormone releasing hormone in calves that differ in genetic merit for milk yield. J. Dairy Sci. 2005;88:1723-1731. [12] Itoh F, Komatsu T, Yonai M, Sugino T, Kojima M, Kangawa K, Hasegawa Y, Terashima Y, Hodate K. GH secretory responses to ghrelin and GHRH in growing and lactating dairy cattle. Domestic Anim. Endocrinol. 2005;28:34-45. [13] Katoh K, Furukawa G, Kitade K, Katsumata N, Kobayashi Y, Obara Y. Postprandial changes in plasma GH and insulin concentrations, and response to stimulation with GHreleasing hormone (GHRH) and GHRP-6 in calves around weaning. J. Endocrinol. 2004;183:497-505. [14] ThidarMyint H, Yoshida H, Ito T, He M, Inoue H, Kuwayama H. Combined administration of ghrelin and GHRH synergistically stimulates GH release in Holstein preweaning calves. Domestic Anim. Endocrinol. 2008;34:118-123. [15] Vernon RG, Sasaki S. Control of responsiveness of tissues to hormones. In: Tsuda T, Sasaki Y, Kawashima R, eds. Physiological aspects of digestion and metabolism in ruminants. San Diego, CA: Academic Press Inc.; 1991:155-182. [16] Kamegai J, Wakabayashi I, Kineman RD, Frohman LA. Growth hormone-releasing hormone receptor (GHRH-R) and growth hormone secretagogue receptor (GHS-R) mRNA levels during postnatal development in male and female rats. J. Neuroendocrinol. 1997;11:299-306. [17] Sun Y, Garcia JM, Smith RG. Ghrelin and growth hormone secretagogue receptor expression in mice during aging. Endocrinology. 2007;148:1323-1329. [18] Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J. Clin. Endocrinol. Metab. 2002;87:2988-2991. [19] Shuto Y, Shibasaki T, Otagiri A, Kuriyama H, Ohata H, Tamura H, Kamegai J, Sugihara H, Oikawa S, Wakabayashi I. Hypothalamic growth hormone secretagogue receptor regulates growth hormone secretion, feeding, and adiposity J. Clin. Invest. 2002;109:1429-1436. [20] Wang HJ, Geller F, Dempfle A, Schäuble N, Friedel S, Lichtner P, Fontenla-Horro F, Wudy S, Hagemann S, Gortner L, Huse K, Remschmidt H, Bettecken T, Meitinger T, Schäfer H, Hebebrand J, Hinney A. Ghrelin receptor gene: identification of several sequence variants in extremely obese children and adolescents, healthy normal-weight and underweight students, and children with short normal stature. J. Clin. Endocrinol. Metab. 2004;89:157-162. [21] Baessler A, Hasinoff MJ, Fischer M, Reinhard W, Sonnenberg GE, Olivier M, Erdmann J, Schunkert H, Doering A, Jacob HJ, Comuzzie AG, Kissebah AH, Kwitek AE. Genetic linkage and association of the growth hormone secretagogue receptor (ghrelin receptor) gene in human obesity. Diabetes 2005; 54:259-267. [22] Malau-Aduli AEO, Niibayashi T, Kojima T, Oshima K, Mizoguchi Y, Komatsu M. Interval mapping of growth quantitative trait loci in Japanese Black cattle using

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

Nucleotide Polymorphisms, Transcriptional Analysis…

[23]

[24]

[25]

[26]

[27]

[28]

[29]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[30]

[31]

[32]

[33]

[34]

29

microsatellite DNA markers and half-sib regression analysis. Anim. Sci. J. 2005a;76: 11-18. Malau-Aduli AEO, Niibayashi T, Kojima T, Oshima K, Mizoguchi Y, Komatsu M. Mapping the quantitative trait loci (QTL) for body shape and conformation measurements on BTA1 in Japanese Black cattle. Anim. Sci. J. 2005b;76:19-27. Colinet FB, Vanderick S, Charloteaux B, Eggan A, Gengler N, Renaville B, Brasseur R, Portetelle D, Renaville R. Genetic location of the bovine growth hormone secretagogue receptor (GHSR) gene and investigation of genetic polymorphism. Anim. Biotech. 2009;20:28-33. Zhang B, Chen H, Guo Y, Zhang L, Zhao M, Lan X, Zhang C, Pan C, Hu S, Wang J, Lei C. Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle. Mol. Biol. Rep. 2009;36:2259-2263. Ma RZ, Russ I, Park D, Heyen DW, Beever JE, Green CA, Lewin HA. Isolation and characterization of 45 polymorphic microsatellites from the bovine genome. Anim. Genet. 1996;27:43-47. Kaji H, Tai S, Okimura Y, Iguchi G, Takahashi Y, Abe H, Chihara K. Cloning and characterization of the 5’-flanking region of the human growth hormone secretagogue receptor gene. J. Biol. Chem. 1998;273:33885-33888. The FANTOM Consortium and the RIKEN Genome Exploration Research Group Phase I and II Team (Okazaki Y, Furuno M, Kasukawa T et al.) Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs. Nature 2002;420:563-573. MacKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan CP, Phillips MS, Smith RG, Van der Ploeg LHT, Howard AD. Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol. Endocrinol. 2005;11:415-423. Komatsu M, Fujimori Y, Sato Y, Okamura H, Sasaki S, Itoh T, Morita M, Nakamura R, Oe T, Furuta M, Yasuda J, Kojima T, Watanabe T, Hayashi T, Malau-Aduli A.E.O., Takahashi H. Nucleotide polymorphisms and the 5'-UTR transcriptional analysis of the bovine growth hormone secretagogue receptor 1a (GHSR1a) gene. Anim. Sci. J. 2010;81:530-50. Komatsu M, Itoh T, Fujimori T, Satoh M, Miyazaki Y, Takahashi H, Shimizu K, Malau-Aduli A. E.O., Morita M. Genetic association between GHSR1a 5’UTRmicrosatellite and nt-7(C>A) loci on growth and carcass traits in Japanese Black cattle. Anim. Sci. J. 2011;82:396-405. Komatsu M, Kojima M, Okamura H, Nishio M, Kaneda M, Kojima T, Takeda H, Malau-Aduli A. E.O., Takahashi H. Gene expression of the growth hormone secretagogue and growth-hormone-releasing hormone receptors in Holstein-Friesian cattle: Age-related changes in the arcuate nucleus, pituitary gland and other tissues. Domestic Anim. Endocrinol. 2011(In press); doi:10.1016/j.domaniend.2011.09006. Nagamine Y, Nirasawa K, Takahshi H, Sasaki O, Ishii K, Minesawa M, Oda S, Visscher PM, Furukawa T. Estimation of the time of divergence between Japanese Mishima island cattle and other cattle populations using microsatellite DNA markers. J. Heredity 2008;99:202-207. Komatsu M, Yasuda Y, Matias JM, Niibayashi T, Abe-Nishimura A, Kojima T, Oshima K, Takeda H, Hasegawa K, Abe S, Yamamoto N , Shiraishi T. Mitochondrial DNA polymorphisms of D-loop and three coding regions, ND2, ND4, ND5 in three

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

30

[35] [36] [37]

[38]

[39]

[40]

[41] [42]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[43]

[44]

[45]

[46] [47]

[48]

[49]

[50]

Masanori Komatsu, Yuki Fujimori, Tomohito Itoh et al. Philippine native cattle: Indicus and Taurus maternal lineages Nihon Chikusan Gakkaiho. 2004;75:363-378. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis ,MEGA Software Version 40. Mol. Biol. Evol. 2007;24:1596-1599. Sasaki Y. Beef cattle breeding and its trend in Japan. Asian-Australasian J. Anim. Sci. 2001;14: (Special Issue),111-122. Smith JD, Melian A, Leff T, Breslow JL. Expression of the human apolipoprotein E gene is regulated by multiple positive and negative elements. J. Biol. Chem. 1988;263: 8300-8308. Guo Z, Shao L, Feng X, Reid K, Marderstein E, Nakao A, Geller DA. A critical role for C/EBPâ binding to the AABS promoter response element in the human iNOS gene. FASEB J. 2003;17:1718-1720. Carmona MC, Hondares E, Rodríguez De La Concepción ML, Rodríguez-Sureda V,* Peinado-Onsurbe J, Poli V, Iglesias R, Villarroya F, Giralt M. Defective thermoregulation, impaired lipid metabolism, but preserved adrenergic induction of gene expression in brown fat of mice lacking C/EBPβ. Biochem. J. 2005;389:47–56. Argente J, Chowen JA, Zeitler P, Clifton DK, Steiner RA. Sexual dimorphism of growth hormone-releasing hormone and somatostatin gene expression in the hypothalamus of the rat during development. Endocrinology. 1991;128:2369-2375. Guilloteau P, Le Huërou-Luron I, Chayvialle JA, Toullec R, Zabielski R, Blum JW. Gut regulatory peptides in young cattle and sheep. J. Vet. Med. 1997; A 44:1-23. Relling AE, Pate JL, Reynolds CK, Loerch SC. Effect of feed restriction and supplemental dietary fat on gut peptide and hypothalamic neuropeptide messenger ribonucleic acid concentrations in growing wethers. J. Anim. Sci. 2010;88:737-748. Hodgeson J. The development of food intake in calves. 4. The effect of the addition of material to the rumen, or its removal from the rumen, on voluntary food intake. Anim. Prod. 1971;13:581-592. Forbes JM. Effects of physiological state and animal productivity. In: Forbes JM, ed. The voluntary food intake of farm animals. London, UK: Butterworth and Co. (Publishers) Ltd;1986:67-85. Block SS, Smith JM, Ehrhardt RA, Diaz MC, Rhoads RP, Van Amburgh ME, Boisclair YR. Nutritional and developmental regulation of plasma leptin in dairy cattle. J. Dairy Sci. 2003;86:3206-3214. McLaughlin CL. Role of peptides from gastrointestinal cells in food intake regulation. J. Anim. Sci. 1982;55:1515-1527. Casanueva FF, Camiňa JP, Carreira MC, Pazos Y, Varga JL, Schally AV. Growth hormone-releasing hormone as an agonist of the ghrelin receptor GHS-R1a. Proc. Natl. Acad. Sci. USA. 2008;105:20452-20457. Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa K, Chihara K. Ghrelin modulates the downstream molecules of insulin signaling in hepatoma cells. J. Biol. Chem. 2002;277:5667-5674. Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard JW, Taub DD. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J. Clin. Invest. 2004;114:57-66. Inoue H, Ogawa W, Ozaki M, Haga S, Matsumoto M, Furukawa K, Hashimoto N, Kido Y, Mori T, Sakaue H, Teshigawara K, Jin S, Iguchi H, Hiramatsu R, LeRoith D, Takeda

Ghrelin: Production, Action Mechanisms and Physiological Effects : Production, Action Mechanisms and Physiological Effects, Nova Science

Nucleotide Polymorphisms, Transcriptional Analysis…

[51]

[52]

[53]

[54]

[55] [56] [57]

[58]

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

[59]

[60] [61]

[62] [63] [64]

31

K, Akira S, Kasuga M. Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo. Nature Medicine. 2004;10:168-174. Leat WMF. Carbohydrate and lipid metabolism in the ruminant during post-natal development. In: Phillipson AT, ed. Digestive physiology and metabolism in the ruminants. Newcastle Upon Tyne, England: Oriel Press Ltd.;1970:211-222. Date Y, Nakazato M, Kangawa K, Matsuo H. Gastro-entero-pancreatic actions of ghrelin. In: Ghigo E, ed. Ghrelin. Norwell, MA, USA: Kluwer Academic Publishers;2004:179-193. Desbois C, Clerc P, Le Huërou-Luron I, Le Dréan G, Gestin M, Dufresne M, Fourmy D, Guilloteau P. Differential tissular expression of the CCKA and CCKB/Gastrin receptor genes during postnatal development in the calf. Life Sci. 1998;63:2059-2070. Lundell I, Blomqvist AG, Berglund MM, Schober DA, Johnson D, Statnick MA, Gadski RA, Gehlert DR, Larhammar D. Cloning of a human receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 1995;270:2912329128. Boldman K, Kriese LA, Van Vleck LD, Kachmen SD. A manual for use of MTDFREML, 1993. 1-115. USDA. Washington DC. Streelman JT, Kocher TD. Microsatellite variation associated with prolactin expression and growth of salt-challenged tilapia. Phisiol. Genomics. 2002;9:1 - 4. Hale CS, Herring WO, Shibuya H, Lucy MC, Lubahn DB, Keisler DH, Johnson GS. Decreased growth in Angus steers with a short TG-microsatellite allele in the P1 promoter of the growth hormone receptor gene. J. Anim. Sci. 2000;78:2099-104. Curi RA, de Oliveira HN, Silveia AC, Lopes C. Effect of polymorphic microsatellites in the regulatory region of IGF1 and GHR on growth and carcass traits in beef cattle. Anim. Genet. 2005;36:58-62. Shimajiri S. Arima N, Tanimoto A, Murata Y, Hamada T, Wang KY, Sasaguri Y. Shortend microsatellite d(CA)21 sequence down-regulates promoter activity of matrix metalloproteinase 9 gene. FEBS Letters. 1999;455:70-74. Li YC, Korol AB, Fahima T, Nevo E. Microsatellites within genes: structure, function, and evolution. Mol. Biol. Evol. 2004;21: 991-1007. McClelland S, Shrivastava R. Medh JD. Regulation of translational efficiency by disparate 5’-UTRs of PPARγsplice variants. PPAR Research Article ID 193413, 8 pages. 2009; doi:10.1155/2009/193413 Kozak M. Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene. 2005;361:13-37. Hofacker IL. Vienna RNA secondary structure server. Nucleic Acids Reserch. 2003;31:3427-3431. Komatsu M, Nishio M, Satoh M, Senda M, Hirooka H. Potential profitability increase of Japanese Black cattle (Wagyu) by raising marbling and carcass weight through planned mating based on DNA testing of QTL alleles. Nihon Chikusan Gakkaiho. 2009;80:157-169.

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In: Ghrelin Editors: H. Yamada and K. Takahashi

ISBN: 978-1-61942-400-5 © 2012 Nova Science Publishers, Inc.

Chapter II

The Role of the Pro-Ghrelin Derived Peptides in the Iris Muscle Regulation: Implications in Glaucoma Pathophysiology Sara Azevedo-Pinto1, Marta Tavares-Silva1, Paulo Pereira-Silva1, A. Leite-Moreira1 and A. Rocha- Sousa1,2,* 1

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Department of Physiology and Thoracic Surgery; Faculty of Medicine, University of Porto; Portugal 2 Department of Ophthalmology; Faculty of Medicine; University of Porto, Portugal

Abstract Ghrelin is a 28 aminoacid peptide first described in the rat’s stomach oxyntic musosa in 1999 by Kojima et al. This peptide derives from the pro-ghrelin and is the endogenous ligand of the growth hormone secretagogues receptor-type 1a (GHSR-1a), being ghrelin’s acylation in the serine 3 residue essential for this linkage. There is also a non acylated ghrelin form, des-octanoil-ghrelin, which does not bind GHSR-1a and represents 90% of the circulating hormone. Ghrelin exerts its actions through different subcellular pathways, being the GHSR-1a related to the IP3-DAG pathway. Besides promoting growth hormone release from the pituitary, ghrelin exerts its actions in several organ systems, namely the endocrine, cardiovascular, musculo-skeletal and the eye, among others. Nowadays, it is accepted that there are other receptors responsible for ghrelin’s action than GHSR-1a. Through an alternative splicing method, pro-ghrelin may also originate another peptide, called obestatin. Obestatin, a 23 aminoacid peptide, was first isolated through bioinformatic techniques, being subsequently described in both rat’s and human

*

Correspondence: Amândio Rocha Sousa, MD PhD. Department of Physiology and Thoracic Surgery; Faculty of Medicine, University of Porto. Al Prof. Hernani Monteiro, 4200-319 Porto, Portugal.

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Sara Azevedo-Pinto, Marta Tavares-Silva, Paulo Pereira-Silva et al. stomach. This peptide has been reported to exert actions opposite to those of ghrelin in several systems, such as the endocrine system. All these pro-ghrelin derived peptides have been proven to exert significant effects in the several components of the ocular tissue. The ghrelin-obestatin system has recently been described to exert an active role on the kinetics of the iris sphincter muscle. On the one hand, ghrelin promotes the decrease of the muscle’s tension, either the actively developed after carbachol pre-contraction, or the basal one. This relaxing effect is not species dependent, being independent from GHSR-1a and from nitric oxide and dependent on prostaglandins’ production. On the other hand, obestatin showed to potentiate the iris’ sphincter muscle cholinergic contraction, although when the muscle stimulation was achieved through electrical field stimulation it seemed to induce a tendency of the developed tension to decrease. The other ocular muscle studied was the iris dilator muscle, a smooth muscle also presenting several pathways of neurohumoral regulation. In this muscle, ghrelin was reported to decrease the norepinephrine induced muscular contraction, through a mechanism dependent on GHSR-1a. Concerning obestatin, it decreased the iris’ dilator muscle basal tension, but showed no effect on the epinephrine induced active tension. The anterior segment of the eye is also influenced by the pro-ghrelin derived peptides. Ghrelin’s mRNA has been observed in the iris posterior segment and in the non pigmented cilliary epithelium. The interaction between this system and the anterior segment has also been reported in glaucoma, an optic nerve damage which presents as the main cause an increase in intra-ocular pressure. Both open angle and pseudoexfoliation glaucoma were associated with increased aqueous humor ghrelin levels, which may imply these peptides in the patophisiology of the disease. Concerning other effects in the eye, obestatin was shown to promote the proliferation of the retinal pigmented epithelium cells, through a pathway dependent on the activation of ERK ½, while ghrelin was implicated in the retina neovascular process.

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Keywords: Ghrelin, GHSR-1a, iris muscle, glaucoma, eye, ocular neovas-cularization

Introduction Ghrelin is an acylated 28 aminoacid peptide that was first discovered by Kojima et al. in 1999 in the rat’s gastric mucosa [1] . It presents with a fatty acid (n-octanoic acid) in the serine 3 residue which, along with the first five aminoacids of the molecule, is essential for ghrelin’s linkage to its receptor, the growth hormone secretagogues receptor type 1a (GHSR1a) [2]. Ghrelin is the GHSR-1a endogenous ligand, promoting growth hormone’s release from the pituitary [1]. The gene encoding ghrelin’s precursor is located on chromosome 3 (3p25-26) [3]. It is composed of 5 exons and 4 introns and leads to the formation of preproghrelin, a 117 aminoacid peptide with 82% homology between species [4]. Preproghrelin is subsequently converted into pro-ghrelin [1]. Through a process of alternative splicing, this gene will originate three different peptides: ghrelin; des-Gln-ghrelin, a 27 aminoacid peptide with the same active potency as ghrelin [5]; and obestatin, a 23 aminoacid peptide primarily found in the human and mouse stomach [6]. After its synthesis, pro-ghrelin is acylated on serine 3 residue, through a post-translational process, a reaction mediated by the enzyme ghrelin octanoyl-acyl transferase (GOAT) [7,8]. Afterwards pro-ghrelin is transported to the Golgi apparatus, where it is cleaved into ghrelin by the prohormone convertase 1/3 [8, 9] . Another form of ghrelin is des-acyl ghrelin, which lacks the n-octanoic acid on the serine 3 residue

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and represents about 90% of the total circulating ghrelin [2, 5,10]. Plasma concentration of acyl-ghrelin is 10-20 fmol/ml, being the total ghrelin concentration of 100-150 fmol/ml (including both acylated and non-acylated forms) [11,12](Figure 1). Concerning its production sites, two thirds of the total ghrelin are produced in the X/A cells of the gastric oxyntic mucosa [13]. The remaining one third is produced by several organs and tissues, namely the intestinal X/A cells, pancreas, kidney, placenta, endometrium, lymphatics, gonads, adrenal glands, thyroid gland, heart, lungs, eye, pituitary gland, hypothalamus, B and T cells and neutrophils [1,14,15,13, 16], and, during the gestational period, in the morula, blastocyst and embryo [17]. It was observed that in some tissues the expression of ghrelin’s gene and its protein were dissociated [18].

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?

?

Figure 1. Products originated from ghrelin’s gene ? through alternative splicing processes.

Once released into the bloodstream, ghrelin circulates associated with triglyceride rich lipoproteins (TRL), high density lipoproteins (HDL), very high density lipoproteins (VHDL) and, to some extent, with low density lipoproteins (LDL), while des-acyl ghrelin circulates as a free peptide in plasma [10,19, 20]. As mentioned above, ghrelin exerts its action through the activation of the GHSR-1a. This receptor was first identified in the pituitary gland and hypothalamus and is distinct from the growth hormone releasing hormone (GHRH) receptor [21, 11, 22]. GHSR-1a is a 366 aminoacid receptor that belongs to the G protein coupled receptors superfamily, having the characteristic seven transmembrane domains [11, 21]. It is encoded by a gene located in chromosome 3q26.2 that, through an alternative splicing process, may also originate a second receptor sub-type, GHSR-1b.This latter sub-type is smaller (289 aminoacids) and only presents with five transmembrane domains [21]. Although GHSR-1b has not been attributed any biological function yet, it has already been identified in several endocrine and non endocrine tissues, such as the heart, thyroid, pancreas, spleen, and adrenal gland [14,23]. It was shown, using the reverse transcriptase – polymerase chain reaction procedure, that GHSR-1b mRNA levels are usually higher than GHSR-1a levels in normal human tissues [11, 24]. Moreover, it was proposed that GHSR-1b may function as a modulator, more precisely a negative regulator, of GHSR-1a activity [25,26].

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Sara Azevedo-Pinto, Marta Tavares-Silva, Paulo Pereira-Silva et al.

Figure 2. Intracellular pathways involved in ghrelin’s effects. (a. Ghrelin binds to the GHSR-1a, a Gq protein coupled receptor, leading to the activation of phospholipase C (PLC). This enzyme converts phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,4,5-inositol triphosphate (IP3) and diacylglyceride (DAG). IP3 promotes Ca2+ release from the sarcoplasmatic reticulum; DAG inhibits K+ channels, inducing the opening of voltage-dependent L-type Ca2+ channels in the cellular membrane. The increase of intracellular Ca2+ results in membrane depolarization. b. Ghrelin activates a tyrosine kinase receptor, leading to the activation of the Ras protein. The double phosphorylation of the Ras protein results in the mitogen-activated protein kinase (MAPK), which needs another phosphorylation to enter the nucleus and regulate cell proliferation. Ghrelin also binds to an unknown receptor which activates ERK1 and ERK2 and Akt/PI3K, resulting in an antiapoptotic effect. c. In the endothelial cells ghrelin stimulates a G-protein-coupled system which activates the guanylate cyclase (GC). This enzyme transforms guanidil triphosphate (GTP) into cyclic guanidil monophosphate (cGMP), that leads to the activation of nitric oxide synthase (NOS), increasing nitric oxide’s (NO) levels. NO then promotes relaxation of the smooth muscle cell by entering it. d. In adipocytes ghrelin binds GHSR-1a and stimulates the peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that regulates genetic transcription and promotes adipogenesis).

Focusing now on the GHSR-1a, the biologically active form of the receptor, it is constitutively active [27] and has two binding sites in its structure: one that is located in the transmembrane domain number 3 and binds ghrelin, as well as other peptide and non peptide synthetic agonists [2, 28]; and another that was proposed to bind adenosine [29]. In the somatotrophes, the activation of the GHSR-1a stimulates the enzyme phospholipase C (PLC), which promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglyceride (DAG) and inositol triphosphate (IP3). IP3 subsequently binds its receptor on the sarcoplasmic reticulum, promoting a transient increase in the intracellular calcium concentration. DAG activates the protein kinase C and promotes the inhibition of potassium channels, leading to the membrane depolarization and posterior opening of voltage dependent

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calcium channels. This process generates a persistent elevation of intracellular calcium levels [30]. Nevertheless, this is not the only subcellular pathway activated by ghrelin. This hormone is also able to stimulate the AMP activated protein kinase [31], the mitogen activated protein kinases (MAPKs) pathway, in particular MAPK p44/p42, also known as the extracellular signal-regulated protein kinases (ERK 1/2) [32-34], the transcriptional factor Elk 1 [33], the Akt/PI3K pathway [32] and tyrosine kinases pathways [34] all routes through which it promotes cellular proliferation. Moreover, it appears to inhibit several inflammatory and pro-apoptotic pathways [35]. Ghrelin also stimulates the activation of the nitric oxide/cGMP pathway, leading to vasodilation [36], as well as the increase of the peroxisomeproliferator activated receptor gamma 2 (PPARγ2) in differentiated adipocytes, promoting adipogenesis through a GHSR-1a dependent pathway [37] (Figure 2). GHSR-1a, like ghrelin, is also widely expressed all over human organ and tissues, although the two distributions do not perfectly overlap [14, 34]. It is markedly expressed in the pituitary gland and in the hypothalamus’ arcuate nucleus [21], being also present in other areas of the central nervous system and in peripheral tissues, such as the thyroid gland, pancreas, spleen, myocardium, adrenal glands [14, 18, 38, 39], testis, ovaries [40], T lymphocytes [15], and the human embryo [17]. Since other ghrelin forms, namely the non acylated isoforms, also express biological activity and ghrelin’s and GHSR-1a’s sites of expression do not perfectly overlap [14], there must be other receptors responsible for this hormone’s actions [14, 41,42]. Moreover, there have been described receptors that bind both ghrelin and des-acyl ghrelin [43]. One of the receptors described to be indirectly involved in ghrelin’s actions is CD36, a multifunctional scavenger receptor expressed in the adipose tissue, platelets, monocytes, macrophages, dendritic cells, microvascular endothelium and myocardium and stimulated by a synthetic ghrelin’s analog, hexaghrelin [44]. Ghrelin has been shown to act in several organs and tissues. The endocrine system is particularly affected by ghrelin’s actions, due to its endocrine and metabolic effects. Ghrelin promotes growth hormone’s release from the pituitary, both independent [30, 45, 46] and dependently [47, 48] of the GHRH. Besides this effect, ghrelin acts as an orexigenic [49,50,51, 52,53] mediator and interferes with the carbohydrates’ and lipid’s metabolism [54, 55, 56]. Concerning the muscular tissue, all three cardiac, smooth and skeletal muscles were described to be affected by ghrelin. In the heart, this hormone has negative inotropic and lusitropic effects [42,57]. The negative inotropic effect occurs as a response to ghrelin, desGln14-ghrelin and des-acyl ghrelin, being more pronounced in the presence of the latter [42]. Des-acyl ghrelin’s effect is modulated by the endothelial cells’ cyclooxygenase, due to the production of prostaglandin I2, and dependent on the endocardiac endothelium, while ghrelin’s effect is not related to these substances, being dependent on calcium activated potassium channels and independent from GHSR-1a [42, 57]. These potassium channels also participate in the negative lusitropic effect generated by ghrelin [57]. This hormone also decreases the afterload and increases cardiac output without altering the cardiac frequency [58]; increases coronary perfusion pressure and the blood flow on its microvasculature [59] and inhibits the cytokines produced after an increase in nitric oxide bioavailability, ameliorating endothelial function [60]. Recently it was reported by Zhang et al. that ghrelin might protect the heart from disease induced by oxidative stress, since it prevented H9c2 cardiac myocytes’ apoptosis. Ghrelin induces the vascular smooth muscle relaxation, which is

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translated into a potent hipotensive effect, dependent on calcium activated potassium channels and associated with a decrease in the nitric oxide’s bioavailability. Both ghrelin and GHSR-1a are expressed in the microvascular endothelial cells, promoting its proliferation, migration and angiogenesis in a process dependent on ERK2. In the skeletal muscle, ghrelin increases the permeability to chloride, which leads to a decrease in membrane’s potential [61] (Figure 3). Ghrelin’s actions also extend to the gastrointestinal tract [62-64], the reproductive system, in both male and female [65], the immune system [15, 60, 66-69], the bone [70-73] and in other areas of the central nervous system [58, 74-76]. Obestatin is a 23 aminoacid peptide that derives from pre-pro-ghrelin. It was first discovered through bioinformatic techniques, having subsequently been isolated from rat’s and human stomach [6]. Obestatin was described as activator of the orphan receptor GPR-39, which belongs to the G protein coupled receptors superfamily [6, 77]. Several obestatin binding sites have been defined, both in rat and in man, namely the stomach, intestine, pituitary and hypothalamus [6]. Besides these sites, GPR-39 mRNA was also markedly identified in other regions of the central nervous system, such as the amygdala, the hypocampus and the auditive cortex [78]. So far, obestatin has been attributed several different effects. Among these are metabolic regulation, since it reduced body weight, although obestatin levels have shown no variation either with satiety, or with fasting periods [6, 79]. Also the gastrointestinal tract appears to be influenced by this hormone, showing, for example, a decrease in the contractility of jejunum smooth muscle [6]. Further studies raised some concerns about obestatin actions and binding to GPR-39 [80, 81]. After this introduction to the systemic actions of ghrelin, des-acyl ghrelin and obestatin, the aim of this article is to review the effects of these three substances in the eye, concerning its different components.

Figure 3. Ghrelin’s actions in the three muscular tissues. a. In skeletal muscle ghrelin increases membrane permeability to chloride, decreasing its potential. b. In cardiac muscle, ghrelin exerts both negative inotropic and lusitropic effects. The inotropic effect is mediated by PGI2 and by calcium dependent potassium channels. It also decreases apoptosis through the inhibition of NF-КB, H2O2 induced Bax activation, increase in Bcl-2 and restoring of mitochondrial membrane’s potential. c. In vascular smooth muscle, ghrelin activates calcium dependent potassium channels, leading to muscular relaxation and subsequent vasodilation.

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Figure 4. Neurohumoral pathways regulating iris’ sphincter muscle contraction. ET-1 – endothelin 1; ETB – endothelin receptor type B; Pg – prostaglandin; Tx – tromboxane; PRGC – calcitonin gene related peptide; PAAC –pituitary adenilate cyclase activator peptide; CCK-cholecystokinin; ATII – angiotensin II; NK2,3 – Neurokinins’ receptor type 2 or 3; AT? – angiotensin receptor unknown; PLC – phospholipase C; PIP2 – phosphatidylinositol 4,5-bisphosphate; IP3 –inositol trisphosphate; DAG – diacylglicerol; AA – araquidonic acid.

Until this date, the ghrelin system, along with obestatin, has been described to be produced by or involved in the function some of the components of the uveal tract, namely the iris and the ciliary body, as well as in the production and drainage of the aqueous humour. Its local production by the eye has also been described, either during development, or even during adulthood.

Iris Sphincter Muscle The iris sphincter muscle is one of the three intra-ocular muscles that exist in the anterior portion of the uvea. It is a smooth muscle and is located in a peripupilar ring, with about 0,751 mm in radius. In this muscle, the parasympathetic nervous system is the main source of innervation, exerting its action through the M3 muscarinic receptors [82], the most common type in human’s iris sphincter muscle, followed by M1 and M5 types [83]. All in all, it also presents some innervation by the sympathetic nervous system [84]. Other systems that participate in the regulation of the kinetics of the iris sphincter muscle are the trigeminal system, through substance P [85, 86]; and the non-adrenergic non-cholinergic system, which comprehends a wide range of mediators, such as the pituitary adenylate cyclase activator [87, 88]; calcitonin gene related peptide [88] neurokinines [89]. Besides this neuronal mediated

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regulation of the muscular kinetics, there are substances like bradykinin [90, 91], prostaglandins F2α, E2 and D2, tromboxane A2 [92], endothelin 1 [93] and angiotensin II [92] that were shown to play a role in the iris’ sphincter muscle kinetics. All these mechanisms and substances, as well as luminous stimulation [94], lead to muscular contraction (Figure 4). The iris’ sphincter muscle relaxation depends on an increase in intracellular cAMP and cGMP levels or on the inhibition of acetylcholine release from the synaptic cleft [95-97]. Systems like the sympathetic nervous system [98, 99] or the vasoactive intestinal peptide [100, 101] lead to increase of cAMP intracellular levels, whilst nitric oxide [102] and natriuretic peptides A and C [97] promote an increase in intracellular cGMP levels. Adrenomedullin is able to increase either cAMP or cGMP intracellular levels, the latter depending on nitric oxide production [103, 104]. Adenosine was shown to inhibit acetylcholine’s release from the synaptic cleft, despite its ability to promote a non-adrenergic non-cholinergic contraction of the iris’ sphincter muscle [105]. Also galanin and somatostatin were suggested to promote a pre-junctional inhibition of acetylcholine’s release [106] (Figure 5). The ghrelin-obestatin system has recently been described as another modulator of the kinetics of the iris’ sphincter muscle [16, 107]. Ghrelin was shown to induce the relaxation of the rabbit’s iris’ sphincter muscle. When applied in a single dose with a concentration of 6 x 10-5 M in an iris’ sphincter muscle precontracted with charbacol, ghrelin promotes a decrease in active tension from 3.45±0.47 mN to 1.27±0.76 mN in the first 1.5-3 minutes, with recovery until the initial active tension in the following 10 minutes.

Figure 5. Neurohumoral pathways regulating iris’ sphincter muscle relaxation. VIP – vasointestinal peptide; P Sub – P substance; ANP – auricular natriuretic peptide; CNP – cerebral natriuretic peptide; NPA – natriuretic peptides receptor type A; ET – endothelin; ETB – endothelin receptor type B; Pg – prostaglandin; Tx – tromboxane; NO – nitric oxide; Gc – guanilate cyclase; cGMP – cyclic GMP; Ac – adenylate cyclase.

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When analyzing the effect of increasing doses, it was shown that concentrations of 10-5 M, 3 x 10-5 M and 6 x 10-5 M lead to percentual decrease of active tension of 10.7±5.6%, 27.2±8.9% and 34.1±12.1% respectively [16]. When the contraction was mediated by electrical field stimulation, and not by charbacol, it was demonstrated that ghrelin decreased by 9.3±3.2 % the active tension developed in a second stimulation, when compared to a first stimulation done without adding any substance [107] (Figure 6). Ghrelin also decreases iris’ sphincter muscle basal tension, since in a muscle without any type of pre-stimulation, this hormone at the concentration of 10-5 M led to a decrease of 21.7±3.7% in basal tension [16, 107]. The relaxing effect of ghrelin over the iris’ sphincter muscle is not mediated by the GHSR-1a. This conclusion was based in two experimental procedures. The first one consisted in the addition of increasing doses of des-acyl ghrelin, a form that does not bind GHSR-1a, to charbacol pre-contracted muscles. Thus, the addition of this substance in 10-5 M, 3 x 10-5 M and 6 x 10-5 M concentrations led to a percentual decrease in active tension of 10.6±3.8%, 20.0±6.1% and 43.3±5.2%, respectively. In the second experimental protocol, Lys3GHRP6, a GHSR-1a antagonist, was used to block the receptor. After adding this substance to a charbacol pre-contracted iris’ sphincter muscle, increasing ghrelin doses (10-5 M, 3 x 10-5 M and 6 x 10-5 M) were tested. These concentrations led to a percentual tensional decrease of 49.9±12.5%, 94.2±19.4% and 118.6±21.1% respectively. Based on these latter values, it is possible that GHSR-1a works as a modulator of ghrelin’s effect, since its blockage seems to potentiate this hormone’s action [16].

% Maximal decrease in active tension

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0 -20 -40 -60

*

*

-80 -100 -120 -140

* Ghr

D-Ghr

Ghr + DLys3-GHRP6

Figure 6. Percentual decrease in active tension developed in charbacol precontraced iris’ sphincter muscles. Ghr – in the presence of ghrelin; D-Ghr – in the presence of des-acyl ghrelin; Ghr – Dlys3GHRP6 – in the presence of ghrelin plus the GHSR-1a inhibitor DLys3-GHRP6. *p