Eicosanoids in Invertebrate Signal Transduction Systems [Core Textbook ed.] 9781400865055

Table of contents :
Contents
Foreword
Acknowledgments
Chapter 1. Introduction: A Theory of the Biological Significance of Eicosanoids
Chapter 2. Eicosanoid Structures and Biosynthesis 11 The Mammalian Model of Eicosanoid Biosynthesis
Chapter 3. Polyunsaturated Fatty Acids
Chapter 4. Eicosanoids in the Reproductive Biology of Invertebrates
Chapter 5. Eicosanoids in Invertebrate Immunity
Chapter 6. Eicosanoids in Invertebrate Ion Transport Physiology
Chapter 7. Emerging Eicosanoid Actions
Chapter 8. Eicosanoids Mediate Ecological Interactions
Chapter 9. A Research Prospectus: Approaching the Frontiers
Abbreviations Used in References
References
Taxonomic Index
Subject Index

Citation preview

EICOSANOIDS IN INVERTEBRATE SIGNAL TRANSDUCTION SYSTEMS

EICOSANOIDS IN INVERTEBRATE SIGNAL TRANSDUCTION SYSTEMS

David W. Stanley

PRINCETON

UNIVERSITY

PRESS

PRINCETON,

NEW

JERSEY

Copyright © 2000 by Princeton University Press Published by Princeton University Press, 41 William Street, Princeton, New Jersey 08540 In the United Kingdom: Princeton University Press, Chichester, West Sussex All Rights Reserved

Library of Congress Cataloging-in-PublicaHon Data Stanley, David W. (David Warren), 1946Eicosanoids in invertebrate signal transduction systems / David W. Stanley p.

cm.

Includes bibliographical references and index. ISBN 0-691-00660-1 (cloth : alk. paper) 1. Eicosanoids—Physiological effect. transduction.

2. Cellular signal

3. Invertebrates—Physiology. QP752.E53S73 572'.57—dc21

I. Title.

2000 99-25842

This book has been composed in Times Roman The paper used m this publication meets the minimum requirements of ANSI/NISO Z39.48-1992 (R1997)

(Permanence of Paper) http://pup.princeton.edu Printed in the United States of America 1

3

5

7

9

1 0

8

6

4

2

This one is for Terri (the girl-child), wife, wise friend; and for Alec (the boy-child), my early morning companion.

Contents Foreword by Ralph W. Howard

ix

Acknowledgments

xi

Chapter 1. Introduction: A Theory of the Biological Significance of Eicosanoids

3

Chapter 2. Eicosanoid Structures and Biosynthesis The Mammalian Model of Eicosanoid Biosynthesis Chapter 3. Polyunsaturated Fatty Acids Essential Fatty Acids C20 Polyunsaturated Fatty Acids in Insects The Complete Biosynthesis of 18:2n-6 in Insects Biosynthesis of C20 Polyunsaturated Fatty Acids Patterns of Polyunsaturated Fatty Acid Metabolism in Insects Chapter 4. Eicosanoids in the Reproductive Biology of Invertebrates Eicosanoids in Insect Reproduction Eicosanoids as the Barnacle Hatching Factor Eicosanoids in Scallop Reproduction Prostaglandins in Crayfish Vitellogenesis Prawns Eicosanoids in the Reproduction of Molluscs Prostaglandins Influence Organismal-Level Events through Their Actions on Cells in the CNS Eicosanoid Actions at the Cellular Level Chapter 5. Eicosanoids in Invertebrate Immunity Eicosanoids in Insect Cellular Immune Reactions to Bacterial Infections Eicosanoids Mediate Clearance of Injected Bacteria from Insect HemolymphCirculation Eicosanoids Mediate Nodulation Reactions to Bacterial Infections Hypothesis: Eieosanoids Mediate Nodulation Reactions to Bacterial Infections in Most, If Not All, Insect Species The Biochemistry of Eicosanoid Systems in Immune Tissues

11 12 34 34 39 40 43 50 55 55 74 79 82

86 87 92 94 109 111 112 117 124 133

viii

CONTENTS

Chapter 6. Eicosanoids in Invertebrate Ion Transport Physiology

152

Chapter 7. Emerging Eicosanoid Actions

173

Eicosanoids in Invertebrate Temperature Biology Eicosanoids in Insect Peptide Hormone Signal Transduction Eicosanoids in Development and Regeneration in Hydroids Chapter 8. Eicosanoids Mediate Ecological Interactions

173 178 183 188

Eicosanoids in Predator Avoidance Eicosanoids in Host-Parasite Interactions Prostaglandin Biosynthesis Inhibitors in Insect Defensive Secretions: Do these Compounds Act in Insect Chemical Ecology?

188 194

231

Chapter 9. A Research Prospectus: Approaching the Frontiers

235

How Do Eicosanoids Work? Understanding Departures from the Mammalian Background The Enzymes Associated with Eicosanoids Eicosanoids and Neurobiology The Molecular Biology of Eicosanoids An Epilogue

236 237 239 241 243 244

Abbreviations Used in References

245

References

249

Taxonomic Index

273

Subject Index

275

Foreword

THE PACE of discovery of new knowledge in the class of biochemicals known as eicosanoids has increased at a rapid rate since von Euler's seminal finding in 1936 that the human prostate gland contains a factor (prostaglan­ dins) that stimulates the contraction of smooth muscle. Indeed, the medicinal and pharmaceutical literature on prostaglandins and other eicosanoids is now so vast that no single human could hope to absorb all of it. The overwhelm­ ing majority of this literature is focused on human and rat models, and con­ siderable understanding has been achieved on how eicosanoids are synthe­ sized and function in these models. An impressive array of eicosanoids has been discovered, including prostaglandins, lipoxins, and leukotrienes. The enzymes responsible for producing these chemicals have been characterized (including X-ray structures), and the modes of action of many eicosanoid biosynthesis inhibitors and eicosanoid receptor antagonists have been estab­ lished. In the last ten years, substantial progress has occurred in characteriz­ ing eicosanoid receptors. The molecular biology and genetic characterization of these systems are also now well under way. This work has already exerted a major impact on the medicinal and pharmaceutical industries. Humans and rats, however, constitute just a tiny fraction of the life forms on this planet. Although a substantial amount of research was generated by the 1969 report that some corals possess remarkable quantities of eico­ sanoids, almost all of this research was directed toward the economic exploi­ tation of the corals rather than toward efforts to understand why the corals had these chemicals. A few biologists, however, did begin to ask themselves such questions and to wonder whether a broader diversity of life might also be using eicosanoids in its biology. Slowly, but surely, it has now been dem­ onstrated that virtually all life forms contain eicosanoids or closely related compounds, and that these compounds do indeed exert important biochemi­ cal, physiological, behavioral, and ecological effects in every system that has been appropriately examined. This book provides the first comprehensive overview and synthesis of the biological importance of eicosanoids in repre­ sentatives of the invertebrate phyla. The focus of this volume is the development of Professor David W. Stan­ ley's "biological paradigm" regarding the biological role of eicosanoids in the evolution of life. Under this model, he postulated that eicosanoids (or closely related biochemicals) were drawn into various roles as signal trans­ duction moieties very early in cellular life. As these early cellular life forms evolved into more complex metazoan organisms, individual cells were al­ ready adapted to use eicosanoids as modulators of events. The number and

X

F O R E W O R D

variety of eicosanoid roles continued to increase throughout all stages of evolution, and as Professor Stanley notes throughout the volume, our under­ standing of many observed biological phenomena is facilitated by the con­ tinuing discovery and understanding of eicosanoid actions in life forms at every stage of phylogenetic history. In agreement with the diversity of life forms, there is an equally diverse collection of eicosanoids and functional roles for these eicosanoids in the taxa that have been examined to date. Although it is tempting to overlay these many eicosanoids and roles over our current understandings of phylogenetic relationships, not enough data have been gathered to do so confidently. Professor Stanley's book does, however, lay an excellent foundation for further efforts in this direction. The chemistry and biochemistry of eicosanoids are complex, and the first few chapters of this volume present the necessary background material for understanding the remaining biological material in the book. In this section, as throughout the volume, extensive details are provided on the actual exper­ iments carried out by various investigators, and critical comments are made on the strengths and weaknesses of the experiments. Subsequent chapters deal with the roles of eicosanoids in reproductive biology, immunity, ion and water transport, and a handful of other physiological functions. A detailed chapter deals with ecological and behavioral functions of eicosanoids. A comparative phylogenetic approach is taken in every case, allowing the reader to see how, quite often, very different organisms have used similar eicosanoids to accomplish similar physiological or ecological functions. Equally important are the cases where this is not true, as they help to shed additional light on the evolutionary history of eicosanoid function. Professor Stanley ends this book with an intriguing chapter on his vision of the future of this research field from both applied and basic outlooks. Although our understanding of eicosanoids in invertebrates lags far behind the mammalian literature, the gap is closing at an ever-increasing pace. In­ deed, it is likely that within a few years, it will no longer be possible for a single author to authoritatively and critically survey the entire field as Pro­ fessor Stanley has done in this volume.

Ralph W. Howard October 1998

Acknowledgments

WRITING a book on an emerging area of scientific inquiry is much more a community activity than the solitary work of a lone writer. I am pleased to offer many thanks to colleagues for encouragement, photographs, sharing new findings, and reading parts of this volume. These individuals include Gary Blomquist, Matt Bentley, Tony Clare, Vincenzo Di Marzo, Elizabeth Hill, Gertrude Hirsch, Wemer Loher, Laurent Meijer, Eric Spazanini, John Steele, and Peter Weller. Special thanks to my friend and colleague, Ralph Howard, for several readings and very constructive criticism, for producing all the chemical structures, and for an Italian dinner with a decent bottle of wine. In addition to their scientific research discussed in various chapters, my graduate students contributed many hours to this project. Rico Rana pro­ vided a final editing, Wyatt Hoback constructed figures, Jon Miller contrib­ uted photographs and figures, Jon Bedick made about a thousand trips to the libraries and once again lent me his artist's hand, Hasan Tunaz and Nor Aliza read chapters. Like most book projects, this one benefits from un­ wavering support from wife and child. Of course writing a book is only part of the process of producing one. Special thanks to Denise Pasternak and Emily Raabe for helping launch this project at Princeton University Press. Thanks also to Deborah Wenger for deft editing.

EICOSANOIDS IN INVERTEBRATE SIGNAL TRANSDUCTION SYSTEMS

CHAPTER 1

Introduction: A Theory of the Biological Significance of Eicosanoids

EICOSANOID is the most general term for all biologically active, oxygenated

metabolites of three C20 polyunsaturated fatty acids, namely 20:3n-6, 20:4n-6, and 20:5n-3. The term was originally coined by Corey et al. (1980) based on the Greek root for twenty. The nomenclature and structures of these acids and their eicosanoid products are detailed in chapters 2 and 3. There are three main groups of eicosanoids: the prostaglandins, the epoxyeicosatrienoic acids, and the lipoxygenase products, including leukotrienes and an array of other compounds. Most of us are probably familiar with prosta­ glandins because they mediate some reactions associated with injury and discomfort. For example, the swelling and inflammation associated with minor injuries are mediated by certain prostaglandins. Aspirin and other an­ algesics used to relieve these symptoms function by inhibiting prostaglandin biosynthesis. With a view to introducing the goal of this book, let us begin with a few background points. First, the study of eicosanoids is deeply rooted in mam­ malian systems. Second, eicosanoids offer powerful insights and are very important clinical tools in human and animal medicine. Third, due to their importance to humans, the literature on eicosanoids in mammalian systems dwarfs the corresponding literature on invertebrates. What follows from these points is a human, or perhaps mammalian-centered, understanding of the significance of eicosanoids. The purpose of this volume is to suggest another, far wider theory on the biological significance of eicosanoids, antic­ ipated in the early reviews of Stanley-Samuelson (1987, 1991). Stanley and Howard (1998) called this theory the "biological paradigm" of eicosanoids. Under this model, we recognize that eicosanoids were drawn into various roles as signal transduction moieties very early in cellular life. As cellular life forms evolved into more complex metazoan animals, the individual cells involved already had considerable experience in the use of eicosanoids as modulators of events. Coordination and communication among cells in meta­ zoan life, of course, is far more complicated than in unicellular life, and eicosanoids were available for recruitment into new biological roles. The numbers and types of roles continued to increase throughout all stages of animal evolution. Under the biological paradigm, we look beyond the wellestablished picture of eicosanoids in mammalian systems to appreciate the

4

CHAPTER 1

many and quite varied eicosanoid actions in all animals. The lengthy history of animal evolution created many opportunities for selecting new, sometimes very subtle, eicosanoid actions. Because eicosanoids were recruited into many biological roles, we also recognize the explanatory power of these compounds. By "explanatory power," we convey the idea that new informa­ tion on eicosanoids will provide important insights into biological systems. Appreciating the biological paradigm of eicosanoids effectively turns the traditional, mammalian-centered approach to physiological and related re­ search inside-out. We can imagine a dichotomy. In a traditional approach to physiology, people study a system, with an interest in discovering which molecules are responsible for regulating events within the system. On the other side of the dichotomy, the biological paradigm offers an alternative approach. Instead of discovering eicosanoid actions through detailed re­ search into specific physiological and pathophysiological phenomena, we can gain tremendous new insights into animal processes from research aimed at discovering and understanding eicosanoid actions. For an example, in chapter 5 we will review work designed to reveal eicosanoid actions in in­ vertebrate immunity. This work yielded the first insights into some of the biochemical events that operate between bacterial infections and cellular re­ actions to the infections in insects. Let us view the biological paradigm through the lens of a brief history of our understanding of eicosanoids. Serious students of the history of science typically approach their schol­ arship through one of three routes. One is the sociology of scientific knowl­ edge, which tries to understand the social structures and processes within the scientific community. A second focuses on the cultural meaning of science within local cultures, and a third approach is concerned with the intellectual content of science within a historical context. The history of any area of science is necessarily a complex matrix. This is especially true for scientific discovery in the twentieth century, during which more than 90 percent of all the world's scientists have lived out their careers. We might believe, as prac­ titioners of science, that a history of any area of science would be treated best by scientists. For a number of good reasons, scientists often are not the best historians of science, and a sophisticated, scholarly history of eico­ sanoids probably should be left to the specialized training and perspectives of historians. The purpose of this brief history is less ambitious than professional histor­ ical research. Instead, my goal is to provide a perspective on the biological paradigm and a context for comprehending the state of our knowledge of eicosanoids in invertebrates. Inquiry into the biologic roles, biochemistry, and chemistry of eicosanoids is less than seventy years old, and the modern phases are much younger. The study of eicosanoids began with investiga­ tions in human reproduction, and only secondarily and much later expanded

S I G N I F I C A N C E O F E I C O S A N O I D S

5

to embrace invertebrates. And we will see that the first work on invertebrates had more to do with eicosanoids in humans than with zoological inquiry. We generally place the original investigations of eicosanoids in the early 1930s. Although they were not so named, prostaglandins were the first group of eicosanoids recorded in the scientific literature. The discovery process began with physiological observations. In their work on human reproductive physiology, Kurzrok and Lieb (1930) found a certain pharmacological activ­ ity in human seminal fluids. This paper probably marks the beginning of our appreciation of the clinical significance of prostaglandins. Kurzrok and Lieb were interested in the biochemistry of semen, and they developed an assay for the influence of semen on the smooth muscle of human uteri. They found that semen stimulated uterine smooth muscle contractions. Soon after this first paper, Goldblatt (1933) postulated a substance in semen responsible for causing smooth muscle contractions. In his 1936 paper, von Euler extended these findings, noting that something in the semen from humans and a few other mammals stimulates smooth muscle (he used the term "plain muscle") contractions and vasodilation. Von Euler also gave us the term "prostaglan­ din", because the substances were associated with the prostate gland. Based on the acidic taste of semen, von Euler thought the pharmacological sub­ stances were probably organic acids. An earlier paper by Jappelli and Scafa (1906) foreshadowed the phar­ macological actions of compounds from the prostate gland. These individ­ uals created aqueous extracts of canine prostates, then injected the extracts into dogs and rabbits. The extracts caused respiratory paralysis and altered heartbeat rates, in some cases killing the experimental animals. The authors concluded that the prostate produces materials of powerful pharmacological actions. These materials are various eicosanoids. These early studies, suggesting that prostaglandins were something to be reckoned with, are of greater historical than strictly scientific interest. Ad­ vances into the biological meaning of prostaglandins had to await a chemical breakthrough, which would not emerge until the early 1960s. Swedish chem­ ist Bengt Samuelsson suggested this would be a suitable problem for his student, Sune Bergstrom. In 1962, some thirty years after the first clinical study, they reported the chemical structures of three prostaglandins, PGE, PGF1, and PGF2 (Bergstrom et al. 1962). They also inferred from the struc­ tures that prostaglandins must be biosynthesized by oxygenation of C20 polyunsaturated fatty acids. These findings stimulated increased interest in prostaglandins and helped accelerate research. While not to be taken in a strictly linear sequence, because a great deal of effort intervened, another major stimulation of research into prostaglandins came from the work of a British pharmacologist, Robert Vane. In a nowclassic, and relatively simple, series of experiments, Vane demonstrated that

6

CHAPTER 1

the conversion of 20:4n-6 into prostaglandins by in vitro enzyme prepara­ tions was inhibited in a dose-dependent way by the common analgesic, aspi­ rin. Aspirin is a potent pharmaceutical product. Many symptoms of illness and injury, including fever, inflammation, swelling, and pain, are attenuated or completely relieved by aspirin. Vane's demonstration that prostaglandin biosynthesis is inhibited by aspirin laid bare the suggestion that prostaglan­ dins exert a wide range of pathophysiological influences within the human body. Samuelsson, Bergstrom, and Vane illuminated a completely new frontier of pharmaceutical research, for which they shared a Nobel prize in the early 1980s. The relationship among 20:4n-6, prostaglandins, and human health became plain, and an industrial and academic research enterprise was launched. The chemical structures of additional prostaglandins and other eicosanoids were soon determined. In the 1970s, the structures of certain hydroxyeicosatetraenoic acids and of leukotrienes were determined (Sam­ uelsson 1983), and structures of lipoxins were determined in the 1980s (Serhan 1994). The presence of eicosanoids in virtually every mammalian tissue and body fluid was progressively uncovered. In a widening array of physi­ ological experiments, additional eicosanoid actions in humans and other mammals were discovered. Eicosanoids were found to influence many organ-level and system-level phenomena, including kidney function, alimen­ tary canal physiology, and a very lengthy list of other elements in human physiology. Possibly some of the most prominent eicosanoid actions involve their influence on mammalian host-defense systems. These eicosanoid ac­ tions will be treated in greater detail in later chapters. Beside an increasing number of eicosanoids actions, as research results accumulated, the overall picture of eicosanoids became more complicated. One complication, for ex­ ample, was finding that some eicosanoid actions seem contradictory. For instance, prostaglandins stimulate contraction in some smooth muscle prepa­ rations, and inhibit contraction in others. Of course, a great deal of research was focused on nonhuman mammals, and the significance of eicosanoids in veterinary medicine emerged. While research accelerated in pace and in discovery, there remained tech­ nical barriers to gaining increased understanding of eicosanoids. Because prostaglandins are very potent biological regulators, they are naturally pro­ duced in very small amounts. It was difficult to obtain sufficient quantities of prostaglandins for physiological studies. This barrier was partly overcome through the efforts of Dr. John Pike and his colleagues at the Upjohn Com­ pany in Kalamazoo, Michigan. Following the discovery that seminal vesicles biosynthesize prostaglandins at relatively high rates, Pike developed a largescale enzymatic prostaglandin biosynthesis system. This system yielded mil­ ligram quantities of prostaglandins, which Pike provided to many research groups. Many papers in the late 1970s and early 1980s attest to the sue-

S I G N I F I C A N C E O F E I C O S A N O I D S

7

cess of his system by acknowledging him as the source of prostaglandins for research. Accurately determining the natural quantities of eicosanoids in biological sources is another daunting technical barrier. Again, because these are bio­ logically potent compounds, they typically occur in very low quantities in tissues. Moreover, in many tissues, the very act of homogenizing tissues stimulates a burst of prostaglandin biosynthesis, making accurate quantita­ tive determinations even more tenuous. Contemporary techniques to quantify eicosanoids include radioimmunoassays, combined gas chromatographymass spectrometry, interfaced liquid chromatography-mass spectrometry, and detection of fluorescent derivatives on high-performance liquid chromatogra­ phy. Compared to those used in the early days, these techniques are reliable and relatively easy. Nonetheless, all remain beset with technical problems and potentials for formation of confounding artifacts. Obtaining some, but not all, prostaglandins for biomedical research be­ came a little more practical soon after the first report of eicosanoids in an invertebrate. Analytical chemists A. J. Weinheimer and R. L. Spraggins were interested in natural products of marine origins. In their fifteenth paper on natural products in coelenterates, they reported an unusual finding. The gorgonian octacoral, Plexaura homomalla, contains high quantities of two prostaglandins within its tissues (Weinheimer and Spraggins 1969). The prostaglandins were a little different from the ones known from mammals, because one was a 15-epi-derivative of PGA2 and the other was its acetate methyl ester. More importantly, at 1.5% to 9% of dry tissue mass, the quan­ tities of these products were astonishingly high. This discovery was greeted with tremendous interest within the prostaglandin community, not because of a scholarly interest in the biology of corals, but because the coral represented a commercial opportunity to harvest prostaglandins. Coral was harvested, the prostaglandins were extracted in large quantities, and they were chemically modified into prostaglandins of biomedical impor­ tance. Of course, harvesting coral on a commercial scale brought along with it concerns for the long-term sustainability of the coral (Theodor, 1977; Berte 1981). There also followed a broad search for other natural, especially marine, sources of prostaglandins. Bundy (1985) reviewed these efforts, which turned up literally hundreds of invertebrate species whose tissues contained what we might call physiological quantities of prostaglandins. Bundy's comment that these small quantities of prostaglandins would not do anybody any good—except the animals in which they were found—ade­ quately reveals the commercial, human-centered tone of the early investiga­ tions into the presence of prostaglandins in invertebrate animals. The main thrust of this volume is about the biological significance, as opposed to the mere presence, of eicosanoids in invertebrates. Detailed list­ ings of species in which eicosanoids have been detected will not contribute

8

CHAPTER 1

much to biological theory. Nonetheless, it is useful to establish the point that eicosanoids have been detected in species representing virtually every major metazoan phylum. As a convenience, Table 1 cites the early publications reporting the presence of prostaglandins and other eicosanoids, without reference to biological studies, in invertebrates. Peered back on from our zoological perspective, the broad-ranging searches for natural sources of prostaglandins represent an early phase in moving from a strictly mammalcentered viewpoint to our biological paradigm for understanding the biology of eicosanoids. E. J. Corey, a Nobel laureate in chemistry, was recognized, not for any single success in synthesizing difficult compounds, but for introducing the idea of designing effective holistic strategies in organic synthesis. While natural product chemists were scouring the outer reaches of invertebrate taxa in search of prostaglandins, Corey turned his attention to the problem of synthesizing prostaglandins and other eicosanoids. In 1980 he and his col­ leagues reported the synthesis of 5-hydroperoxyeicosatetraenoic acid (Corey et al. 1980). This breakthrough was the first of many successes in synthesiz­ ing eicosanoids, and it effectively erased the barrier of obtaining sufficient quantities of prostaglandins and other eicosanoids for physiological and pharmacological research. Compounds that were once available only as gifts from John Pike are now routinely purchased from competing commercial

TABLE 1-1 The original report of eicosanoids in a coral launched a great deal of exploration into the presence of eicosanoids in other invertebrates. The early reports on eicosanoids in invertebrate phyla are cited here. Christ and Van Dorp 1972 Cnidaria Mollusca Annelida Arthropoda Nomura and Ogata 1976 Echinodermata Arthropoda Mollusca Annelida Cnidaria Gromov et al. 1983 Mollusca Korotchenko et al. 1983 Echinodermata

2 1 1 3

species species species species

2 5 2 3 2

species species species species species

1 species 9 species

S I G N I F I C A N C E O F E I C O S A N O I D S

9

firms at nearly negligible prices. This important advance in chemistry obvi­ ated the searches for natural sources of eicosanoids as commercially promis­ ing activities. Given the manifold biological actions of prostaglandins and other eico­ sanoids in mammals, and the information on the occurrence of at least some eicosanoids in many invertebrate species, it now seems but a small step to begin asking about the potential biological significance of eicosanoids in invertebrates. Once again, the context of the first biological experiments with prostaglandins in any invertebrate was human-centered. During the 1970s, the late U. E. Brady and his colleagues considered the possibility of pros­ taglandin actions in the reproductive physiology of house crickets, Acheta domesticus (chapter 4; Destephano et al. 1974; Destephano and Brady 1977). They succeeded in demonstrating the synthesis of prostaglandins by repro­ ductive tracts isolated from male crickets, and eventually showed that certain prostaglandins release egg-laying behavior in newly mated females. The context of these investigations had to do with insects as human pests: Brady and his colleagues had an interest in looking for novel approaches to devel­ oping new insecticide targets. The 1970s yielded other indications of eicosanoid actions in invertebrates, to be addressed in more detail in later chapters. Dalton (1977a, b) suggested that eicosanoids influence salivary gland function in blow flies, and Loher (1979) reported on a preliminary experiment in which he showed that certain prostaglandins release egg-laying behavior in another cricket species, Teleogryllus commodus. Insects make up the largest group of animals, and in­ sects live in a special relationship with humans. It is not surprising, then, to see a great deal of our information about experimental biology of inverte­ brates emerging from studies of various insect systems. In the 1970s and 1980s, however, a formidable conceptual barrier slowed progress toward the research frontier of C20 polyunsaturated fatty acids and eicosanoids in the largest group of invertebrates, insects. It was rather well understood in those early days that insect tissue lipids differed in fatty acid compositions from all other animal tissue lipids then known. Specifically, the contemporary wis­ dom held that in comparison to other animals, insect tissue fatty acid compo­ sitions were very much simpler. More importantly for our purposes, insects were thought to characteristically lack 20:4n-6. The idea that 20:4n-6 is gen­ erally not present in insect lipids developed from several bits of information. One of them comes from the numerous reports of analytical studies of insect tissue fatty acid compositions, virtually all of which did not detect 20:4n-6. Periodically, the mass of analytical studies were compiled into lengthy tables listing known fatty acid compositions. The tables assembled by Fast (1970) are probably the most useful gateways into this literature. These reviews neither included nor commented on the absence of eicosanoid-precursor polyunsaturated fatty acids in insect lipids. Taken with other information,

10

CHAPTER 1

there was a general acceptance of the idea that insect fatty acid compositions included fewer components than other animal systems, and C20 polyunsatu­ rated fatty acids were not present in insect lipids. The conceptual barrier can be highlighted in a question: How can organisms lacking in eicosanoid-precursor polyunsaturated fatty acids have very much to do with eicosanoids in biologically important ways? Later work demonstrated the presence of these components, albeit at low levels, in many insect species. We will also see several important eicosanoid actions in insects. With this barrier tumbling down, progress on the biological significance of eicosanoids in insects and other invertebrates is moving forward apace. Let us return for a moment to the biological paradigm of eicosanoids. Chapter titles indicate that eicosanoids act in fundamental, crucial areas of invertebrate physiology, including reproduction, ion transport, and immunity, to mention the most well-understood points. These chapters document details of eicosanoid actions in animals representing all major phyla. Seen from a phylogenetic perspective, then, one dimension of the biological paradigm guides us in understanding the significance of eicosanoids echoes far beyond mammalian physiology and pathophysiology. The biological paradigm em­ braces yet another dimension, which I foresee will take on increasing impor­ tance in the years to come. One of the largest chapters in this volume is devoted to the significance of eicosanoids in ecological interactions. In some settings, prey animals derive a level of protection from potential predators by generating large quantities of prostaglandins. We will also see that some host-parasite relationships, perhaps many more than now appreciated, are mediated by prostaglandins, as well as other eicosanoids. It follows that the biological significance of eicosanoids is not limited to modulating events within organisms. The biological paradigm is a new, robust theory on the significance of eicosanoids in animals. Although the topic is outside the scope of this vol­ ume, the theory may well extend to the plant kingdom as well. Plants pro­ duce lipoxygenase products upon wounding and other challenges. Moreover, jasmonic acid, a signal moiety active in many aspects of plant biology, is a cyclopentane compound that looks suspiciously like a prostaglandin. Plants generally produce these compounds by oxygenation of Cl8 polyunsaturated fatty acids, rather than the C20 components used in eicosanoid biosynthesis. We are beginning to see hints of overlap in the roles of oxygenated polyun­ saturated fatty acids in plants and animals. As more information and clarity emerge, we may well find that the biological paradigm of eicosanoids is large indeed.

CHAPTER 2

Eicosanoid Structures and Biosynthesis

As SEEN in most specialized fields of scholarly activity, research in eicosanoids has generated a language and set of conventions that are commonly helpful in conveying the concepts encompassed by eicosanoids. Our under­ standing of eicosanoids was developed from research on the chemistry, bio­ chemistry, and biology of these molecules in mammalian systems. This re­ search has produced a very large corpus of literature which, for convenience, we can refer to as the "mammalian model." The mammalian model is the source of most of the language used in discourse on eicosanoids. Probably because research on eicosanoid actions in mammals preceded inquiry into invertebrates by a couple of decades, a great deal of the language is based on the mammalian model. At the end of this chapter, I will stress the point that the background from mammals can be very useful in developing our inter­ ests in eicosanoid systems in invertebrates. However, in this chapter and elsewhere in this book, we will see several instances in which the mam­ malian model is misleading with respect to invertebrates. In other instances, we simply do not have enough information to judge how well invertebrate systems fit into the mammalian background. This chapter is meant to provide a brief overview of several elements of the mammalian model, including the structures of known eicosanoids, and outlines of their biosynthetic pathways. The information in this chapter is drawn from a host of especially useful reviews, cited here as a convenient entry to the literature (Samuelsson et al. 1978, Samuelsson 1983, Needleman et al. 1986, Spector et al. 1988, Pace-Asciak and Asorta 1989, McGiff 1991, Holtzman 1992, Smith 1992, Negishi et al. 1993, Young 1994, Ford-Hutchin­ son 1994, Serhan 1994, Otto and Smith 1995, Metters 1995, Clark et al. 1995, Negishi et al. 1995b, Smith et al. 1996). Recognizing the impressive volume of published information, a comprehensive review of the topic is beyond the scope of this chapter. Before launching into the following overview, let us set a few conventions for this volume. Many of the eicosanoids and the enzymes responsible for the biosynthesis of eicosanoids have rather long nomenclature, such as hydroxyeicosatetraenoic acid. Accordingly, the terms have been abbreviated, often in various ways, over the years. The result is a complex alphabet soup, some of which has to be relearned on each reading of a review or journal

12

C H A P T E R 2

article. I suppose that some day a stable argot of abbreviations will emerge, which should serve to facilitate facile communication and be approachable to scientists new to the field. Meanwhile, most of the terms in this volume are spelled out, with the hope that the extra length in words will be offset by a facilitated introduction to a new field. Some abbreviations, such as DNA, are universally recognized by biologists and biochemists, and these are used without definition. I prefer to use a standard chemical shorthand for fatty acids, especially arachidonic acid, which is commonly written as 20:4n-6. Three polyunsatu­ rated fatty acids, 20:3n-6, 20:4n-6, and 20:5n-3, are potential direct sub­ strates for eicosanoid biosynthesis. Most of the available information on eicosanoid biosynthesis is focused on the metabolism of 20:4n-6. Indeed, eicosanoid biosynthesis is often taken to be synonymous with "arachidonate metabolism." Another common term refers to the so-called arachidonate cas­ cade. I believe the word "cascade" is used incorrectly in this context. For the most part, a cascade refers to falling water, either as a waterfall or a stream of water. A cascade also refers to a succession of stages in a process, and it may be in this sense that some people imagine the formation of eicosanoids as an arachidonate cascade. However, we shall see that 20:4n-6 does not actually enter a cascade. To the contrary, many biologically active eico­ sanoids are the products of single enzyme steps. Alternatively, we shall also see that 20:4n-6 can be taken into a range of metabolic pathways. Perhaps the sheer number of potential metabolic fates gives a sense of a cascade. Again, this is not consistent with the meaning of the word. Hence, eico­ sanoid biosynthesis, or arachidonate metabolism, which are more accurately descriptive of the processes, are more appropriate terms.

THE MAMMALIAN MODEL OF EICOSANOID BIOSYNTHESIS Phospholipase A2 Is the First Step in Eicosanoid Biosynthesis Eicosanoid biosynthesis is thought to begin with hydrolysis of 20:4n-6 from the sn-2 position of cellular phospholipids (Dennis 1994, 1997). Polyunsatu­ rated fatty acids are generally associated with the sn-2 position of phospho­ lipids, whereas saturated or monounsaturated fatty acids are associated with the SH-1 position. This asymmetry in the organization of fatty acids within phospholipids was partly responsible for suggesting that phospholipase A 2 might be a regulatory step in eicosanoid biosynthesis. Although five or six types of phospholipase A 2 are recognized (Dennis 1994, 1997), we will consider only two major classes of this enzyme. The secretory phospholipases A 2 include those found in snake and arthropod venoms, those found in synovial fluids in some cases of pathology, and those

E I C O S A N O I D S T R U C T U R E S / B I O S Y N T H E S I S

13

associated with digestion. Most of these enzymes are low-molecular-weight (about 14 kDa) proteins stabilized by five to seven disulfide bridges. They require the presence of mM concentrations of calcium for full catalytic activ­ ity. The other major class consists of the intracellular, or cytosolic, phospholipases A2. These are high molecular weight (about 85 kDa) proteins that achieve full catalytic activity in the presence of μΜ calcium concentrations. Some of the cytosolic phospholipases A 2 are of particular interest because they show a marked preference for substrate with 20:4n-6 in the sn-2 position. These enzymes are thought to be the first step in eicosanoid biosynthesis. Cytosolic phospholipases A 2 have been purified from several mammalian sources (Dennis 1994, 1997), including rat mesangial cells, human mono­ cytes, human platelets, leukocytes, Swiss mouse 3T3 cells, glomerular mes­ angial cells, mouse keratinocytes, and mouse peritoneal macrophages. These enzymes are variously stimulated by proinflammatory cytokines, tumor nec­ rosis factor, lipopolysaccharide, and mitogens. The stimulations result in the translocation of the enzyme from the cytosol to the cellular membranes, where 20:4n-6 is selectively released from phospholipids. While the concen­ tration of free 20:4n-6 is maintained at submicromolar levels by a reacylation pathway, stimulation produces a rapid increase in free 20:4n-6. This increase is specific to 20:4n-6, and the concentrations of other fatty acids do not increase. The cytosolic phospholipases A2 are upregulated by several mechanisms. One is a calcium-dependent translocation to the membrane fractions of cells, as just mentioned. Another is phosphorylation by protein kinases. The en­ zymes are activated and deactivated in phosphorylation/dephosphorylation cycles. In other cases, a G protein is involved in activation of phospholipase A2. Finally, some of these enzymes are activated by transcriptional activa­ tion, resulting in increased levels of phospholipase A2 protein. Recognizing that several activation mechanisms exist, the cytosolic phospholipases A2 represent the first step in eicosanoid biosynthesis. In chapter 5, we will review the evidence for cytosolic phospholipase A2 in the fat body and hemocytes of several insect species. These enzymes may similarly represent the first step in eicosanoid biosynthesis in some inverte­ brate systems.

Three Major Pathways of Eicosanoid Biosynthesis Figure 2-1 provides an overview of the major eicosanoid biosynthetic path­ ways. The cyclooxygenase pathways yield the prostaglandins and thrombox­ anes. The lipoxygenase pathways convert 20:4n-6 into various hydroperoxyeicosatetraenoic acids and hydroxyeicosatetraenoic acids. These species are themselves biologically active; they are also potential substrates for further

14

C H A P T E R

2

Ο=—]

•COOH

OOH •COOH

'COOH HO

0H

OH

OH ,COOH

H< •COOH

Ή .COOH

OH FIGURE 2-1. An overview of 20:4n-6 metabolism as understood from the mammalian background. Three polyunsaturated fatty acids, 20:3n-6, 20:4n-6, and 20:5n-3, are potential substrates for eicosanoid biosynthesis. Of these, the metabolism of 20:4n-6 is most well studied. Chemical structures are denoted by numerals. 1 = a cellular phospholipid. 2 = hydrolyzed 20:4n-6. 3 = pro­ staglandin E2. 4 = 5-hydroperoxyeicosatetraenoic acid. 5 = leukotriene B4. 6 = 11,12-epoxyeicosatrienoic acid. 7 = lipoxin A. Upper-case letters indi­ cate major enzyme systems responsible for eicosanoid biosynthesis. A = phospholipase A2; B = cyclooxygenase and associated enzyme steps; C = cytochrome P450 epoxygenase; D = lipoxygenase.

E I C O S A N O I D S T R U C T U R E S / B I O S Y N T H E S I S

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metabolism to leukotrienes and still other biologically active products. The epoxygenases are cytochromes P450, which yield the various epoxyeicosatrienoic acids. Let us consider each of these pathways in more detail.

The Cyclooxygenase Pathways Prostaglandins are C20 carboxylic acids with a five-membered ring variously substituted at C-9 and C-11, and two aliphatic chains featuring a substitution at C-15 and one, two, or three double bonds. Three polyunsaturated fatty acids, 20:3n-6, 20:4n-6, and 20:5n-3, are potential substrates for the cyclooxygenase pathways, although 20:4n-6 is the most common substrate among mammals. The prostaglandins always have two fewer double bonds than their parental polyunsaturated fatty acids, giving rise to the 1-, 2-, and 3-series prostaglandins (Fig. 2-2). Prostaglandins are defined by the substitu­ tions at C-9 and C-11. PGE, for example, features a keto function at C-9 and a hydroxyl function at C-11. Specific prostaglandins are identified by com­ bining the number and letter designations. PGE1, to continue the example, is the product of the cyclooxygenase pathway that features one double bond at C-13. The parental fatty acid is 20:3n-6. Figure 2-3 indicates that prostaglandin biosynthesis requires three en­ zyme steps. The steps are similar for all three fatty acid substrates; we will focus on 20:4n-6 because this is the most well-studied substrate. The first step is the cyclooxygenase step, which catalyzes the bis-oxygenation of 20:4n-6 to form the endoperoxide PGG2- This step involves two separate oxygen molecules (Smith et al. 1991). One oxygen interacts with C-11, and the other interacts with C-15, yielding the endoperoxide. The endoperoxide undergoes a two-electron reduction at C-15 which is catalyzed by a perox­ idase activity. The peroxidase activity yields PGH2, from which other, bio­ logically active prostaglandins are produced. The cyclooxygenase and perox­ idase activities are juxtaposed in a single protein. This protein was known as prostaglandin endoperoxide synthase, or PGH synthase, for many years. Of course, nomenclature has changed, and it is now often seen as COX, an abbreviation for cyclooxygenase. A couple of interesting features of the PGH synthase help us understand prostaglandin biosynthesis. For one, the PGH synthase is inactivated before all available substrate is converted into product. This is referred to as a "suicide" inactivation, and is thought to be an intrinsic property of the en­ zyme. PGH synthase is inactivated after about 1,300 to 1,400 catalytic oper­ ations (Smith et al. 1991, Smith and Marnett 1991). The suicide step is a feature of the cyclooxygenase, because the peroxidase activity remains after the cyclooxygenase has faded. Suicide inactivation may serve to set an upper limit on cellular capacity for prostaglandin biosynthesis.

16

C H A P T E R

Fatts' Acids

2

Prostaglandins

COOH

/=\/=V^^uui •COOH

COOH

.COOH

COOH

Ring Features of Prostaglandins

HC

HO PGA

PGB

PGD

PGE

PGF

FIGURE 2-2. The relationship between parental polyunsaturated fatty acids and their respective 1-. 2-, and 3-series prostaglandin products. Each pros­ taglandin series features two fewer double bonds than the parental polyunsatu­ rated fatty acid. The lower panel displays the ring features of five prostaglan­ dins. where R represents the aliphatic chains shown on the complete structures. 1 = 20:3n-6: 2 = PGE1: 3 = 20:4n-6: 4 = PGE2; 5 = 20:5n-3: 6 = PGE3.

A second interesting feature of the mammalian PGH synthase lies in the intracellular localization of the protein. PGH synthase is a glycoprotein asso­ ciated with membrane fractions—mainly the endoplasmic reticulum and, to some extent, the nuclear membrane—of cellular preparations. The enzyme was originally thought to feature transmembrane domains; however, current

17

E I C O S A N O I D S T R U C T U R E S / B I O S Y N T H E S I S

/=V=V\^u •COOH

20-

I

Cyclooxygenase

00H 2 e-

Hydroperoxidase COOH

Isomerase

Thromboxane synthase COOH

COOH

Isomerase COOH

Prostacyclin synthase COOH

Reductase

COOH

FIGURE 2-3. The pathways responsible for prostaglandin biosynthesis involve three enzymatic steps. In the first step, 20:4n-6 (structure 1) is converted to the unstable endoperoxide, PGG2 (structure 2). A hydroperoxidase step reduces PGG2 to the more stable PGH2 (structure 3). Both enzymatic activities are associated with a single protein known as PGH2 synthase. PGH2 serves as a substrate for biosynthesis, by the indicated enzymes, of the classical pros­ taglandins, including PGE2 (structure 4), thromboxane B2 (structure 5), PGD2 (structure 6), PGI 2 (prostacyclin, structure 7), and PGF 2a (structure 8).

18

C H A P T E R

2

thinking places the enzyme virtually entirely within the lumen of the endo­ plasmic reticulum (Otto and Smith 1995). The mammalian model as so far reviewed held sway until early 1991, when it became clear that some cells express another form of the PGH syn­ thase. The two forms were named PGH synthase-1 and PGH synthase-2. All the preceding remarks are based on our understanding of PGH synthase-1. (I imagine that the more brief terms, COX-I and COX-2, will eventually be­ come the standard nomenclature for these proteins.) In either case, the mam­ malian model is a little bit more complex than once thought. The following comments form a comparison of the two isozymes. COX-I is thought to serve as a housekeeping isozyme—that is, it is re­ sponsible for biosynthesizing prostaglandins active in the homeostasis of normal physiology. This enzyme is found in most mammalian tissues, although not in all cells within a tissue, where it is constitutively expressed. The prostaglandins formed under normal physiological conditions are thought to act at the extracellular level, by interacting with G-protein linked recep­ tors located on the outer surface of cells. In this model, COX-1 is thought to release PGH2 into the cytosol, where it is converted by other enzymes into biologically active prostaglandins. The active species then exit the cells, probably assisted by a prostaglandin transporter (Kanai et al. 1995). Otto and Smith (1995) called COX-2 a differentiative isozyme. This en­ zyme produces prostaglandins for inflammatory processes, ovulation, and mitogenesis. In contrast to COX-1, this isozyme is not expressed in most mammalian cells (Smith et al. 1996); however, it can be rapidly induced in fibroblasts, endothelial cells, monocytes, and ovarian follicles. COX-2 is an inducible, rather than constitutive, enzyme, and its expression is increased by ten- to eighty-fold by proinflammatory or mitogenetic factors, such as cyto­ kines and tumor-promoting phorbol esters. While also associated with the membrane fractions of cells, COX-2 is mainly associated with the luminal surface of nuclear membranes. It may release PGH2 into the nucleus, and the PGH2 or another prostaglandin derived from it may interact with nuclear proteins to influence gene expression. Hence, COX-I and COX-2 may repre­ sent two unrelated pools of active enzyme within the same cell, each with separate biological functions (Otto and Smith 1995). There is no information on the forms of the enzymes responsible for pros­ taglandin biosynthesis in invertebrates. Because COX-2 is a proinflammatory enzyme, there is a tremendous economic significance attached to the possi­ bility of developing pharmaceutical compounds that can inhibit COX-2 but not COX-1. Indeed, before the presence and significance of two isozymes was recognized, Smith and Marnett (1991) speculated, on a strictly eco­ nomic basis, that PGH synthase may be the most important enzyme in the world because so much money is invested in the discovery and use of anti­ inflammatory drugs.

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1 9

Another glance at Figure 2-3 will reveal the third enzymatic step in the biosynthesis of prostaglandins. PGH2 is the substrate for several enzymes responsible for converting PGH2 into the active prostaglandins. Prostaglan­ dins D, E, and F are formed by three groups of enzymes, respectively, the PGD synthases, the PGE synthases, and the PGF synthases (Urade et al. 1995). PGD synthase is responsible for isomerization of PGH2 to PGD2. There are three types of PGD synthase. A glutathione-independent form is associated mainly with brain tissue. A glutathione-dependent form is associ­ ated with the spleen, and another form is found in the liver. PGE synthase is responsible for converting PGH2 to PGE2. Membrane-associated and soluble forms of the enzyme have been found. The PGD and PGE synthases are associated with various tissues, and they may be responsible for the specific populations of prostaglandins detected in any given system. The biosynthesis of PGF2a is a bit more complicated. This prostaglandin may emerge from three different pathways. As shown in Figure 2-3, it was thought that PGH2 could be directly reduced to PGF2a. Besides this direct mechanism, Watanabe and his colleagues purified a PGD2 11 -ketoreductase from bovine lung (Urade et al. 1995). This enzyme expresses dual activity and can convert PGH2 and PGD2 into PGF2a. Another PGD2 11-ketoreduc­ tase has been purified from bovine liver. This enzyme exhibits a higher pref­ erence for PGD2 than the lung enzyme just mentioned. For a third mecha­ nism of PGF2a synthesis, human placenta expresses a PGE2 9-ketoreductase that converts PGE2 into PGF2a. Two other prostaglandins are formed directly from PGH2. The structure of thromboxane A2 is a little different from the other prostaglandins (Fig. 2-3), and sometimes the thromboxanes are called "prostanoids." Thromboxane A2 is produced by major host-defense cells, including platelets, macrophages, and monocytes. It is also seen in the kidney and liver. Among other biolog­ ical actions in mammals, thromboxane A2 induces platelet aggregation and vascular and bronchiolar smooth muscle constriction. This cyclooxygenase product is seen in increased levels in pathophysiological conditions, such as unstable angina and human septic shock. The cellular actions that thrombox­ ane A2 is responsible for inducing are mediated through receptors. The thromboxane A2 receptor is also activated by PGH2, and the receptor is sometimes denoted as the thromboxane A2/prostaglandin H2 receptor (Halushka et al. 1989, 1995). Thromboxane synthase is the enzyme responsible for converting PGH2 to thromboxane A2. The enzyme is associated with the microsomal fraction of cells, and it has been purified and cloned from a variety of sources (Tanabe and Ullrich 1995). The human platelet thrombox­ ane synthase has a molecular weight in the range of 53 to 59 kDa. These enzymes are part of the cytochrome P450 superfamily. Prostacyclin is also called PGI2 (Fig. 2-3). This prostaglandin seems to act in a yin-yang relationship with thromboxane A2. While thromboxane A2

20

C H A P T E R

O

2

HQ_

'COOH HO

'COOH

0H

0

0H 2

(

'COOH

'COOH 0Η 3

O

0Η 4

FIGURE 2-4. Two prostaglandins are formed by rearranging classical pros­

taglandins, rather than modifications of PGH2. PGA2 (3) is formed by a nonenzymatic rearrangement of PGE2 (1). A12-PGJ2 (4) is formed by a nonenzymatic dehydration of PGD2 (2).

promotes platelet aggregation and smooth muscle contraction, PGI 2 exerts the opposite effects. PGI 2 receptors are abundant in many tissues, including the thymus, lung, aorta, and spleen. The receptors are apparently not present in the brain, stomach, liver, intestine, or kidney (Hirata et al. 1995). PGI 2 synthase is the enzyme responsible for converting PGH 2 into PGI 2 , and this enzyme is also a member of the cytochrome P 450 superfamily (Tanabe and Ullrich 1995). Two other prostaglandins, A 12 -PGJ 2 and PGA 2 (Fig. 2-4), are not formed directly from PGH 2 (Negishi et al. 1995a). PGA 2 is produced through a nonenzymatic rearrangement of PGE 2 . PGJ 2 is a nonenzymatic dehydration product of PGD 2 that is converted to the A 12 -PGJ 2 in the presence of serum albumin. Most prostaglandins, including PGE 2 , PGF2CT, PGD 2 , PGI 2 , and thromboxane A 2 express their actions through specific receptors located on cell surfaces. PGA 2 and A 12 -PGJ 2 operate through a different mechanism. These prostaglandins are thought to be actively moved into cells via a trans­ porter protein. An intracellular carrier molecule facilitates transport into the nucleus, where the prostaglandins bind with thiol groups of nuclear proteins. The prostaglandin-protein complex then interacts with DNA, resulting in the expression of genes. Among their other biological actions, the A and J series prostaglandins induce expression of genes for heat shock proteins in many normal and tumor cells. In overview, the mammalian model tells us that prostaglandin biosynthesis most generally involves a three-step pathway, beginning with a cycloox-

E i C O S A N O I D S T R U C T U R E S / B I O S Y N T H E S I S

21

ygenase step, which is followed by an endoperoxidase step. These two steps probably occur in most mammalian cells. The specific cellular profiles of prostaglandins probably result from the actions of the synthases responsible for converting PGH2 into any particular suite of products. Of course, our appreciation of the mammalian model of eicosanoid biosynthesis involves other major groups of eicosanoids, to which we now turn our attention.

The Lipoxygenase Pathways

Sir John Vane shared in the 1982 Nobel Prize in Physiology or Medicine for his pioneering discoveries in the pharmacology of prostaglandins and other eicosanoids. In one of his many contributions to the area, Vane developed the idea that aspirin and similar drugs exerted their anti-inflammatory actions by inhibiting the biosynthesis of prostaglandins from 20:4n-6. Soon after this, it was recognized that the anti-inflammatory effects of corticosteriods were expressed in a manner different from the anti-inflammatory effects of aspirin. Bengt Samuelsson, another participant in the 1982 Nobel prize, in­ ferred from these findings that 20:4n-6 may enter metabolic pathways other than the cyclooxygenase pathways. Polymorphonuclear leukocytes are central to many mammalian inflamma­ tory events, and Samuelsson and his colleagues used these cells to investi­ gate the possibility that 20:4n-6 could be metabolized into products other than prostaglandins. They discovered that the main pathways in leukocytes yielded 5-hydroxyeicosatetraenoic acid and a series of products later called leukotrienes (Oates 1982, Samuelsson 1983). This work launched an entire field of research into the 20:4n-6 lipoxygenase pathways. The mammalian 20:4n-6 lipoxygenases produce a series of six products (Fig. 2-5). These are 5-, 8-, 9-, 11-, 12-, and 15-hydroperoxyeicosatetraenoic acids. Each of the lipoxygenases is named according to the carbon that is oxygenated. Thus, 5-lipoxygenase yields 5-hydroperoxyeicosatetraenoic acid, and so forth (Pace-Asciak and Asotra 1989). In analogy to the abbreviation COX, the lipoxygenases are abbreviated by the term LOX, de­ rived from lipoxygenase. Thus, we frequently see expressions such as 5-LOX. This logical nomenclature is not perfect, however, because the posi­ tional specificity of some lipoxygenases is more connected to the substrate and reaction conditions than to a property of the enzyme. Moreover, some lipoxygenases exert dual or multiple positional specificities (Kuhn and Thiele 1995). Although certain biological actions are ascribed to the hydroperoxide de­ rivatives of polyunsaturated fatty acids, these products are rarely seen in biological fluids. The lipoxygenase products are quickly taken into various metabolic systems, which yield other biologically active products. In one of

22

CHAPTER

2

FIGURE 2 - 5 . Structures of hydroperoxyeicosatetraenoic acids and hydroxyeicosatetraenoic acids formed from 20:4n-6 (1). These products are produced by specific lipoxygenases, which are identified by the positional specificity of the introduced oxygen. For example, 5-hydroperoxyeicosatetraenoic acid (2) is the product of 5-lipoxygenase. The hydroperoxy fatty acids are rapidly reduced to their corresponding hydroxy fatty acids by glutathione peroxidases. The remaining structures are identified by the position of the introduced oxygen, (3) = 15-hydroperoxy and 15-hydroxyeicosatetraenoic acid; (4) = 8-hydroperoxy and 8-hydroxyeicosatetraenoic acid; (5) = 12-hydroperoxy and 12hydroxyeicosatetraenoic acid; (6) = 9-hydroperoxy and 9-hydroxyeicosatetraenoic acid; (7) = 11-hydroperoxy and 11-hydroxyeicosatetraenoic acid.

the most c o m m o n fates, the hydroperoxide products are quickly reduced to the corresponding hydroxyeicosatetraenoic acid by various glutathione peroxidases, which are abundant in most m a m m a l i a n cells. T h e hydroxyeicosatetraenoic acids are biologically active (Spector et al. 1988). For example, t w o products, 5- and 12-hydroxyeicosatetraenoic acids, induce degranulation of h u m a n neutrophils. Similarly, 12-hydroxyeicosatetraenoic acid is a potent

EICOSANOID STRUCTURES/BIOSYNTHESIS

23

chemoattractant for polymorphonuclear leukocytes. Several hydroxyeicosatetraenoic acids are active in various pathophysiological events, including proinflammatory processes. We will see in later chapters that hydroxyeicosanoids exert very important biological actions in invertebrates. One of the lipoxygenase products, 5-hydroperoxyeicosatetraenoic acid, serves as a substrate for biosynthesis of the leukotrienes (Fig. 2-6). The root leukotriene is leukotriene A4, an unstable epoxide of 5-hydroperoxyeicosat­ etraenoic acid. The enzyme responsible for this is called leukotriene A4 syn­ thase. The 5-lipoxygenases from human leukocytes and mouse mast cells also express leukotriene A4 synthase activity, and many scientists believe that both enzymatic steps are carried out by the same bifunctional enzyme. Leukotriene A4 is an unstable substance whose main function is serving as substrate for biosynthesis of other leukotrienes. There are two main path­ ways (Pace-Asciak and Asotra 1989). The first is catalyzed by leukotriene A4 hydrolase, which yields leukotriene B4 (Fig. 2-6). This leukotriene is a pro­ inflammatory mediator of host defense reactions in mammals. It activates polymorphonuclear leukocytes, myeloid cells, and mast cells. Leukotriene B4 also induces neutrophils to adhere to endothelial cell walls (Metters 1995). Alternatively, leukotriene A4 can be modified into the cysteinyl leuko­ trienes, also called peptidoleukotrienes (Fig. 2-6). Leukotriene C4 synthetase catalyzes two modifications of leukotriene A4. First, the epoxide is opened, then glutathione is added in covalent linkage to C-6, yielding leukotriene C4. This leukotriene can be converted to leukotriene D4 by a single transpep­ tidase step, which catalyzes hydrolysis of the terminal amino acid residue from leukotriene C4. Leukotriene D4 undergoes another transformation to leukotriene E4 by hydrolyzing the glycine residue from leukotriene D4. This step is catalyzed by a dipeptidase. Leukotriene F4 can be formed by adding an amino acid residue. The cysteinyl leukotrienes make up the slow reacting substance of an­ aphylaxis (Samuelsson 1983). The major biological action of these com­ pounds is contraction of smooth muscles associated with respiratory and vascular systems, and with the alimentary canals of mammals. These actions are mediated through specific receptors, of which two types are known (Met­ ters 1995). These are designated Cys-LT1 and Cys-LT2 receptors. Unlike the prostaglandin receptors, which exhibit marked specificity for each pros­ taglandin, the receptors for the cysteinyl leukotrienes are much less fastid­ ious. Both types of receptors have equal affinity for leukotrienes C4 and D4. The receptors have less affinity for leukotriene E4. We have no information on receptors for leukotriene F4. The leukotrienes have not yet been consid­ ered in invertebrates. Beside the hydroperoxy- and hydroxyeicosatetraenoic acids and leuko­ trienes, the lipoxygenase pathways can yield another suite of compounds, none of which are known from invertebrates. I provide the structures of

24

CHAPTER

2

LTE4 FIGURE 2 - 6 . The biosynthesis and structures of leukotrienes. The pathways begin with conversion of 20:4n-6 (AA) to 5-hydroperoxyeicosatetraenoic acid by a 5-lipoxygenase. Leukotriene A 4 synthase is responsible for creating the unstable epoxide, leukotriene A 4 (LTA4), which serves as a substrate for leukotriene biosynthesis. In a one-step pathway, leukotriene hydrolyase yields leukotriene B 4 (LTB4). The alternative pathway yields the peptidoleukotrienes, leukotrienes C 4 (LTC4), D 4 (LTD4), and E 4 (LDE4).

25

E I C O S A N O i D S T R U C T U R E S / B I O S Y N T H E S I S

OOH

OOH

HO

O

O Hepoxilin A3

Hepoxilin B3

OOH

OOH

H HO

OH

Trioxilin A3

HO

OH

Trioxilin B3

FIGURE 2-7. The structures of hepoxilins and trioxilins.

some of these compounds (Figs. 2-7 and 2-8) as a form of speculation. It can easily be imagined that leukotrienes and other lipoxygenase products not yet studied in invertebrates may well serve very important functional roles in these animals. One group of lipoxygenase products is known as "hepoxilins" (Pace-Asciak and Asotra 1989). The 12-lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid, is the direct precursor of the hepoxilins (Fig. 2-7). The hepoxilins may be nonenzymatic rearrangements of 12-hydroperoxyeicosatetraenoic acid, in which the hydroperoxide group is converted to one ep­ oxide group and one hydroxy group. There are two known hepoxilins, hepoxilin A3 and B3. The epoxide groups of these two products are then enzymatically hydrolyzed to their corresponding trihydroxy derivatives by an epoxide hydrolase. These trihydroxy compounds are called trioxilin A3 and B3. The hypoxilins act to release insulin from pancreatic islets (PaceAsciak and Martin 1984). Serhan and his colleagues are responsible for identifying a new family of lipoxygenase products known as lipoxins (Fig. 2-8). The following descrip­ tion of lipoxin biosynthesis and biological actions is drawn from Serhan's recent review (Serhan 1994).

C H A P T E R 2

26

.COOH

.COOH

OH

HO

Lipoxin A

Lipoxin B

OH

FIGURE 2-8. Two members of the lipoxin family.

Lipoxins exert potent biological actions in vertebrate microcirculation. The two main lipoxins, lipoxins A 4 and B 4 , sometimes exert opposite effects, one vasoconstricting and the other vasodilating. Lipoxin A 4 is a vasodilator in hamster cheek pouch and rat kidney microcirculation. In the hamster cheek pouch model, the lipoxins appear to act independent of other eicosanoids. On the other hand, the vasodilatory action of lipoxin A 4 in the rat kidney may be mediated, in part, by stimulating eicosanoid biosynthesis, which secondarily influences hemodynamics. Lipoxins also stimulate vaso­ dilation in cerebral arterioles—again, by direct action not involving other eicosanoids. Lipoxins clearly act via a number of different cellular mecha­ nisms, according to the particular system under study. We may infer that these eicosanoids have been independently recruited into a variety of regula­ tory mechanisms during vertebrate evolution, a point we will revisit shortly. Lipoxins also regulate some cellular events in leukocytes. The cytotoxic actions of natural killer cells are inhibited by lipoxins. These eicosanoids also stimulate lipid remodeling in polymorphonuclear leukocytes. Lipoxins also stimulate other cellular processes involved in cell-cell interactions. For example, they stimulate colony formation with mononuclear cells. Perhaps the most important lipoxin actions relate to their roles as stop signals in vertebrate immune reactions. Serhan (1994) proposed the chalone hypothesis. Chalone is derived from a Greek root meaning to relax, and the term is used to describe compounds that exert inhibitory effects on cells. In the inflammatory process, a wide range of proinflammatory mediators is gen­ erated. These include cytokines, a variety of eicosanoids, and so forth. Serhan's point was that the influence of all these proinflammatory compounds must be internally regulated in a way that creates a self-limiting inflamma­ tory reaction to challenge. He suggested that lipoxins may serve to inhibit proinflammatory events, and thereby allow healing reactions to emerge. The main evidence supporting this model is discovery of an increasing number of inhibitory lipoxin actions. The inhibitory actions include reduced leukotriene binding to T-lymphocytes, inhibition of leukotriene-induced chemotoxins in human neutrophils, inhibition of leukotriene-induced inflammation in the

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27

cheek pouch microcirculation model, and inhibition of polymorphonuclear leukocyte adherence. Lipoxins exert other chalone actions, but the point is moving away from the main thrust of this brief overview. All support Serhan's hypothesis. Lipoxins express their actions through three cellular mechanisms (Serhan 1994). Human neutrophils express cell surface receptors specific for lipoxin A4. Other cells, polymorphonuclear leukocytes for example, express recep­ tors that are shared by lipoxin A4 and the cysteinyl leukotrienes. Lipoxins can also act within their originating cells. In this mode of action, the lipoxins interact with certain forms of protein kinase C, thereby modulating events within the cell. Two main biosynthetic pathways are responsible for producing lipoxins (Fig. 2-8). In one, free 20:4n-6, or 20:5n-3, can be converted to 15-hydroperoxyeicsatetraenoic acid by a 15-lipoxygenase. A 5-lipoxygenase can con­ vert this to an epoxytetraene, from which all the lipoxins can be synthesized. Hence, the epoxytetraene is a central intermediate in lipoxin biosynthesis. Lipoxins A4 and B4 are products of specific epoxide hydrolases. Alter­ natively, human eosinophils are able to add the tripeptide glutathione to C-6 of the epoxytetraene, through a glutathione-S-transferase. This reaction is similar to the formation of the cysteinyl leukotrienes, and the lipoxin product is termed lipoxin C4. With additional metabolic steps, also similar to leukotriene metabolism, selective peptidases yield lipoxins D4 and E4. We have no information relative to the biological significance of these cysteinyl lipoxins in eosinophils. These lipoxins are not produced in human neutrophils or platelets, suggesting that the cysteinyl lipoxins may be important in eo­ sinophil biology. Another pathway to the central epoxytetraene begins with conversion of 20:4n-6 to leukotriene A4 via a 5-lipoxygenase. The leukotriene A4 is the root substrate from which all other leukotrienes are formed. However, leuko­ triene A4 can undergo another metabolic fate. Nearly 50% of the leukotriene A4 produced by some cells, such as polymorphonuclear leukocytes, can be released from the cells. Neighboring cells can take up the released leuko­ triene A4 and convert it into the C4 and B4 leukotrienes. This process is known as transcellular metabolism, and it is thought to serve as a mechanism for rapidly increasing the total production of leukotrienes. Human platelets are able to take up leukotriene A4 from the medium, and transform the leu­ kotriene into lipoxin A4. This is accomplished through a 12-lipoxygenase, which is able to use the epoxide-containing leukotriene A4 as a substrate. The transcellular leukotriene A4 route may be a very important source of lipoxins in human platelets. Lipoxins have not been considered in studies of invertebrate eicosanoid systems, although there is reason to suspect that they may occur in these animals. The occurrence and biological significance of lipoxins and other

28

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lipoxygenase products are not restricted to mammalian systems. Rowley (1996) and his colleagues have shown that blood cells from fish species are competent to produce substantial amounts of lipoxins. This finding was part of a broader investigation of eicosanoid biosynthesis in fish blood cells. Pettitt et al. (1989a) first reported on leukotriene B 4 biosynthesis by blood cells from the rainbow trout, Salmo gairdneri. They later found that lipoxins are the major lipoxygenase products in rainbow trout macrophages (Pettitt et al. 1989b, 1991). Using an in vitro assay system, they showed that lipoxin A 4 stimulated migration in rainbow trout neutrophils (Sharp et al. 1992). They inferred from this work that the migration-inducing action of lipoxins has a long evolutionary history. From the perspective of our focus on eicosanoids in invertebrate signal transduction systems, the evolutionary distance from fish to mammals is not particularly impressive. However, fish represent very early stages of vertebrate evolution. We may infer from this work on fish blood cells that at least some invertebrate systems may also produce and respond to lipoxins.

The Epoxygenase Pathways For many years, the mammalian model of 20:4n-6 metabolism, or eicosanoid biosynthesis, was thought to include the two major pathways just discussed in the preceding two sections of this chapter. This picture changed, begin­ ning in the mid-1980s, to include a third pathway of 20:4n-6 metabolism (Fig. 2-9). The third pathway is usually called the epoxygenase pathway, although the designation is often couched in quotation marks meant to rec­ ognize that it is nothing more than a convenient expression for cytochrome P 450 -dependent monooxygenases. The following remarks on this pathway are drawn from reviews by Fitzpatrick and Murphy (1989) and McGiff (1991). The epoxygenase pathway yields four epoxide metabolites of 20:4n-6 (Fig. 2-9). These are 5,6-epoxyeicosatrienoic acid and its counterparts with the epoxide-linking carbons 8,9; 11,12; and 14,15. These four epoxides can be further metabolized by epoxide hydrolases to their corresponding vicinal diols. Vicinal refers to neighboring, and is meant to convey the point that the hydroxy! substitutions are associated with adjacent carbons. The cytochrome P 450 system can also catalyze two other types of reac­ tions. One reaction yields 5-, 8-, 9-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids. These are formed without regional- or stereospecificity, although liver microsomes may contain isozymes that do act regiospecifically. Another reaction is hydroxylation at C-19 and C-20, yielding 19and 20-hydroxyeicosatetraenoic acids. These products may be involved in hypertension.

E I C O S A N O I D S T R U C T U R E S / B I O S Y N T H E S I S

Ο

O OOH

COOH

1

2

OOH

O

29

3

COOH

O

4

FIGURE 2 -9. Structures of

epoxyeicosatetraenoic acids. (1) = 5 ,6-epoxyeicosatrienoic acid; (2) = 8,9-epoxyeicosatrienoic acid; (3) 11,12-epoxyeicosatrienoic acid; (4) = 14,15-epoxyeicosatrienoic acid.

The cytochrome P450 system is substantially different from the cyclooxygenase and lipoxygenase systems discussed in the earlier sections. The cy­ tochrome P450S are monooxygenases—that is, they split the oxygen-oxygen bond in oxygen molecules and transfer one oxygen to their substrate. In contrast, cyclooxygenases and lipoxygenses are dioxygenases, inserting both oxygen atoms in molecular oxygen into their substrates. We saw that hydroxyeicosatetraenoic acids can be produced via lipoxygenase and monooxygenase pathways. These details are more important to the pharmaceutical industry than to us; however, we will see in later chapters that some inverte­ brates produce substantial levels of hydroxyeicosatetraenoic acids. As it stands, we can not yet be certain of the pathways involved in these syn­ theses. The epoxyeicosatrienoic acids exert biological actions in several mam­ malian tissue systems. In the hypothalamus, 5,6-epoxyeicosatrienoic acid and its corresponding diol may be involved in release of somatostatin. Sim­ ilarly, 14,15-eicosatrienoic acid may regulate release of glucagon from pan­ creatic islets. The diol metabolite of 11,12-epoxyeicosatrienoic acid inhib­ its renal sodium-potassium pumps. As seen in our consideration of lipoxins, the number of discovered biological roles of the epoxygenase products is ra­ pidly growing. The brief treatment in this chapter is meant to alert us to the possibility that some of the eicosanoids known from mammals, while not yet discovered in invertebrates, may be of considerable importance in these animals.

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An Invertebrate Prostaglandin Biosynthesis Pathway To complete this chapter on the structures and biosynthesis of eicosanoids, let us recall a key point in the history of eicosanoids. The first discovery of eicosanoids in invertebrates emerged from studies of the chemistry of coelenterates. Weinheimer and Spraggins (1969) reported on the presence of two prostaglandin derivatives in the Caribbean octocoral, Plexaura homomalla. A flurry of research on the chemistry and ecology of this coral fol­ lowed, and it was soon noted that the coral produces prostaglandins by a pathway fundamentally different from the known mammalian background (Corey et al. 1975). In mammals, prostaglandin biosynthesis proceeds through PGG 2 to PGH 2 and thence to an active product (Fig. 2-3). In experiments with radioactive PGG 2 and PGH 2 , it was found that bovine seminal vesicles could readily convert these intermediates to PGE 2 and PGF 2a . In similar experiments with several different coral preparations, however, none of the radioactive intermediates was converted into prostaglandins. The authors concluded that prostaglandin biosynthesis in the coral must follow from a different biosynthetic pathway. This small note launched a very lengthy research program aimed at revealing the coral prostaglandin biosynthetic pathway. The proposed pathway for prostaglandin biosynthesis in the coral begins with a lipoxygenase, rather than cyclooxygenase, step (reviewed by Gerwick 1993; Fig. 2-10). In coral, 20:4n-6 undergoes 15-lipoxygenation, followed by a peroxidase step, to yield a 15-hydroxyeicosatetraenoic acid. A subse­ quent 8-lipoxygenation produces 8-hydroperoxy-15-hydroxyeicosatetraenoic acid. This intermediate is converted into an allene oxide by an enzyme not previously known in animals, allene oxide synthase. The allene oxide is then converted into PGA 2 . Brash et al. (1991) also described biosynthesis of an allene oxide of 20:4n-6 in starfish oocytes. We will see in chapter 4 that a lipoxygenase product of 20:4n-6 is involved in development of starfish oocytes. The oocytes produce 8-hydroxyeicosatetraenoic acid, and Brash et al. (1991) set out to determine whether a lipoxygenase was involved. They learned that the oocytes express an 8(R)-lipoxygenase that converts 20:4n-6 to 8-hydroperoxyeicosatetraenoic acid, which is consistent with the mammalian lipoxygenase pathway to this product. To their surprise, however, they found the major metabolites of the 8-hydroperoxide were an allene oxide and a new C-13 aldehyde. As mentioned just above, the allene oxide synthetase was previously unknown in animal biochemistry. This enzyme is abundant in plant cells, where it is involved in the pathway that converts 18:2n-6 into jasmonic acid, a prostaglandin-like molecule that serves as a signal transduction moiety in plant development and host defense reactions. The biological significance of the

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31

OOH

OOH -COOH

2

-COOH

3

'COOH

FIGURE 2-10. An alternative pathway for the biosynthesis of prostaglandins in some invertebrates. In this pathway, prostaglandin biosynthesis begins with lipoxygenation, rather than cyclooxygenation of 20:4n-6 (1). After a sec­ ond lipoxygenase step, the 8-hydroperoxy-15-hydroxyeicosatetraenoic acid (2) is converted to an allene oxide (3), from which PGA 2 (4) is formed.

allene oxide and C-13 aldehyde in starfish oocytes remains unclear; how­ ever, it does seem to be a step in prostaglandin biosynthesis (Brash et al. 1991). In their research on the natural product chemistry of the soft coral, Clavularia viridis, two Japanese groups discovered a new class of prostaglandin derivative. Kobayashi et al. (1982) called these prostanoids claviridenones and Kikuchi et al. (1982) named them clavulones (Fig. 2-11). Although we are unclear as to the biological significance of these compounds to the coral, we do know that they have biological activity on various chemical screening procedures and, hence, they received considerable attention in the 1980s. Several variations on the original clavulones, the accepted term for these compounds, have been isolated from the coral (Gerwick 1993). The bio-

32

CHAPTER

2

4 FIGURE 2-11. Structures of oxylipins from the soft coral Clavularia (1) = clavulone I; (2) = clavulone II; (3) = clavulone III; (4) claviridenone a.

viridis.

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synthesis of these products also is thought to proceed via an allene oxide intermediate (Gerwick 1993). Hence, the allene oxide pathway for pros­ tanoid biosynthesis occurs in at least two groups of coral. Gerwick (1993) noted that the term eicosanoid is limited to oxygenated metabolites of specifically C20 polyunsaturated fatty acids. He suggested that a new term, oxylipin, was required to serve as a broader name for all oxygenated compounds that are formed from fatty acids of any chain length by reactions involving at least one step of a monooxygenase- or dioxygenase-dependent oxygenation. This broad mantle informs our appreciation of many fatty acid-derived products in various animal systems. Another term, phytooxylipins, similarly describes a very wide array of oxygenated fatty acids that serve important roles in plant defense reactions (Blee 1998). We may infer that various forms of oxygenated fatty acids are crucial media­ tors in the life histories of most organisms. The discoveries of the enzyme allene oxide synthetase in coral and starfish oocytes is of tremendous interest in understanding eicosanoids in inverte­ brate signal transduction systems. I once mentioned that if taken with due skepticism, the large body of information that makes up the mammalian model can serve as a rough guide to our studies of eicosanoid actions in invertebrates (Stanley-Samuelson 1987). The skepticism is an important in­ gredient because the mammalian model can be misleading at crucial points in our inquiry. The finding that an unknown number of invertebrates, so far represented by a few coral species, are able to biosynthesize prostaglandins via a pathway completely unknown in mammals illustrates the potential dif­ ferences between the eicosanoid systems of invertebrates and the mam­ malian model. The thrust of these remarks lies in recognizing that in many cases, trying to squeeze new information from invertebrate systems into the confines of the mammalian model will confound, rather than clarify, our understanding.

CHAPTER 3

Polyunsaturated Fatty Acids BY THE early 1980s, a fairly conventional view of fatty acid biochemistry had been established, as outlined below. Virtually all organisms, aside from viruses and a few other tiny forms found in the vicinity of the life/not-life threshold, were thought capable of biosynthesizing the common saturated and monounsaturated fatty acids (structures and nomenclatures of fatty acids are displayed in Fig. 3-1). The ubiquitous fatty acid synthase, a multienzyme complex, is responsible for these biosyntheses. In addition to these components of complex lipids, plants were recognized for their ability to biosynthesize two polyunsaturated fatty acids: 18:2n-6 and 18:3n-3. Plants were not able to produce longer-chain polyunsaturated fatty acids. While animals were not able to produce these two compounds independently, they were able to metabolize the C18 polyunsaturates into longer-chain, and more unsaturated, fatty acids (via elongation-desaturation pathways shown in Fig­ ures 3-2 and 3-3). The longer-chain components include 20:3n-6, 20:4n-6, and 20:5n-3, which are found in most animals as part of cellular and sub­ cellular biomembranes and also serve as substrates for eicosanoid biosynthe­ sis. Insects were thought to differ from the general animal background by the absence of the C20 polyunsaturates. The 1980s were dynamic years for lipid biochemists, and the major pillars supporting this comfortable view deteriorated in the light of new evidence. The purpose of this chapter is to provide a detailed appreciation of fatty acid biochemistry, especially in insects, as it is now understood. The central theme is recognizing the presence and metabolism of polyunsaturated fatty acids as an essential element of eicosanoid biosynthetic systems. Most of this chapter will focus on insects, primarily because insects have received more attention than other invertebrates in this area and because insects have been the source of new discovery. To be sure, the presence and metabolism of polyunsaturated fatty acids in other invertebrates is very im­ portant. For one point, many invertebrates are prominent food sources sup­ plying dietary n-3 fatty acids, which have a visible connection to human health. For another, marine invertebrates produce and maintain very high proportions of C20 polyunsaturates in tissue phospholipids. ESSENTIAL FATTY ACIDS Burr and Burr (1929, 1930) are credited with defining the concept of essen­ tial fatty acids. It is useful to distinguish between physioloigcal and nutri-

P O L Y U N S A T U R A T E D

FATTY

ACIDS

35

18:0 Octadecanoic acid (stearic acid)

18:1 n-9 Z-9-octadecenoic acid (oleic acid)

18:2n-6 Z,Z-9,12-octadecadienoic acid (linoleic acid)

18:3n-3 Z,Z,Z-9,12,15-octadecatrienoic acid (alpha-linolenic acid)

20:3n-6 Z,Z,Z-8,11,14-eicosatrienoic acid (dihomo-gamma-linolenic acid)

20:4n-6 Z,Z,Z,Z-5,8,11,14-eicosatetraenoic acid (arachidonic acid)

>0:5n-3 Z,Z,Z,Z,Z-5,8,11,14,17-eicosapentaenoic acid FIGURE 3 - 1 . Nomenclature and structures of saturated and unsaturated fatty acids. C o m m o n names often reflect the major source of fatty acids; for example, oleic acid is a predominant component of olive oil. In the shorthand notation, the number to the left of the colon represents the number of carbons in the acyl chain, and the number to the right of the colon indicates the number of double bonds in the chain, all in (Z) configuration. The n-3, n-6, and n-9 designate positions of the first double bond, counting f r o m the methyl terminus of the acyl chain. These designations also represent metabolic families of fatty acids. Except for conversion of 18:1 n-9 to 18:2n-6 by plants and certain insect species, polyunsaturated fatty acids cannot be converted from one family to another.

36

CHAPTER 3 Fatty Acid Synthase

2000 O 2000 α 1500

>. 1000

O

0 02

0.2

SOO

0.08 0.25

20

0.5

0.75

1.0

2.0

3.0

4.0

5.0

10.0

Incubation time (mln)

Radioactive substrate concentration (μΟΙ)

~ 2000

CD 200

ECL 150

0.25

0.5

0.75

1.0

1.5

2.0

5.0 10.0

Protein concentration (mg/ml)

25

30

35

40

Incubation temperature (0C)

FIGURE 5-10. The influence of the four indicated parameters on eicosanoid biosynthesis by microsomal enriched preparations of fat body from tobacco hornworms. The histogram dis­ plays the biosynthesis of individual prostaglandins, and the line represents total prostaglan­ din biosynthesis. Each point is the mean of three separate experiments. The error bars, where visible, indicate 1 SEM. Reprinted from Insect Biochemistry and Molecular Biology, Volume 24, Stanley-Samuelson and Ogg, Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta, pages 481-491, Copyright 1994, with permission of Elsevier Science.

biosynthesis in in vitro reactions, and possibly in intact cells. Early in the reactions, the rapid bursts of biosynthesis would exceed prostaglandin degra­ dation, thereby favoring product accumulation. During the second phase of the reaction, the suicide inactivation of cyclooxygenase would lead to de­ creased prostaglandin biosynthesis, and product degradation would exceed product formation. The overall result of this asymmetry in the reaction prog­ ress would be registered as higher product accumulation in shorter reaction periods, which decreases during longer reaction periods. In our work with the tobacco hornworm fat body, we observed an initial

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burst of prostaglandin biosynthesis, which peaked at about one minute. Thereafter, we recorded decreasing product accumulation over the next nine minutes. These findings are congruent with the mammalian background, and also with the time course of prostaglandin biosynthesis seen in housefly preparations. Wakayama et al. (1986b) also observed rapid prostaglandin biosynthesis during two-minute incubations. After the first two minutes, there was a very gradual increase in product formation over the following fifty-eight minutes. To the contrary, though, Brenner and Bernasconi (1989) reported a linear increase in PGE2 biosynthesis over a sixty-minute time course. In their characterization of prostaglandin biosynthesis by spermatophore contents from males of the Australian field cricket, T. commodus, Tobe and Loher (1983) also found a linear increase in synthesis over sixtyminute reaction periods. The systems in which longer reaction times promote increased product formation may point to important differences between the well-established mammalian background and the emerging information on invertebrate eicosanoid systems. PGA2 is the predominant product in tobacco hornworm fat body 20:4n-6 metabolism. Unlike the other major prostaglandins, PGA2 biosynthesis does not follow from direct enzymatic conversion of PGH2 (chapter 2). PGH2 can be converted to PGE2, and formation of PGA2 can follow this by sponta­ neous, nonenzymatic rearrangement of the PGE2. We were concerned that the relatively high levels of PGA2 biosynthesis in the fat body preparations could be artifacts of PGE2 rearrangements during the analytical workup fol­ lowing the biosynthesis reactions. We ruled this out by incubating routine fat body preparations with radioactive PGE2. Products of these reactions were extracted from the reaction mixtures and separated following our usual pro­ cedures. After two-minute reaction periods, about 90% of the radioactivity associated with PGE2 was recovered in chromatographic fractions corre­ sponding to PGE2, and less than 3% was recovered in fractions representing PGA2. The results of these exercises indicate that our analytical procedures do not yield PGA2 as an artifact. We suggested that the PGA2 produced by the fat body preparations emerges by way of an unknown intermediate (Stanley-Samuelson and Ogg 1994). All of the biological investigations into the roles of eicosanoids in cellular and humoral immunity described in this chapter were based on the use of pharmaceutical inhibitors of eicosanoid biosynthesis as probes of putative eicosanoid biosynthesis pathways in the invertebrate species tested so far. Some of the more or less standard eicosanoid biosynthesis inhibitors have been shown to inhibit eicosanoid biosynthesis in insects and other inverte­ brates; however, the influence of these inhibitors on the tobacco hornworm eicosanoid system remained to be seen. In these experiments, the routine fat body preparations were incubated in the presence of selected doses of the pharmaceutical products; then, the reaction products were extracted and an-

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alyzed as usual. We found the fat body preparation to be very sensitive to the cyclooxygenase inhibitors indomethacin and naproxen. At the low dosage of 0.1 μΜ. indomethacin and naproxen reduced total prostaglandin biosynthe­ sis by about 807c. Higher dosages virtually abolished prostaglandin bio­ synthesis. Along with inhibition of prostaglandin biosynthesis, reactions with naproxen yielded substantial levels of a radioactive product we tentatively identified as the lipoxygenase product 15-hydroxyeicosatetraenoic acid. The influence of naproxen on increased lipoxygenase activity obtained in a dosedependent manner. Hence, in the presence of increasing naproxen concentra­ tions. the decreasing cyclooxygenase activity was attended by increasing lipoxygenase activity. While indomethacin effectively inhibited cyclooxy­ genase activity, there was no attending increase in lipoxygenase activity. We presume the indomethacin also inhibits the insect lipoxygenase, as seen in some mammalian preparations. HEMOCYTES

We also documented eicosanoid biosynthesis by tobacco hornworm hemocyte preparations. We used fifth instars taken from our routine semisterile culture for these experiments. The larvae were anesthetized by chilling, and hemolymph was collected by pericardial puncture and immediately diluted in Manduca saline buffer. Hemocytes from 16 to 18 insects were pooled to obtain 5 mg to 7 mg of protein for each experiment. Pelleted pools of hemocytes were washed, then homogenized by sonification. Again, microsomalenriched preparations were prepared by centrifugation. The eicosanoid biosynthesis assays followed the protocols just described for fat body pre­ parations. The hemocyte preparations yielded two major products on our chroma­ tographic systems. One is the cyclooxygenase product PGA2, and the other is the lipoxygenase product 15-hydroxyeicosatetraenoic acid. The identifica­ tion of 15-hydroxyeicosatetraenoic acid is based on a single thin-layer chro­ matographic step, and undoubtedly that fraction cloaks more than one lipox­ ygenase product. For this reason, I will refer to this product simply as total lipoxygenase activity. As in the fat body preparations, the highest eicosanoid biosynthesis oc­ curred in short incubation periods. The two-minute incubations yielded about 2 pmol/mg protein/hour of PGA2 and about 7 pmol of total lipoxygenase activity. After longer incubations, five- and ten-minute periods, we recovered virtually no cyclooxygenase products and slightly reduced yields of total lipoxygenase products. The influence of protein concentration was expressed in different ways for cyclooxygenase and lipoxygenase activities. The opti­ mal protein concentration for cyclooxygenase activity was about 1.5 mg/ml. Alternatively, lipoxygenase activity increased in a linear way with increasing

I N V E R T E B R A T E IMMUNITY

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protein concentration from 0.25 to 2 mg/ml. Temperature, however, similarly influenced lipoxygenase and cyclooxygenase activities, with optimal eico­ sanoid biosynthesis at 30°C. We considered the influence of two eicosanoid biosynthesis inhibitors on eicosanoid biosynthesis by the hemocyte preparations. The cyclooxygenase inhibitor, naproxen, reduced cyclooxygenase activity by 88% at 0.1 mM and by 96% at 1.0 mM. Contrary to results with the fat body, the hemocyte lipoxygenase activity was not enhanced in the presence of naproxen. If any­ thing, lipoxygenase activity was reduced by about 20% in the presence of 1.0 mM naproxen. Reactions carried out in the presence of the lipoxygenase inhibitor, esculetin, also yielded reduced cyclooxygenase and lipoxygenase product formation. At 0.1 mM, esculetin nearly totally inhibited all eico­ sanoid biosynthesis. These inhibitors support the point that the eicosanoid biosynthesis observed on thin-layer chromatography plates is not an artifact of substrate auto-oxidation. However, the influence of these eicosanoid bio­ synthesis inhibitors is quite instructive. While these are well-characterized pharmaceutical compounds in mammalian systems, their actions in inverte­ brate systems can differ in important ways from the mammalian background. For example, indomethacin, a cyclooxygenase inhibitor, potently inhibited cellular defense reactions and cyclooxygenase activity in hornworm fat body preparations. This compound did not inhibit prostaglandin biosynthesis in preparations of male reproductive tracts from the cricket A. domesticus (Destephano et al. 1976). More to the point, naproxen is taken as a specific cyclooxygenase inhibitor. Our results with the hornworm fat body prepara­ tion would suggest that this is so in invertebrates as well, because low dos­ ages of naproxen (0.1 μΜ) effectively inhibited cyclooxygenase, but not lipoxygenase, activity. These results suggest that the influence of eicosanoid biosynthesis inhibitors should be investigated in each species, and each tis­ sue within a species, to ensure that the inhibitors act in known ways. This comment is quite relevant to our own work on invertebrate immunity. On the basis of a single experiment with naproxen, one might conclude that cyclooxygenase activity is essential to cellular immune reactions to bacterial challenge. However, the results of our biochemical experiments with he­ mocyte preparations make it clear that naproxen may act by inhibiting lipox­ ygenase as well as cyclooxygenase activities. This is one of the reasons we carry out inhibitor experiments with a substantial number of separate inhibitors. We also considered the subcellular localization of cyclooxygenase and lipox­ ygenase activities in hemocytes. In these exercises, we prepared microsomalenriched fractions as usual. These are the 11,500 g supernatant fractions of hemocyte sonicates. The 11,500 g pellet fractions are the mitochondrial frac­ tions. The 11,500 g supernatant fractions were then centrifuged at 100,000 g, creating a microsomal and a cytosolic fraction. We assessed eicosanoid bio-

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synthesis in the mitochondrial, microsomal, and cytosolic fractions. The cyclooxygenase activity was unevenly distributed among the three cellular fractions: about 5% in the mitochondrial fraction, 58% in the microsomal fraction, and about 36% in the cytosolic fraction. Most of the lipoxygenase activity (87%) was recovered in the cytosolic fraction, and the remaining 13% in the microsomal fraction. Eicosanoid-biosynthesizing enzymes are rather uniformly distributed within mammalian cellular fractions, with cyclooxygenases almost exclusively associated with endomembrane fractions of cells. We will return to this in chapter 10, where we will consider in more detail the shortcomings in our understanding of eicosanoid biochemistry in invertebrates.

Remodeling Fatty Acids among Complex Lipids in Hemocytes Recording very low proportions of 20:4n-6 in the tobacco hornworm fat body and hemocytes is consistent with the general picture of polyunsaturated fatty acids in terrestrial insects, as discussed in chapter 3. These low propor­ tions invite questions about insect fatty acid biochemistry. Do hemocytes actively maintain low proportions of 20:4n-6 and other eicosanoid-precursor polyunsaturated fatty acids? We investigated this issue by tracing the incor­ poration of four radioactive fatty acids into hemocyte lipids (Gadelhak and Stanley-Samuelson 1994). For these experiments, hemocytes were collected by pericardial puncture, then diluted in buffer. About 3.2 X IO 6 hemocytes were incubated in each incorporation experiment. We used four radioactive fatty acids, 18: ln-9, 18:2n-6, 20:4n-6, and 20:5n-3, in separate incorporation experiments. The reactions were started by adding one radioactive fatty acid to an Eppendorf tube containing hemocytes in 1.0 ml of diluted hemolymph. Individual tubes were incubated at 30°C in a shaking water bath for five, twenty, forty-five, or one hundred twenty minutes. The reactions were stopped by centrifuging the tubes at 200 g for ten minutes at 4°C. The cells were then gently washed three times to remove adherent radioactive fatty acids from the cell surfaces. This treatment does not disrupt the cells (Ogg et al. 1991). The hemocytes were then homogenized by sonication, and total lipids were extracted. The extracts were separated via thin-layer chromatography, resolving the phos­ pholipids. monoacylglycerols, diacylglycerols, and triacylglcyerols. Radioac­ tivity in bands corresponding to each fraction was assessed by liquid scin­ tillation counting. In some experiments, the phospholipid fraction was eluted from the gel, and rechromatographed on another thin-layer solvent system to resolve selected phosphoglycerides. Again, radioactivity in bands corre­ sponding to phosphatidylethanolamine, phosphatidylcholine, and phospha-

INVERTEBRATE IMMUNITY

145

tidylserine/phosphatidylinositol (these two fractions do not resolve on the system we used) was measured. Consistent with the general background of animal lipid biochemistry, the hornworm hemocytes incorporated all four of the radioactive fatty acids into cellular complex lipids. Of the four, 18:2n-6 was most efficiently incorpo­ rated into phospholipids, while 18:ln-9 and 18:2n-6 were efficiently incorpo­ rated into triacylglycerols. About 1% to 3% of the starting radioactivity in 18:ln-9 was recovered in diacylglycerols and monoacylglycerols. Very little of the other fatty acids were incorporated into these two fractions. The incor­ poration patterns were generally congruent with the fatty acid compositions of hemocytes. When exposed to longer incubation periods, the incorporated fatty acids are redistributed among lipid fractions in hornworm hemocytes (Gadelhak and Stanley-Samuelson 1994). This redistribution is due to selected hydro­ lysis of some components from phospholipids, allowing incorporation of other components. Chilton and Murphy (1986) documented this remodeling process in human neutrophils, showing that after initial incorporation into ester-linked phospholipids, radioactive 20:4n-6 was selectively remodeled into ether- and plasmalogen-linked phospholipid pools over time. We did not consider these phospholipid fractions in our analysis because they have not yet been sufficiently detailed in insect systems. We began by considering the remodeling of incorporated fatty acids from phospholipids to triacylglyerols (Gadelhak and Stanley-Samuelson 1994). After five-minute incubation periods, 100% of the radioactivity associated with 20:4n-6 and 20:5n-3 was recovered in the phospholipid fraction. After longer incubations, the radioactivity recovered in the phospholipid fraction declined, with concomitant increases in radioactivity in the triacylglycerols. For 20:4n-6, the radioactivity recovered in phospholipids declined to about 97% at twenty minutes, and to about 83% by 120 minutes. This directional shift was not seen with incorporated 18:2n-6 and 18:ln-9, although some of the radioactivity associated with these fatty acids was shifted between these two major cellular lipid fractions. For a variety of reasons, animal cells do not tolerate the presence of free fatty acids. Just to mention a couple, free fatty acids can disrupt mitochon­ dria function by breaking down proton gradients required for oxidative phos­ phorylation, and 20:4n-6 can exert regulatory actions on intracellular signal transduction systems. When cells are challenged by exogenous free fatty acids, their fatty acid incorporation systems are activated immediately to clear the unesterified components from the environment. During this early phase of incorporation, the free fatty acids are incorporated into complex lipids and certain other biochemical pathways in more or less unselective ways. In the minutes following this phase, cells typically begin a slower process of selective remodeling, aimed to yield fatty acid compositions that

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CHAPTER 5

are optimal for each lipid fraction within the biological context of each cell type. Hence, the low proportions of 20:4n-6 seen in hemocytes and other tissues in terrestrial insects may well result from selective remodeling processes. We derived a similar picture from an analysis of selected phospholipid fractions. After five-minute incubation periods, most of the radioactivity as­ sociated with 20:4n-6 was recovered in phosphatidylcholine. With longer incubations, the proportions of radioactivity recovered in phosphatidyl­ choline declined, with attending increased radioactivity in phosphatidylethanolamine. After 120-minute incubations, slightly more radioactivity asso­ ciated with 20:4n-6 was detected in phosphatidylethanolamine, rather than phosphatidylcholine. Again, these results support the idea that polyunsatu­ rated fatty acids are selectively remodeled among the complex lipid fractions in hornworm hemocytes. We have not yet carried out similar analyses of other insect tissues; nonetheless, similar remodeling dynamics are to be expected. Again, these processes help understand the low proportions of 20:4n-6 and other eicosanoid-precursor polyunsaturated fatty acids in terres­ trial insects.

The Pharmacology of an Eicosanoid Biosynthesis Inhibitor All investigations of the roles of eicosanoids in insect immune reactions to bacterial infections have so far relied on variations of a fairly simple strat­ egy. Basically, the influence of one or another of the many available eicosanoid biosynthesis inhibitors on a parameter of the defense reactions was recorded. In our first exploration, we recorded the influence of inhibitors on the ability of tobacco hornworms to clear bacterial infections from hemolymph circulation. Later, we recorded the influence of the inhibitors on num­ bers of microaggregates and mature nodules formed in response to bacterial challenge. Downer and his colleagues similarly assessed the roles of eico­ sanoids on other cellular parameters. The research results reported so far present a cheerfully bright picture of the identification of an important signal transduction system in invertebrate immunity. However, we should cast a darkening shadow upon this mural. The use of eicosanoid biosynthesis inhib­ itors, while potentially quite effective as an exploratory probe, is attended by several serious concerns (Stanley-Samuelson 1994b). For one, as just mentioned in the section on eicosanoid biosynthesis, we have little enough biochemical information on the influence of eicosanoid biosynthesis inhibitors, which have been very thoroughly characterized in many mammalian systems, on invertebrate eicosanoid-biosynthesizing en­ zymes. We saw, for example, that indomethacin potently inhibits prostaglan­ din biosynthesis in tobacco hornworm fat body preparations, but not in

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house cricket male reproductive tracts. Similarly, Wakayama et al. (1986b) reported that feeding adult houseflies, Musca domestica, on food laced (at 10% by weight) with eicosanoid biosynthesis inhibitors, including indo­ methacin, had no influence on eicosanoid biosynthesis by in vitro prepara­ tions of the housefly tissues. Plainly, we cannot be certain, on the basis of information from mammalian systems, that any given eicosanoid biosynthe­ sis inhibitor actually inhibits eicosanoid biosynthesis in an invertebrate ex­ perimental system. A second concern lies in the pharmacology of the compounds we use in invertebrates. In simple terms, we have very little knowledge of the move­ ments or metabolic fates of eicosanoid biosynthesis inhibitors in inverte­ brates. Again, we saw that indomethacin did not inhibit eicosanoid biosyn­ thesis in house cricket male reproductive tract preparations. Alternatively, Murtaugh and Denlinger (1982) maintained a group of house crickets on diets amended with indomethacin. After several days on these diets, they found reduced levels of PGE2 and PGF2a in testes from the experimental males and spermathecae from mated females. These findings suggest that indomethacin can move from the alimentary canals to at least two tissues in house crickets. They also suggest the possibility of substantial biochemical differences in the action of these compounds in whole animals and enzyme preparations. We addressed these issues by investigating the pharmacology of indo­ methacin in tobacco hornworms. Using the usual fifth instars, we injected radioactive indomethacin into the hemocoels of the animals, then traced its movement, excretion, and metabolism. In the first experiment, we followed the clearance of radioactive indo­ methacin from hemolymph circulation. As in all injections, the needle of the syringe was inserted into the intersegmental suture between the last two spiracles, taking care to keep the needle parallel to the body wall. This is important to avoid injuring or penetrating the alimentary canal. After three-, ten-, twenty-, and thirty-minute incubation periods, hemolymph was col­ lected by pericardial puncture. The volume of hemolymph was determined; then, the indomethacin and its potential metabolites were extracted from the hemolymph. The extraction solvent was evaporated and the dried extract was dissolved in ethanol. An aliquot of each sample was transferred to a vial for liquid scintillation counting. We found that more than 99% of the radioac­ tivity associated with injected indomethacin was cleared from hemolymph circulation within the first three minutes after injection. We found that the indomethacin was rapidly taken up by hornworm tis­ sues. In these experiments, the tissues were isolated, then processed for indo­ methacin extraction. Again, an aliquot of the extract was transferred to liquid scintillation counting vials. The remainder of each sample was applied to thin-layer chromatography plates to separate the injected indomethacin from

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potential metabolites. Radioactive indomethacin was recovered from all tis­ sues analyzed, including integument, ventral nerve cord, silk gland, fat body, Malpighian tubules, and gut epithelium. The greatest amount of radioactivity was recovered from the fat body, the largest tissue. On the other hand, when normalized to wet tissue weight, most radioactivity was recovered from the silk glands. These results indicate that indomethacin is probably distributed among all tissues in this insect: however, as seen in mammals, the compound is not uniformly distributed. Less radioactivity was recovered from the inte­ gument and nerve cord, while more was recovered from the fat body, silk gland and Malpighian tubules. This is also consistent with the pharmacology of indomethacin in mammals. The extraction system we used to recover indomethacin and its metabo­ lites from hornworm tissues provided information on the metabolic fate of indomethacin. Indomethacin is highly soluble in chloroform and other or­ ganic solvents, and it is virtually completely insoluble in water. Rigorous multiple extraction procedures yielded about 90% of the recovered radioac­ tivity in the collected organic phases. The remaining 10% of the radioac­ tivity was detected in the aqueous phases. This 10% represents polar metab­ olites of indomethacin. taken to indicate that about 10% of the injected indomethacin was metabolized into water-soluble products. We used the thin-layer chromatography system to determine whether the radioactivity recovered from the hornworm tissues was associated with indo­ methacin metabolites of intermediate polarity—that is. products that are sol­ uble in organic solvents, but still more polar than indomethacin. We saw no evidence for indomethacin metabolism in most tissues, including integu­ ment. nerve cord, fat body. Malpighian tubules, and gut epithelium. Virtually all of the radioactivity recovered from these tissues co-chromatographed with authentic indomethacin on thin-layer chromatography. Contrary to re­ sults with other tissues, the silk gland produced at least one polar product of indomethacin. About 75% of the radioactivity recovered in the organic phases of silk gland extracts co-chromatographed with indomethacin, and about 25% remained at the origin of the thin-layer plates. These data indicate that most hornworm tissues are not competent to metabolize indomethacin to a great extent. Hence, the material taken up into virtually all hornworm tissues is present as indomethacin. We then recorded the excretion of radioactive indomethacin. In this exper­ iment. radioactive indomethacin was injected into the hemocoels of fifth instars. The insects were held in individual cups, and frass pellets were col­ lected every two hours for the following forty hours. Indomethacin was extracted from the frass. and the radioactivity in an aliquot of each sample was determined by liquid scintillation counting. We recovered about 56% of the injected material over the forty-hour incubation period. A trace of radio­ activity appeared in the frass as early as two hours after injection, however.

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almost half of the injected radioactivity was recovered between four and twelve hours. These data show that substantial amounts of injected indo­ methacin remains in the hornworm tissues, in its original form, during the first twelve hours after treatment. As a practical point, the active pharma­ ceutical product was present in the hornworm throughout the time course of our immunology experiments. We analyzed the products extracted from the frass on thin-layer chroma­ tography. These exercises showed the presence of three to five indomethacin metabolites in frass, accounting for about 30% of the recovered radioactivity. Reasoning that a factor in the frass, perhaps enteric microbes, was responsi­ ble for the apparent metabolism, we incubated radioactive indomethacin with freshly collected frass pellets. After forty-eight-hour incubation periods, the indomethacin and possible products were extracted from the frass and analyzed on radio-high-performance liquid chromatography. These data showed that about 10% of the starting material was metabolized into more polar products. Similar incubations in buffer did not yield any indomethacin metabolites. We inferred from this result that one or more factors in horn­ worm frass are responsible for indomethacin metabolism prior to excretion. Let us juxtapose three points of information. One, indomethacin impairs the ability of tobacco hornworms and other insect species to form nodules in response to intrahemocoelic bacterial challenges. Two, indomethacin inhibits cyclooxygenase and lipoxygenase activities in fat body preparations. Three, indomethacin remains in hornworm tissues in unaltered form for several hours following injection. We may assert on the basis of these points that indomethacin is an appropriate probe for assessing the roles of eicosanoid biosynthetic pathways in the cellular immunity of tobacco hornworms. I say tobacco hornworms, rather than insects or invertebrates, because there are substantial differences in the pharmacology of indomethacin among mam­ malian species. For example, dogs and guinea pigs require about twenty minutes to clear 50% of injected indomethacin doses from blood circulation (Yesair et al. 1970), whereas rats require about four hours (Hucker et al. 1966). The distribution of indomethacin among tissues within mammals also differs among species. Rats maintain higher concentrations of indomethacin in blood circulation than in tissues at all times after injection. In guinea pigs, indomethacin is concentrated from blood into the liver, kidney, and small intestine. The excretion of indomethacin also differs among mammals (Hucker et al. 1966, Yesair et al. 1970). Nearly all injected indomethacin is excreted in feces in dogs, while rabbits excrete most injected indometh­ acin in urine. Guinea pigs and humans excrete about half of injected indo­ methacin in urine, and the remainder in feces. These findings emphasize differences in distribution, metabolism, and excretion of injected indometh­ acin among mammals. We can expect similar differences among inverte­ brates. Indeed, we have already drawn attention to the different biochemical

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effects of indomethacin on eicosanoid biosynthesis. Overall, this work points to the importance of understanding the pharmacology of eicosanoid bio­ synthesis inhibitors in our work on assessing the roles of eicosanoids in invertebrates.

From Tobacco Hornworms to Other Insect Species A theme of this volume lies in recognizing the great potential for many departures from the standard eicosanoid model, as represented by the litera­ ture on eicosanoids in mammalian systems. A springboard for this line of thinking is the substantial variation in eicosanoid systems among mam­ malian species and among tissues within a mammalian species. Relative to invertebrates, as noted in chapter 2, some invertebrates use completely dif­ ferent biosynthetic pathways to produce prostaglandins. Other invertebrates generate bizarre eicosanoids, such as the prostaglandin 1.15-lactone. Such departures from the standard model are to be expected among invertebrates, and I think more will emerge as research activities continue to generate new information. Despite appearances formed by the literature cited in this vol­ ume, our understanding of eicosanoids in invertebrates remains thin and un­ evenly distributed. In this light, it remains important to document the pres­ ence of eicosanoid-biosynthesizing systems in invertebrates thought to use eicosanoids. On this reasoning, we investigated at least some aspects of eicosanoid biosynthesis in several insect species in which eicosanoids have been evoked. In our work on the tenebrionid beetle, Z atratus. Miller et al. (1996) documented the presence of trace amounts of 20:4n-6 in six tissues, includ­ ing midgut. Malpighian tubules, integument, fat body, hindgut, and head. In a more detailed study of the fatty acids in lipids from two tissues, the fat body and Malpighian tubules, we recorded more than trace amounts of 20:3n-6, 20:4n-6. and 20:5n-3 in phospholipids, showing that proportions of these components change with life stage and tissue (Howard and StanleySamuelson. 1996). We also showed that microsomal-enriched fat body prep­ arations express a cyclooxygenase and lipoxygenase activity. We recorded biosynthesis of PGA2, PGE2. PGF2a. PGD2, and a hydroxyeicosatetraenoic acid. Similarly, we recorded the presence of 20:4n-6 in fat body phospholipids from true army worms and black cutworms (Jurenka et al. 1997). The fat body from these larvae also expressed an intracellular phospholipase A2 that can hydrolyze 20:4n-6 from cellular phospholipids. At about 26 pmol/mg protein/hr. the phospholipase A2 activity in the black cutworm fat body was somewhat lower than the activity recorded for hemocyte preparations from tobacco hornworms (Schleusener and Stanley-Samuelson 1996). The true

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armyworm phospholipase A2 expressed even lower rates of catalytic activity (about 4 pmol/mg protein/hr). However, these exercises were carried out to document the presence of the enzyme, and the results do not represent opti­ mal activities. We also showed that fat body preparations from both species are able to convert radioactive 20:4n-6 into PGA2, PGD2, PGE2 and PGF2c,. We conducted a similar line of documentation exercises, showing the presence of an eicosanoid-biosynthesizing enzyme in the fat body from silk­ worms, B. mori (Stanley-Samuelson et al. 1997). We recorded the presence of 20:4n-6 at about 1.5% of fatty acids associated with phospholipids iso­ lated from the silkworm fat body. A fat body intracellular phospholipase A2 can hydrolyze 20:4n-6 from the sti-2 position of phospholipids, although the catalytic rate was, again, not so high as seen in tobacco hornworm hemocyte preparations. The silkworm fat body is also competent to produce eicosanoids. Unlike the fat body from the other insects just mentioned, a lipox­ ygenase product, one or more of the hydroxyeicosatetraenoic acids, was the major product. Miller et al. (1999) recorded traces of 20:4n-6 in the fat body of adult crickets, G. assimilis. We also detected biosynthesis of three eicosanoids by fat body preparations, PGA2, PGE2, and a hydroxyeicosatetraenoic acid. As in the silkworm fat body preparations, the lipoxygenase product(s) was the major product produced by the cricket fat body preparations. The point of these exercises, again, is to document the presence of an eicosanoid biosynthesizing system, albeit at a superficial level, in insects thought to use eicosanoids in cellular immune signal transduction mecha­ nisms. Even at this superficial level of analysis, however, we recorded sub­ stantial differences in the eicosanoid systems among these few insect spe­ cies. More detailed characterizations of these elements of eicosanoid biosyn­ thesis in these species will undoubtedly reveal more differences. As the eicosanoid hypothesis gains more support, the biochemistry of these systems will merit considerably more research attention.

C H A P T E R 6

Eicosanoids in Invertebrate Ion Transport Physiology

IT IS GENERALLY thought that cellular life evolved in watery environments. Cells live in more or less aqueous environments, and virtually all cells have evolved physiological mechanisms to maintain homeostasis of ion and water balance. A couple of examples illustrate this point. Some bacteria actively transport protons outward across their membranes, maintaining lower intra­ cellular proton concentrations relative to their extracellular environments. The potential energ\ in the resulting proton gradient is used to drive active transport mechanisms to import solutes (Darnell et al. 1990). Within mam­ mals. circulating lymphocytes respond to water loss by active influx of so­ dium and chloride ions. The resulting osmotic pressure gradient results in water moving into the cell, thereby restoring homeostasis (Grinstein et al. 1985). The various mechanisms involved in moving ions and water across cellular membranes are ubiquitous phenomena. These cellular events are responsible for maintaining homeostasis of water and solutes at both the cellular and the organismal levels. The cells of metazoan animals are in approximate osmotic balance with their extracellular fluid compartments. Terrestrial animals tend to lose water to their environ­ ments. and they have mechanisms to restrict and offset water losses (Prosser 1973). Freshwater animals tend to maintain their extracellular fluid compart­ ments hyperosmotic to their environments. Marine invertebrates are typically isosmotic to seawater. Still other animals live in waters that are subject to rapid changes in osmotic concentration, such as estuaries and rocky pools in intertidal zones. The osmotic homeostasis of all animals is subject to fre­ quent challenges at cellular and organismal levels. Many homeostatic mechanisms are expressed at the organismal level. Ter­ restrial insects reduce water loss with a small layer of hydrocarbons and other lipids on their integument. Some animals drink water to offset losses, while others can absorb water from humid atmospheres. The respirator}' sur­ faces of terrestrial animals are internalized. And some animals are able to sustain extreme dehydration. For example. Ian ae of the midge PolypedUum \ anderphmki. live in ephemeral pools on rocks in Africa. These pools evap­ orate in the dry season, and the lan ae can tolerate nearly complete dehydra­ tion until the following rain\ season (Hinton 1960). Cellular mechanisms for maintaining water balance are integrated into organismal level reactions to

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osmotic challenge. Vertebrate kidneys and the Malpighian tubules of insects and certain other invertebrates, for example, are influenced by diuretic and antidiuretic hormones to eliminate or conserve body water or solutes. Let us consider a very brief sketch of the mechanisms involved in solute transport, drawn from Alberts et al. (1994). The lipid bilayers of cellular membranes are fairly permeable to water and relatively impermeable to bio­ logically important ions, such as chloride, sodium, potassium, magnesium, and calcium. These ions are transported across membranes by two classes of membrane-associated proteins, namely channels and carrier proteins. Chan­ nels are transmembrane proteins that form hydrophilic pores through mem­ branes. The pores are generally selective, allowing specific ions, such as potassium or calcium, to pass through. Carrier proteins also bind specific solutes, which are transported due to conformational changes in the carriers. All channel-mediated and some carrier-mediated transport actions are driven solely by local electrochemical gradients, known as passive transport or facilitated diffusion. Some carrier proteins are associated with an energy source, and these carriers transport solutes against sometimes steep electro­ chemical gradients. Some carrier proteins move a specific solute across biomembranes, and others act as coupled transporters. In coupled transport, the same protein is responsible for movement of one solute, coupled with move­ ment of another solute. Symports move the two solutes in the same direc­ tion, and antiports move the two solutes in opposite directions. The ubiqui­ tous sodium-potassium antiport is a coupled transporter responsible for actively pumping sodium ions out of cells, and potassium ions into cells. Passive and active transport are also responsible for moving water across membranes. Again, membranes are fairly permeable to water, and local os­ motic pressures drive water movements. This brief glimpse allows us to regard homeostasis of water and solute concentrations at the organismal and cellular levels as the outcome of regulated actions of specific intracellular proteins. Eicosanoids are among the regulatory elements. As usual, most of our knowledge of the roles of eicosanoids in the physiology of water and solute homeostasis comes from studies on mam­ malian systems, particularly the kidney. Nephrons are the operative cells in kidney water and solute transport, and eicosanoid biosynthesis and actions vary along the nephrons (Bonvalet et al. 1987). In one section of the nep­ hron, the cortical collecting tubule, PGE2 is the major eicosanoid product. In experiments with isolated nephrons, exposure to PGE2 on the basolateral side of the cortical collecting tubule resulted in a nearly 70% reduction in the transport of sodium from the lumenal to basolateral side of the tubule. Subsequent experiments showed that PGE2 inhibited action of the basolateral membrane sodium-potassium antiport in the cortical col­ lecting tubule. Hence, the influence of eicosanoids on the mammalian kid­ ney can be appreciated in terms of their actions, typically expressed

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through intracellular messenger systems, on specific proteins responsible for membrane transport. The first indications that eicosanoids acted in the physiology of fluid se­ cretion or ion transport in invertebrates came from two papers by Dalton (1977a. b). Fluid secretion by salivary glands isolated from the blowfly, Calliphora erythrocephala. can be stimulated by exposure to the biogenic am­ ine. serotonin. In this system, the serotonin acts as an external hormone, interacting with cell surface receptor sites. The serotonin-receptor site inter­ action stimulates increased concentrations of cAMP within the salivary gland cells (Berridge 1970. Prince et al. 1972). and the increased intracellu­ lar cAMP concentrations lead to increased secretion of a isosmotic po­ tassium-rich fluid. Dalton (1977a) investigated the influence OfPGE1 on sali­ vary gland physiology. He found that treating isolated salivary glands with low doses (10~' to 10 9 M) of PGE1 did not alter their basal fluid secretion rates. On the other hand, such low doses of PGE1 attenuated the usual stimu­ latory influence of serotonin on fluid secretion rates. As just mentioned, it was thought that serotonin stimulated fluid secretion rates by causing in­ creased intracellular cAMP concentrations. If this was so. the attenuating influence of PGE1 could be due to inhibiting the enzyme responsible for biosvnthesizing cAMP. adenylate cyclase, or due to stimulating the activity of phosphodiesterase, the enzyme that inactivates cAMP through a single catabolic step. Dalton (1977b) suggested that PGE1 downregulates adenylate cyclase, with no influence on phosphodiesterase. It appeared that at least one eicosanoid. PGE1. played an important physiological role in fluid secretion physiology in an invertebrate. Of course, this idea suffered from the usual shortcomings. There was very little evidence for polyunsaturated fatty' acids or eicosanoids in invertebrates at the time, and no evidence on this point for blowfly salivary glands. Per­ haps more important, there was virtually no context, aside from the mam­ malian background, for appreciating the work on salivary glands from an invertebrate. In any case, work on eicosanoids in blowfly salivary gland fluid secretion has not progressed. John Phillips and his colleagues have developed a lengthy line of research on transport physiology in the locust rectum. Interest in the physiology of the rectum stems from appreciation of excretory processes in insects, which involve two organ systems (Chapman 1982). The Malpighian tubules are secretory structures, responsible for forming a primary urine that is isosmotic to the hemolymph. Selective resorption of water and solutes, according to the instantaneous condition of the animal, takes place in the rectum. Hence, kidney function in insects is distributed. Hanrahan (1978) reported that treat­ ing isolated recta with either 20:4n-6. PGE1. PGE2 or PGF2a (dosages not reported) led to increased chloride transport. The eicosanoids may stimulate increased intracellular cAMP concentrations, which in turn lead to increased

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chloride transport (Phillips 1980). Inquiry into the roles of eicosanoids in the locust rectum, as seen in the blowfly work, flagged after these reports. In this case, however, we will see that the issue was taken up again fifteen years later. Eicosanoids exert regulatory actions on ion transport in other inverte­ brates. In his research on ion homeostasis in the freshwater mussel, Ligumia subrostrata, Dietz and his colleagues recorded a diurnal rhythm in the blood concentration of sodium, from which they inferred that an endocrine mecha­ nism regulates sodium transport physiology in mussels (Yeider and Dietz 1978). They formed the hypothesis that prostaglandins may be part of the regulatory mechanism. To test this idea, they acclimatized mussels in artificial pond water. In their first line of experiments, the mussels were injected with either ethanol for controls, or with selected drugs (Graves and Dietz 1979). After an equili­ bration period, the mussels were placed in separate containers spiked with radioactive sodium. The researchers used flame photometry to determine to­ tal amounts of sodium in the experimental containers. Changes in total so­ dium were taken as net flux. The reduction in radioactive sodium in the baths was taken to represent the influx into the mussels. Outflux was calcu­ lated as the difference between the two measures. The authors recorded a slight positive influx of sodium in control animals, as expected in freshwater mussels. Injections of low doses of PGE2 resulted in nearly 80% reductions in sodium uptake by the mussels. The prostaglan­ din treatments did not significantly influence sodium efflux, and the authors concluded that PGE2 regulates the organismal influx of sodium through its influence on sodium uptake. This idea is supported by the results of experi­ mental mussels treated with the cyclooxygenase inhibitor, indomethacin. The sodium influx approximately doubled in these mussels. Together, these ex­ periments suggest that PGE2 regulates sodium influx. The authors considered the presence of prostaglandins in the mussels. They collected blood samples, then extracted prostaglandins by standard pro­ cedures. The extracts were separated on thin-layer chromatography. After making the separated spots visible, they found spots exhibiting the chroma­ tographic behaviors of 20:n-6 and authentic eicosanoids, including PGE2. Two other spots may have been keto metabolites of the prostaglandins. While it appears that the mussels have prostaglandins in their tissues, there are a couple of unresolved issues with this work. For one, we do not know how much blood was used in these exercises, and we have no quantitative information on the PGE2 that was isolated. For another, the authors spiked the animals with radioactive 20:4n-6 to gauge their extraction efficiencies. However, they did not try to assess prostaglandin biosynthesis by separating the extracted radioactivity on their thin-layer chromatography system. This is the first report on the physiological action of eicosanoids in mod-

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ulating ion transport in an invertebrate (Graves and Dietz 1979). The oppo­ site effects of injected indomethacin and PGE2, taken with preliminary deter­ mination of prostaglandins in the mussel tissues, effectively launched their hypothesis that prostaglandins influence ion transport in a mollusc. The authors advanced their case by recording sodium uptake by gills iso­ lated from L. subrostrata (Dietz and Graves 1981). Sodium transport rate of untreated gills was about 1.3 μπιοΐ/gram dry gill/minute, on par with the estimated transport rates in intact mussels. Incubating the gills in the pres­ ence of serotonin increased the sodium transport rate in a dose-dependent manner (10 6 to IO-4 M) to a maximum of 80% over control rates. More­ over, incubations in the presence of cAMP, in the form of dibutyryl cAMP, also resulted in increased sodium uptake to a maximum of about 60% more than control rates. Drawing on their findings with prostaglandins, the authors speculated that the link between serotonin and prostaglandins may be their influence on intracellular cAMP concentrations (Dietz and Graves 1981). In a subsequent paper (Graves and Deitz 1982), the authors provided addi­ tional details associated with their first work on prostaglandins (Graves and Deitz 1979). They used intact mussels, Carunculina texasensis and L. sub­ rostrata, in the experiments just covered for their first work. They found that the influences of indomethacin on increased sodium influx and of PGE2 on sodium efflux were expressed in a dose-dependent manner. Again, they re­ corded increased sodium uptake in cAMP-treated mussels. They also demon­ strated that cGMP had no influence on sodium transport, indicating that the cAMP effect was specific. They advanced the validity of their hypothesis by recording the biosynthesis of prostaglandins from radioactive 20:4n-6. These exercises indicated biosynthesis of PGE2 during five-minute incubations. Longer incubations yielded unclear results; however, the inactive dehydrogenated products of PGE2 and PGF2oi may be present in their chromatograms. Their chromatograms also indicate the possibility of a lipoxygenase product, although this was not discussed. These findings add support to their hypoth­ esis that prostaglandins are involved in regulation of sodium uptake by freshwater mussels. Saintsing and Dietz (1983) continued this line of research on sodium transport in L. subrostrata. They found that several eicosanoid biosynthesis inhibitors, including the phospholipase A2 inhibitor dexamethasone, and the cyclooxygenase inhibitors meclofenamate and indomethacin, stimulated so­ dium influx. As seen earlier, PGE2 or PGF2a injections inhibited sodium uptake by inhibiting sodium influx. The prostaglandins did not influence so­ dium efflux. The inhibitory influence of PGE2 injections could be reversed by treating the mussels with dibutyryl cAMP. The authors also provided evidence of the interactions between serotonin and prostaglandins. As seen before, serotonin injections stimulated increased sodium uptake. The serotonin effect was substantially increased, by about

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60%, by treating mussels with a cocktail of serotonin and meclofenamate. It appears that serotonin increases sodium transport though its influence on intracellular cAMP concentrations. Because additional exogenous cAMP, ad­ ministered as dibutyryl cAMP, which is readily taken into cells, stimulated sodium uptake, it can be inferred that serotonin acts by stimulating adenylate cyclase activity. PGE2 apparently downregulates adenylate cyclase activity. The influence of the cyclooxygenase inhibitor, meclofenamate, would be to attenuate the downregulation by PGE2. The hypothesis that prostaglandins influence sodium uptake was tested more rigorously by investigating the biochemistry of 20:4n-6 metabolism in mussel gills (Saintsing et al. 1983). They determined the fatty acid composi­ tion of total lipid extracts prepared from isolated gills, finding that 20:4n-6 comprised about 14% of total fatty acids. They also showed that gill homogenates were able to convert radioactive 20:4n-6 into three peaks of radioac­ tivity, generated by scanning thin-layer plates. One matched the chroma­ tographic behavior of PGE2, one corresponded to PGF 2a , and the third was an unlabeled peak that might represent one or more lipoxygenase products. Their published radio-chromatograms indicated that boiled control prepara­ tions did not convert radioactive 20:4n-6 into eicosanoids, and that incuba­ tions in the presence of meclofenamate inhibited cyclooxygenase, but not the apparent lipoxygenase activity. Dietz and his colleagues later showed the gill produces two major lipoxygenase activities, 5- and 12-hydroxyeicosatetraenoic acids, although the physiological significance of these products is unknown (Hagar et al. 1989). The authors also used radioimmunoassays to determine the presence of PGE2 and PGF2ct in blood withdrawn from the mussels (Saintsing et al. 1983). The data from these experiments are presented in a table that does not quite correspond to the experimental protocol. In these experiments, mussels were acclimatized to pond water or to deionized water. The authors first determined the net sodium flux of each mussel. They then determined the quantities of prostaglandins in blood drawn from the mussels. The pond water mussels experienced very low sodium fluxes, and the PGE2 concentra­ tions in the blood of these animals averaged 0.39 ng/ml. Sodium flux rates were stimulated in the deionized water mussels, in which the PGE2 concen­ trations were reduced by nearly 50% relative to the pond water mussels. Similar exercises with PGF 2a indicated that sodium flux rates were not con­ nected to changes in blood PGF 2a concentrations. They also found reduced prostaglandin concentrations in blood from mussels that had been treated with eicosanoid-biosynthesis inhibitors two or three hours prior to analysis. Saintsing et al. (1983) considered the possibility that sodium flux rates and blood PGE2 concentrations were inversely related by plotting sodium flux rates as a function of PGE2 concentrations. They obtained a significant linear relationship, from which they concluded that the two variables are inversely

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related. As expected, the blood concentrations of PGF2a were not related to sodium flux rates. We can infer from this series of papers that eicosanoids, specifically PGE2, are major players in regulating sodium flux rates in gills of a freshwater mussel. Certainly, there is plenty of opportunity to investigate the intracellu­ lar relationship among serotonin, PGE2, and cAMP in more detail. As it stands, however, it appears that PGE2 acts in ion transport physiology in this representative mollusc. Their hypothesis is supported by another line of work on a marine bivalve. Freas and Grollman (1980) reported on their investigations into the influ­ ence of magnesium deprivation and hypoosmotic stress on the release of prostaglandins from gill tissue of the marine bivalve, Modiolus demissus. For these experiments, the animals were collected, then acclimated to artifi­ cial seawater in the laboratory. The authors prepared isolated gill tissues for most of their experiments. The tissues were incubated in various forms of artificial seawater, and the release of prostaglandins into the seawater was monitored by radioimmunoassay. To show the presence of prostaglandins in the mussel tissues, six tissues were isolated and extracted for prostaglandins. Prostaglandin concentrations were determined by radioimmunoassay. This work was conducted in the late 1970s, before antibodies specific to each of the major prostaglandins became commercially accessible. The available antibody was specific to PGB. The standard protocol for determin­ ing prostaglandin quantities involved separating PGE and PGA by column chromatography, then chemically converting these prostaglandins into PGB. The radioimmunoassays were then performed using PGB1 to generate stan­ dard curves. This protocol is beset with pitfalls, and the authors put effort into assuring the validity of their results. They used radioactive PGE to de­ termine the extent of converting PGE to PGB. Instead of using the PGB supplied with the commercial radioimmunoassay kits, they used PGE1, which they converted to PGB, to generate their standard curves. They also conducted assays on seawater they spiked with known amounts of pros­ taglandins. Their extra efforts add validity to their results. They first showed that isolated gills could release prostaglandins into arti­ ficial seawater. Tissues were incubated with added 20:4n-6 and secreted sub­ stantial amounts of PGA and PGE into the medium, while tissues incubated without 20:4n-6 did not. They also incubated tissues in the presence of stan­ dard co-factor mix (1 mM 5-hydroxytryptamine and 1 mM glutathione) plus added substrate. These tissues released about the same amount of PGE (1500 pg/ml seawater), but virtually no PGA. The authors thereafter refer simply to release of immunoreactive pros­ taglandins, but we cannot be sure whether they mean the measured PGE or the sum of PGA and PGE. Nonetheless, their next experiments revealed the influence of the quality of seawater on prostaglandin secretion.

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In the first experiment, gills were acclimatized in 100% seawater, then transferred to 75%, 50%, or 25% artificial seawater and incubated for 30, 60, or 120 minutes. These treatments placed the gills in hypoosmotic challenge. The gills did not respond to the 75% and 50% treatments. Contrarily, the shift from 100% to 25% seawater resulted in substantially increased secre­ tion of prostaglandins into the surrounding water. Freas and Grollman (1980) concluded that hypoosmotic challenge caused the biosynthesis and release of prostaglandins. M. demissus is a euryhaline osmoconformer, and the authors drew on the literature to document the ability of these mussels to withstand osmotic challenges in this range for more than three weeks. In the next series of experiments, the authors developed five separate forms of artificial seawater, all of equal osmotic strength. These were the normal artificial seawater, and sodium-, calcium-, potassium-, and magne­ sium-free artificial seawater. In separate experiments, isolated gills were incubated for sixty minutes in each seawater preparation. The release of prostaglandins was significantly increased in gills incubated in the magne­ sium-free seawater. It would appear that two challenges resulted in increased release of prostaglandins from the mussel gills, a hypoosmotic challenge and a specific ionic challenge. The authors considered the possibility that the apparent hypoosmotic challenge was merely a reflection of reduced magne­ sium in the 25% seawater incubations. Gill tissues were incubated for sixty minutes in 20% artificial seawater and in 20% artificial seawater supple­ mented with magnesium to model the natural magnesium concentration in seawater. In this experiment, the gills incubated in 20% seawater released more prostaglandins than the gills incubated in 20% magnesium-supplemented seawater. Hence, the release of prostaglandins in response to the hypoosmotic challenge is independent of the release in response to the mag­ nesium challenge. The authors used their radioimmunoassay procedure to determine the presence of prostaglandins in several mussel tissues, including gill, upper visceral mass, lower visceral mass, mantle, the mantle edge at the siphon, and the posterior adductor muscle. All tissues yielded prostaglandins; how­ ever, there were substantial differences among the tissues. High levels of prostaglandins were recovered from the gill and upper visceral mass, and low levels from the lower visceral mass and mantle. The mantle edge and muscle yielded intermediate results. Gills pretreated by incubating with indomethacin and, separately, with aspirin yielded very low amounts of pros­ taglandins. Freas and Grollman (1980) concluded that the mussels are com­ petent to biosynthesize prostaglandins. The prostaglandins are produced and secreted from gills in response to osmotic stress and magnesium shortage. Of course, the roles of the prostaglandins in the other tissues remain to be uncovered. In mammalian systems, the E-, F-, D-, and !-prostaglandins act through

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cell surface receptors. The prostaglandin receptors are linked to intracellular events through G proteins (Smith 1989). We now have a great deal of know­ ledge on eicosanoid receptors in mammalian systems, sufficient to generate a sophisticated classification of eicosanoid receptors (Coleman et al. 1990, Negishi et al. 1993). Freas and Grollman (1981) provided the first report on prostaglandin receptors in an invertebrate system. They extended their work on the marine bivalve, M. demissus, by showing that the gill expressed spe­ cific receptors for PGA2. They developed a fairly classical receptor binding assay for this work. Gills were isolated from the mussels, then equilibrated in seawater for one hour to recover from the surgical procedure. The tissues were homogenized, roughly filtered, then centrifuged at 12,000 g. The resulting supernatant frac­ tion was the receptor source. The supernatants were incubated with radioac­ tive prostaglandin and a range of unlabeled prostaglandin. To terminate the reactions, cold buffer was added to the reaction mixture, and the entire solu­ tion was filtered. The filters were rinsed and radioactivity retained by the filters was assessed by liquid scintillation counting. The authors calculated total binding as the total amount of radioactivity bound to the supernatant, minus binding in a blank tube. Nonspecific binding was calculated by deter­ mining the amount of radioactivity bound to the supernatant in the presence of 100 g of unlabeled prostaglandin, minus blank values. Specific binding was the difference between total binding and nonspecific binding. They considered two radioactive ligands, PGE2 and PGA2. They reported evidence for specific PGA2, but not PGE2 binding sites. Total binding and specific binding for PGA2 was dependent on pH, with maximal binding around pH 8.5 to 9.0. In time course studies, they recorded increased specific binding with time up to thirty minutes, after which binding reached a plateau. The binding of PGA2 was reversible by adding unlabeled PGA2 to incuba­ tion tubes. The prostaglandin binding sites in the gill preparation were spe­ cific to PGA2. Incubations in the presence of unlabeled PGF2a did not influ­ ence the PGA2 binding, and similar experiments with unlabeled PGE2 only slightly displaced PGA2 binding. These data support the idea that the mussel gill expresses specific, saturable, reversible PGA2 binding sites. Adding calcium or magnesium to the reaction mixtures enhanced the binding of PGA2 to the supernatant fractions (Freas and Grollman 1981). In these experiments, the gill preparations were dialyzed before the binding assays were carried out. Then, in separate series of experiments, binding assays were conducted in the presence of calcium and magnesium in varying concentrations. Calcium increased binding in the range of 4 to 20 mM, and magnesium in the range of 3 to 110 mM. The natural concentrations of these ions in seawater (IOmM for calcium and 54 mM for magnesium) fall within these concentration ranges. Freas and Grollman (1981) speculated that

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changes in the concentrations of these ions in nature may influence pros­ taglandin binding sites in the gill tissue. In this report, the authors also registered the presence of prostaglandin binding sites in other mussel tissues, including the mantle, siphon, adductor muscle, upper visceral mass, and lower visceral mass. Compared in terms of total binding/mg tissue protein, the adductor muscle bound considerably more PGA2 than the other tissues, while the upper visceral mass bound much less. They also report a small, but not quite negligible, amount of PGE2 binding in these tissues, except for the gill, which did not bind PGE2. We may conclude that most tissues in this marine mollusc express prostaglandin binding sites. Our understanding of eicosanoid receptors in mammalian systems has ad­ vanced far beyond the information available at the time of the Freas and Grollman (1981) paper. We now know that mammalian systems do not ex­ press cell surface receptors for the cyclopentenone prostaglandins, including A12-PGJ2 and PGA2. These prostaglandins are taken into cells via a specific transporter, and they accumulate in the nucleus. Once in the nucleus, they bind to thiol groups of nuclear proteins. The prostaglandin-protein conju­ gates stimulate protein synthesis (Negishi et al. 1995b). In light of this infor­ mation, the PGA2 binding sites in gill and other tissues in a marine bivalve take on considerable interest. We do not know that PGA2 occurs naturally in the mussel, and can not yet speculate on the biological significance of a PGA2 binding site in its tissues. Coupled with the early work on an insect salivary gland and the locust rectum, these findings with a freshwater and a marine mussel amount to a strong statement that prostaglandins are involved in the physiology of ion and water movement in invertebrates. The work on PGA2 binding sites does not add clarity to the roles of eicosanoids in ion transport, but it represents the first effort to identify prostaglandin binding sites or receptors in an inver­ tebrate animal. In chapter 9 we will see that this remains one of only two papers on the topic. Research in this area has progressed, and in the 1990s there emerged more evidence on the roles of eicosanoids in invertebrate ion and water transport. Drawing on the work just described, Petzel and Stanley-Samuelson (1992) set forth the hypothesis that prostaglandins modulate basal fluid secretion rates in Malpighian tubules of female yellow fever mosquitoes, Aedes aegypti. We tested the hypothesis using a series of fluid secretion assays (Petzel 1993). Female mosquitoes were anesthetized on ice; then, the alimen­ tary canals were drawn into mosquito saline. All five tubules were prepared by cutting them at their junction with the alimentary canal. The hindgutMalpighian tubule complexes were then transferred to a drop of saline. These preparations were then placed under a pool of light white paraffin oil. Using glass hooks fabricated to approximately the diameter of the tubules,

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the open ends of the tubules were pulled into the oil. Secreted fluid formed a droplet on the open end of the tubules, which was visible in the oil. The droplet was measured with an ocular micrometer every five minutes for thirty minutes, and the volume of the secreted fluid was calculated from the measurements. Each experiment was conducted in a series of six consecutive thirty-min­ ute periods. The first period was an equilibration period, and no measure­ ments were taken. The experimental treatment sequences were done in the following periods, which were designated 1 through 5. The first period was a saline control period, and the next two were experimental periods. These periods were started by exchanging 10 μΐ of saline with 10 μΐ solution of an eicosanoid biosynthesis inhibitor. The last two periods, 4 and 5, were cAMP stimulation periods. Petzel et al. (1987) showed that cAMP stimulates in­ creased fluid secretion in mosquito Malpighian tubules. The purpose of the cAMP stimulation periods was to ensure that the tubules were in a healthy state, able to respond to added cAMP with increased fluid secretion rates. In all experiments, the cAMP treatment resulted in increased fluid secretion rates. We first determined the influence of saline and 0.5% ethanol, the drug vehicle, on fluid secretion. Fluid secretion rates were steady at about 0.8 η17 minute through periods 1 through 3. Added cAMP in period 4 approximately doubled the fluid secretion rates. We then assessed the influence of eicosatetraynoic acid, an analogue of 20:4n-6 with triple bonds in place of the usual double bonds. This compound inhibits most enzymes that process 20:4n-6, including phospholipase A2. lipoxygenase, and cyclooxygenase. In the pres­ ence of this compound at 100 μΜ, fluid secretion decreased from about 1.0 nl/minute in the saline period to 0.55 nl/minute in period 2 and to 0.4 nl/ minute in period 3. These findings supported the idea that eicosanoids were involved in modulating basal fluid secretion rates in mosquito Malpighian tubules. Similar experiments with the lipoxygenase inhibitor esculetin and the epoxygenase inhibitor SKF-525A, did not influence fluid secretion rates. Al­ though epoxygenase products act in the ion transport physiology of mam­ malian kidneys (Escalante et al. 1991), we inferred that products of these pathways were not directly involved in maintaining basal fluid secretion in mosquito Malpighian tubules. Experiments with the cyclooxygenase inhibi­ tor indomethacin, however, produced a different picture. In the presence of 100 μΜ indomethacin, fluid secretion decreased from 0.9 to 0.5 nl/minute. The influence of indomethacin was expressed in a dose-dependent manner. Indomethacin did not influence fluid secretion rates at 1 μΜ; at 10 μΜ, fluid secretion decreased from 1.2 to 0.9 nl/minute. On the basis of these results, it appeared that prostaglandins, but not li­ poxygenase or epoxygenase products, are involved in maintaining basal fluid

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secretion rates in mosquito Malpighian tubules (Petzel and Stanley-Samuelson 1992, Stanley-Samuelson and Petzel 1993). The idea that prostaglan­ dins act in mosquito Malpighian tubules raised the issue of the occurrence and metabolism of 20:4n-6 in these tissues. This topic has a longer history for mosquitoes than other invertebrates (see chapter 3). Petzel et al. (1993) determined the presence of 20:4n-6 and PGE2 in mos­ quito Malpighian tubules. Although we had determined the presence of 20:4n-6 in extracts of whole adult mosquitoes (Stanley-Samuelson and Dadd 1981), there was no specific information on 20:4n-6 in mosquito Malpighian tubules. We isolated pools of about 250 Malpighian tubules, then processed them for lipid extraction. For two pools of tubules, the fatty acids associated with total lipid extracts were hydrolyzed from complex lipids, then esterified to fatty acid methyl esters. For other pools of tubules, the phospholipids and triacylglycerol fractions were isolated from the total lipid extracts by thinlayer chromatography. After forming methyl esters of the fatty acids associ­ ated with each fraction, the fatty acid compositions were determined on gas chromatography. The chemical identities of the fatty acids were confirmed by gas chromatography-mass spectrometry. In the total lipids prepared from Malpighian tubules, 20:4n-6 composed about 7.8% of the total fatty acids. Similar results obtained with isolated phospholipids; however, no 20:4n-6 was detected in the triacylglycerols. Hence, as seen in extracts of whole mosquitoes (Stanley-Samuelson and Dadd 1983), 20:4n-6 is associated with cellular phospholipids. Two phospholipid fractions, phosphatidylcholine and phosphatidylethanolamine, are quantitatively the major glycerophospholipids. Two other frac­ tions, phosphatidylinositol and phosphatidylserine, are quantitatively smaller, but are important fractions. We isolated the phospholipids from total lipid extracts of Malpighian tubules. The isolated phospholipids were then applied to thin-layer chromatography plates, and three major fractions were isolated: phosphatidylcholine, phosphatidylethanolamine, and an unresolved fraction composed of phosphatidylinositol plus phosphatidylserine. After trans­ methylation, the fatty acids associated with each major glycerophosphopholipid were determined. We detected no 20:4n-6 in the unresolved fraction, and only traces in phosphatidylethanolamine. About 2% of the phospha­ tidylcholine fatty acids were present as 20:4n-6. Hence, we concluded that the 20:4n-6 in mosquito Malpighian tubules is associated with a specific phospholipid fraction. This may be important in eicosanoid biosynthesis be­ cause the 20:4n-6 could be released from a specific pool, then channeled to the cyclooxygenase pathways (Otto and Smith 1995, Smith et al. 1996). Mosquito Malpighian tubules are composed of about 200 cells per tubule, which imposes severe restrictions on the ability to determine the presence of prostaglandins by chemical methods in these tissues. We approached the goal of determining prostaglandins in Malpighian tubules via immunohisto-

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chemistry (Howard et al. 1992, PetzeI et al. 1993). Malpighian tubules were isolated from adult female mosquitoes, then fixed. The fixed tubules were incubated in the presence of 0.4% Triton X-100 with a primary antibody, rabbit anti-PGE2. Following the sixteen-hour incubation, the tubules were rinsed and incubated for another sixteen hours with a secondary antibody, goat anti-rabbit IgG. The secondary antibodies were complexed with horse­ radish peroxidase, and the enzyme activity was made visible by suspending the tissues in 0.02% hydrogen peroxide and the chromogen 3,3'-diaminobenzidine. We conducted several control experiments. Method specificity was evalu­ ated by omitting the primary antibody to show that the staining was due to the antibody, and not to another component of the tissue or reagents. Sim­ ilarly, leaving out the secondary antibody showed that staining was the result of secondary antibodies. We assessed the antibody specificity by preincubating the primary antibody with either PGE2, the appropriate antigen, or other prostaglandins. Preincubation with the natural antibody reduced staining, while preincubation with the other prostaglandins did not. Immunohistochemical procedures can be misleading due to nonspecific interactions be­ tween antibodies and cell components, inadvertent coloration appearing as staining, and other pitfalls. The control experiments described here are rou­ tine procedures in this line of work, and with appropriate outcomes they add considerable verisimilitude to the idea of localizing prostaglandins in spe­ cific cells. Figure 6-1 shows a representative immunohistochemical visualization of PGE2 in Malpighian tubules from the mosquito A. aegypti. The repeated dark brown staining pattern indicates the presence of PGE2 in principal, but not stellate, cells of the tubule. The principal cells are responsible for secret­ ing fluid into the lumen of the tubule. We have observed a similar staining pattern for PGE2 in Malpighian tubules of the yellow mealworm, Tenebrio molitor (Howard et al. 1992). PGF2ct is also present in Malpighian tubules from the mosquito and the mealworm, visualized by similar procedures using a primary antibody to PGF2a. The PGF2a staining patterns are quite different from the patterns obtained for PGE2. For PGF2a, the staining is not restricted to the principal cells, but seems to be more or less evenly distrib­ uted among both major Malpighian tubule cell types. This work demonstrates the presence of 20:4n-6 in mosquito Malpighian tubules. The distribution of the 20:4n-6 is unusual in that it is associated with a single glycerophospholipid, phosphatidylcholine. We also obtained evidence for the presence of prostaglandins in the tubules of these two insect species. These points do not firmly establish a physiological role for the prostaglandins; however, when taken with the positive results of the phar­ maceutical treatments, there is fairly strong circumstantial evidence that

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FIGURE 6-1. Immunohistochemical localization of PGE 2 in the principal, but not the stellate, cells in a Malpighian tubule from an adult female mosquito, Aedes aegypti. Reprinted from Insect Biochemistry and Molecular Biology, volume 23. Petzel et al.. Arachidonic acid and Prostaglandin E 2 in Malpighian tubules of female yellow fever mosquitces, pp. 431-437, Copyright 1993, with permission from Elsevier Science.

prostaglandins modulate the basal fluid secretion rates in mosquito Malpighian tubules. In their work on mosquito nutrition, Dadd and Kleinjan (1984, 1988) anticipated that the essentiality of dietary 20:4n-6, in part, was related to the biosynthesis of prostaglandins. First, they showed that supplementing the larval growth medium with nonsteroidal inflammatory drugs and 20:4n-6 produced the same symptoms of essential fatty acid deficiency. The symp­ toms could be eased by increasing the amount of 20:4n-6 in the medium (Dadd and Kleinjan 1984). Second, they found that the symptoms of essen­ tial fatty acid deficiency were partly attenuated by adding PGF2q to deficient medium (Dadd and Kleinjan 1988). The ameliorating influence was specific

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to PGF2a, because similar treatments with other prostaglandins were not ef­ fective. Dadd and Kleinjan concluded that one biological role of 20:4n-6 in mosquitoes is to serve as a substrate for prostaglandin biosynthesis. They were not able to suggest specific physiological roles of the prostaglandins. Petzel et al. (1987) showed that basal fluid secretion rates in mosquito Malpighian tubules are influenced by changes in intracellular cAMP concen­ trations. We initiated a preliminary investigation of the possibility that pros­ taglandins stimulate increased cAMP biosynthesis in the tubules. For these experiments, the tubules were isolated from adult female mosquitoes, then incubated in mosquito saline. PGE2 (100 μΜ) was added to the saline and after selected incubation periods, the tubules were frozen in liquid nitrogen. The frozen tissues were then processed for extracting and quantitating cAMP using a commercial radioimmunoassay kit. These studies showed that PGE2 stimulated a four- to fivefold increase in intracellular cAMP concentrations. This work remains preliminary, and has so far been presented only as an abstract (Parrish et al. 1992). In a more direct test of the idea that prostaglan­ dins influence fluid secretion rates, we conducted routine fluid secretion as­ says. In these experiments, tubules were equilibrated as usual in mosquito saline; then, PGE2 was added to the saline. Again, this is preliminary work; however, PGE2 stimulated increased fluid secretion in mosquito Malpighian tubules. We infer from these findings that prostaglandins are among the reg­ ulatory elements in mosquito Malpighian tubule physiology. Van Kerkhove et al. (1995) conducted a similar line of work on Malpighian tubules from workers of the ant, Formica polyctena. The Malpighian tubules from this species are considerably smaller than the mosquito tissues. In earlier work on tubules from the ant, Van Kerkhove and her colleagues had developed slightly different techniques to assess fluid secretion (Van Kerkhove et al. 1989). A single Malpighian tubule was placed in a 50 μΐ bathing droplet on the stage of an inverted microscope. The droplet was covered with oil to avoid evaporating the saline during the experiment. Fluid secretion was determined by collecting tiny drops of secreted fluid every ten minutes, and placing the fluid into oil. The sizes of the droplets were mea­ sured with a microscope, and fluid secretion rates were calculated from the sizes. The ten-minute segments were combined into four experimental pe­ riods; a saline control period, an experimental period, a washout period, and a cAMP stimulation period. Again, all cAMP stimulation steps indicated that the tubules were able to increase fluid secretion at the end of the experi­ ments. Fluid secretion rates are highly variable in these tubules, and data were expressed as a percentage of the fluid secretion rate determined in the third ten-minute saline control period. Our findings matched the earlier findings with mosquito Malpighian tu­ bules. Exposing the tubules to the 20:4n-6 analogue eicosatetraynoic acid or

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to indomethacin (100 μΜ) significantly reduced the fluid secretion rates, while treatments with esculetin and SKF-525A had no influence on basal fluid secretion rates. Again, these pharmaceutical experiments yield rather circumstantial evidence on the point. With due recognition of the inherent shortcomings, however, these results certainly support the view that pros­ taglandins modulate the basal fluid secretion rates in ant Malpighian tubules. Taken with the work on mosquito tissues, I suggest that prostaglandins may serve as important modulators in all insect Malpighian tubules. The early work by Philips (1980) implicated prostaglandins in the physiol­ ogy of the locust rectum. Fournier and his colleagues advanced this work recently (Radallah et al. 1995). They used an everted sac preparation of the locust rectum to assess the influence of 20:4n-6 and PGE2 on water resorp­ tion. The everted sac is formed by tying one end of the rectum onto a cathe­ ter tube. Test compounds could then be injected into the sac, thereby expos­ ing the hemolymph side of the preparation to the compounds. The sacs were typically equilibrated in saline for one hour, then the recta were filled with 10 μΐ of saline, amended with drugs for experimental preparations or not amended for control preparations. The sacs were incubated in saline for a second hour, and fluid resorption rates to the lumen of the sac preparations, were determined. PGE2 stimulated increased fluid resorption in a dose-dependent manner. Maximal stimulation, 59% over controls, obtained at about IO-9 M PGE2. They could also stimulate increased fluid resorption by adding 20:4n-6, which resulted in a 79% increase in resorption at 10 6 M 20:4n-6. Contrarily, similar experiments with aspirin and indomethacin also stimulated dose-dependent increases in fluid resorption. It would appear that prostaglan­ dins and inhibitors of prostaglandin biosynthesis exert similar influences on the rectal preparations. The authors offered several speculations on this ap­ parent contradiction. Perhaps the most reasonable is that indomethacin re­ stricts biosynthesis of all cyclooxygenase products. Many prostaglandins exert inhibitory influences on cellular processes. The putative inhibitory product may exert greater influence on overall fluid transport dynamics than the stimulatory product. If this is so, inhibiting the biosynthesis of a possible inhibitory prostaglandin may create an apparent stimulation of fluid transport. The authors also considered the influence of 20:4n-6 and PGE2 on se­ lected intracellular signal transduction moieties. They used a microfluorimetric technique to consider the influence of 20:4n-6, PGE2, and indometha­ cin in influx of calcium into rectal cells. Rectal tissue was loaded with the calcium indicator indo-1, then observed by a fluorescent microscope. PGE2 and 20:4n-6 stimulated large increases in calcium influx, which relaxed to baseline within minutes. As in the fluid transport experiments, indomethacin

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similarly provoked increased calcium influx. Pretreating the tissues with nif­ edipine, which blocks L-type calcium channels, completely blocked the ef­ fects of 20:4n-6 and PGE 2 on calcium transport. Another intracellular signaling system known from studies on mammalian systems involves the hydrolysis of phophatidylinositols. In this system, upon stimulating cells with an extracellular ligand, such as a hormone or eicosanoid. a calcium-sensitive phospholipase C hydrolyzes the inositol moiety from the phospholipid, leaving a diacylglycerol and an inositol. The inositols are typically phosphorylated, with either one, two, three, or four phosphates. The diacylglycerol may activate some protein kinases C. while the inositol phosphates, particularly the triphosphate form, may cause the release of cal­ cium from intracellular stores (Berridge 1993). Radallah et al. (1995) showed that treating the locust rectum with 20:4n-6 or PGE 2 stimulated sub­ stantial increases in phospholipase C activity. Once again, aspirin and indomethacin similarly induced increased phospholipase C activity. This appar­ ently depends on the influx of calcium because the L-type calcium channel blocker strongly attenuated the influences of 20:4n-6 and PGE 2 on phospho­ lipase C activity. They showed that the phospholipase C activity is associ­ ated with release of inositol phosphates. The data in this paper (Radallah et al. 1995) are convincing on the idea that 20:4n-6 and at least one eicosanoid. PGE 2 , are involved in modulating fluid transport in the locust rectum. The locust rectum is probably under primary regulation of the antidiuretic hormone known as neuroparsin. Neuroparsin is a peptide hormone released from the corpora cardiaca, which stimulates fluid resorption in the rectum. The hormone apparently acts through a G-protein linked cell surface receptor, and it stimulates increased phospholipase C activity. I guess that the hormone also stimulates the release of 20:4n-6 from cellular phospholipids, which leads to increased PGE 2 . The PGE 2 probably acts in an autocoidal mechanism to coordinate the cellular reactions to the central hormone. If this is so, PGE 2 acts in similar ways in the insect rectum and segments of the mammalian kidney (Bonvalet et al. 1987). Prostaglandins also modulate fluid secretion rates in salivary glands iso­ lated from the lone star tick, Ainblyomma americanum (Qian et al. 1997). Ticks are obligate ectoparasites, living on vertebrate blood. Their salivary glands are major osmoregulatory organs during their lengthy host-parasite interaction. The salivary glands concentrate the nutrients associated with ver­ tebrate blood by forming a copious salt-rich saliva, which is injected back into their hosts during feeding. The salivary glands appear to be under ner­ vous control, and the neurotransmitter dopamine stimulates fluid secretion in isolated salivary glands (Sauer et al. 1995). Qian et al. (1997) investigated the influence of prostaglandins on dopamine-stimulated fluid secretion. In their first experiments, they exposed pairs of salivary glands, then tied

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off the major duct. The pairs then served as matched experimental and con­ trol glands. The glands were weighed, then exposed to eicosanoid bio­ synthesis inhibitors (10 μΜ) or to the solvent carrier for controls for fifteen minutes. The glands were then exposed to dopamine (10 μΜ) for five min­ utes. The glands were then rinsed, weighed, and taken through another round of incubations. Compared to controls, treatments with the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine and the cyclooxygenase inhibitor aspirin resulted in about 30% to 40% reductions in dopamine-stimulated fluid secretion rates. Longer incubations produced greater reductions, and the influence of both inhibitors was expressed in a dose-dependent manner. The influence of oleyloxyethyl phosphorylcholine was reversed by incu­ bating the inhibitor-treated glands with 100 μΜ PGE2 or its stable analog 17-phenyl trinor PGE2. The prostaglandin treatments did not reverse the in­ hibitory influence of the cyclooxygenase inhibitors aspirin and diclofenac. Dopamine stimulates fluid secretion through G protein-coupled receptors that lead to increased intracellular cAMP concentrations (Sauer et al. 1995). Qian et al. (1997) considered the influence of prostaglandin biosynthesis inhibitors and of prostaglandins on salivary gland cAMP concentrations. Iso­ lated glands were exposed to selected pharmaceuticals, then processed for cAMP determinations by a commercial radioimmunoassay. They found that the phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine and the cy­ clooxygenase inhibitor indomethacin inhibited dopamine-stimulated in­ creases in intracellular cAMP concentrations by about 25%. In the presence of oleyloxyethyl phosphorylcholine, PGE2 and its analog stimulated 20% to 40% increases in the oleyloxyethyl phosphorylcholine-inhibited cAMP con­ centrations. The prostaglandins did not reverse the influence of cycloox­ ygenase inhibitors on intracellular cAMP concentrations. The eicosanoid system did not influence fluid secretion or intracellular cAMP concentrations in the absence of dopamine stimulation. The authors' results provide strong support for their hypothesis that eicosanoids modulate dopamine-stimulated fluid secretion rates in tick salivary glands. However, the most important part of this seminal work lies in the first identification of physiologically functional receptors in an invertebrate system. They prepared salivary gland membrane fractions by centrifugation, using the final 11,500 g pellet for classical binding assays. The membrane frac­ tions were incubated in the presence of radioactive PGE2. After three-hour incubations, buffer was added to the reactions and the solution was filtered through microglass filters. The filters were rinsed, and specific binding was determined, although details are omitted from the publication. They first showed that compared to the 100,000 g pellet and supernatant, greatest spe­ cific binding obtained with the 11,500 g preparation. Specific binding to the 11,500 g fraction was sensitive to amount of membrane protein (up to 200 g), pH (optimal binding at pH = 8.5), and amount of radioactive PGE2.

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The binding was specific to PGE2, because PGF2a, PGD2 and thromboxane A2 did not effectively displace the radioactive PGE2. By contrast, unlabeled PGE2 very effectively displaced the radioactive PGE2. Hence, their results indicate that the salivary glands express a saturable, reversible, and specific PGE2 receptor. The results of Scatchard plot analysis suggested a single, high-affinity receptor. The authors generated strong evidence that the salivary gland receptor is functionally coupled to a stimulatory G protein. Binding assays conducted in the presence of GTP and its stable analog GTP7S yielded significantly re­ duced PGE2 binding. Similar assays in the presence of ATP and GDP did not influence PGE2 binding. Moreover, pretreating the membrane preparations with cholera toxin blocked the effect of GTP7S on PGE2 binding. Cholera toxin is a bacterial product that inhibits stimulatory G proteins. This finding indicates the salivary gland PGE2 receptor is linked to a Gs protein. Recognizing that PGE2 influence intracellular cAMP concentrations, Qian et al. (1997) determined the influence of PGE2 on adenylate cyclase activity. Their results showed that PGE2 did not directly influence the enzyme. The authors concluded that the tick salivary gland expresses a functional PGE2 receptor that does not directly regulate adenylate cyclase. They speculated that the PGE2 receptor may influence calcium mobilization. In a subsequent paper, Qian et al. (1998) confirmed their speculation. They found that thirty-second exposures of dispersed salivary gland tissue with PGE2 resulted in increased amounts of intracellular inositol 1,4,5-triphosphate. In parallel experiments, they preloaded salivary gland tissue with radioactive calcium. Similar exposures to PGE2 resulted in the release of radioactive calcium from intracellular stores. Both prostaglandin actions were expressed in a dose-dependent way, from 1 nM to 10 μΜ PGE2. The biological significance of the salivary gland PGE2 receptor is linked to secre­ tion, or exocytosis, of anticoagulant proteins, which are thought to facilitate blood feeding. Qian et al. (1998) demonstrated this by incubating dispersed salivary gland tissue in the presence of PGE2, after which they recorded increased release of anticoagulant proteins. Four subtypes of PGE2 receptors are known (EP1, EP2, EP3, and EP4), recognized on the basis of their responses to prostaglandins and various pharmaceutical compounds (Coleman et al. 1990). Because PGE2 treatments influenced calcium mobilization in salivary gland preparations, Qian et al. (1998) tested the idea that the PGE2 receptor in tick salivary glands is an EPl-Iike receptor. They incubated salivary gland preparations in the pres­ ence of AH 6809, a compound that antagonizes mammalian EPl receptors. Secretion of anticoagulant proteins was inhibited in these preparations, sup­ porting the idea that the salivary gland PGE2 receptor is an EPl receptor responsible for regulating the secretion of proteins. This work provides very convincing evidence for a functional receptor

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active in tick salivary gland physiology. We will see later (chapter 9) that studies on eicosanoid receptors and biochemical modes of action remain a major research frontier. Taken together, work on blowfly salivary glands, locust rectum, insect Malpighian tubules, the gills of a freshwater and a marine mussel, and tick salivary glands provide evidence for the biological roles of eicosanoids in water and solute transport in representatives of the two largest invertebrate phyla, the arthropods and the molluscs. It is easy to suppose that eicosanoids may similarly act in many other invertebrates, especially those in phyla not represented by the studies so far. This supposition is supported by findings from nonmammalian verte­ brates. The earliest vertebrates were probably jawless aquatic animals, which arose sometime in the Precambrian Era. This line gave rise to the agnathans, or jawless vertebrates, and the gnathostome fishes. Although agnathans were very diverse animals early in the Paleozoic Era, there are now only about sixty living species of these animals, all either lampreys or hagfishes. Al­ though they are called hagfish. the similarities of these animals to true fish are only superficial. From our point of view, it is interesting that eicosanoids exert important physiological actions in a living representative of the earliest vertebrates. Wales (1988) investigated the influence of several prostaglandins in urine flow and other physiological parameters in the hagfish, Myxine glutinosa. The fish were cannulated in lateral sinus, and hagfish Ringer's solution was infused into the sinus. Experimental compounds were introduced via the can­ nula. Urine was collected by palpation. The background, control-level urine flows in these hagfish were between 540 and 660 ml/hour/kilogram. PGE1, PGE2, PGA2, and thromboxane B2 in doses of 10 g/kilogram evoked marked reductions in urine output, although the actual volume changes were not presented in quantitative terms. The urine flow in hagfish is a reflection of the glomerular filtration rate because the hagfish kidney is incapable of net fluid resorption. Glomerular filtration is a function of perfusion pressure, and the eicosanoid-induced reductions in urine flow may result from reduced filtration. These four prostanoids also influenced the hagfish blood pressure. The control levels in resting hagfish are between 3.5 and 3.75 mm of mer­ cury, and the prostaglandin treatments produced about 50% reductions in blood pressure. While the effects of these compounds was not specific to a single prostaglandin, PGF2a treatments did not influence urine flow rates nor blood pressure. None of the experimental treatments influence the concentra­ tions of four electrolytes, sodium, potassium, calcium, and magnesium, in blood or urine. It appears that some eicosanoids exert a vasodepressive effect in hagfish, and the effect can be registered in terms of reduced blood pressure and urine flow. The eicosanoid effects were not specific to a single compound, al-

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though they were expressed in a dose-dependent way. Additional informa­ tion will be required before we can uncritically accept the roles of eicosanoids in hagfish, but Wales' data amount to an intriguing hypothesis. Eicosanoids modulate water and solute transport in several nonmammalian vertebrates. Frog skin is a commonly used model of ion transport physiology. Bjerregaard and Nielsen (1987) showed in detail that PGE2 stim­ ulates ion and water secretion in isolated frog skin. Also using frog skin and toad urinary bladder models, Yorio et al. (1991) showed that PGF2ot treat­ ments reduce proton transport. Prostaglandins are present in toad urinary bladder preparations, and concentrations of 6-keto-PGFla, the stable meta­ bolic product of PGF2a, are increased in experimental acidotic toads. In an­ other study on frog skin, Gerencser (1993) showed that PGF2a elicits sus­ tained decreases in the short-circuit current. The current is carried by a net sodium flux, and the author inferred that PGF2a influences sodium transport in frog skin. There is a rich literature on eicosanoids and transport physiology in amphib­ ians. A comprehensive review of this important work is not appropriate for this volume on invertebrates. However, I draw on these few examples to press a point. Eicosanoids act in transport physiology in mammals, and many other vertebrates. We have just seen that eicosanoids also influence transport physi­ ology in a wide range of invertebrates. I suggest that eicosanoids have been recruited into roles in ion transport physiology very early in animal evolution. Eicosanoids may well regulate ion transport in all animals.

C H A P T E R 7

Emerging Eicosanoid Actions

As OUR understanding of eicosanoid actions in invertebrates expands, we catch an occasional glimpse of newly emerging discoveries. This chapter is devoted to two such areas. In one, it appears that eicosanoids somehow modulate thermoregulatory actions in several invertebrate species. In the other, eicosanoids act in the regulation of certain intracellular metabolic events. Let us begin with temperature regulation in invertebrates, which has an uneven history.

EICOSANOIDS IN INVERTEBRATE TEMPERATURE BIOLOGY Many ectothermic vertebrate animals are able to regulate their body tempera­ tures, and generate fevers, via various behavioral mechanisms (Prosser 1973, Casey 1981). A mechanism may be as simple as moving in and out of shade to maintain a fairly stable body temperature. Some animals exhibit more subtle behaviors to regulate their body temperatures, such as assuming partic­ ular postures relative to the sun. Due to the enormous heat capacity of water, the body temperatures of most aquatic animals are similar to the temperature of their surrounding medium. Nonetheless, some aquatic animals are able to thermoregulate behaviorally by selecting microenvironments according to water temperatures. Most of the work in this area has involved vertebrates; however, there are a few papers suggesting that some invertebrates are able to thermoregulate and to generate behavioral fevers in this way. Casterlin and Reynolds (1977) reported on behavioral fevers in crayfish, Cambarus bartoni bartoni. In their initial experiments, individual crayfish were placed in tanks for twenty-four hours, then allowed to establish base­ line temperature preferences in something like a laboratory temperature gra­ dient pool over the following twenty-four hours. The animals were then in­ jected, via the gill chambers, with a suspension of killed gram-negative bacteria, Aeromonas hydrophila. Control crayfish were injected with sterile saline. The animals were then allowed to readjust their body temperature by selecting different positions along a temperature gradient. The preferred temperature preference for untreated crayfish was about 22°C. The preferences of crayfish injected with saline was not different from

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those of untreated animals. However, the crayfish treated with the bacteria moved into slightly warmer water, increasing their body temperatures by about 1.8°C above the control animals. The authors interpreted this informa­ tion in terms of the adaptive significance of fever responses to bacterial infections. Drawing on the literature on mammals, Casterlin and Reynolds (1978) considered the possibility that prostaglandins would stimulate fever in treated crayfish, C. bartoni. They followed the protocols just described for the ex­ periments with bacteria. After the crayfish had settled in their preferred tem­ peratures, groups of control animals were either injected with pyrogen-free saline, or handled but not injected. Separate groups of experimental animals were injected with 50, 100, or 500 g of PGE1 dissolved in the saline. The control crayfish did not depart from their selected temperature of about 22°C. The experimentals generated behavioral fevers by moving into warmer water. The fevers were expressed in a dose-dependent way, from about I0C increase in response to the lowest dose to about 3.4°C with the highest dose. Casterlin and Reynolds (1978) concluded that PGE1 exerts some influence on thermoregulatory neurons. Casterlin and Reynolds (1979) worked to extrapolate their findings to other aquatic arthropods. They conducted similar experiments with the American lobster, Homarus americanus, the pink shrimp, Penaeus duorarum, and the horseshoe crab, Limulus polyphemus. All experimental ani­ mals exhibited a fever reaction to prostaglandin injections. The authors con­ cluded that their findings reveal new experimental models for study of the neuropharmacological mechanisms of fever. Unfortunately, their experimental results do not facilitate critical thought on the subject. All experiments with eicosanoids involved injections of only one compound, PGE1. The recorded fever responses following PGE1 injec­ tions could very well have been simple reactions to an irritating chemical. In the absence of experiments with other prostaglandins and selected fatty acids, there is no way to judge the influence of eicosanoids on fever in these invertebrates. Moreover, we might suppose that the fever response to in­ jected bacteria would be negated by treating the animals with various cyclooxygenase inhibitors. Plainly, there is opportunity in this area. Cabanac and Le Guelte (1980) produced a similar paper describing the influence of PGE1 injections on behavioral fever in two terrestrial arthro­ pods, the scorpions Buthus occitanus and Androctonus astralis. The authors constructed a thermogradient using a box of sand with a resistor at one end. Thermocouples were implanted into the cephalothoraces of the animals, which were then allowed to settle in a preferred temperature zone. Experi­ mental animals were injected with PGE1 dissolved in saline, and control animals were injected with saline. The temperatures of the scorpions were continuously recorded.

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The authors observed dose-dependent increases in preferred temperatures, in some cases as high as 20°C above the temperatures of controls or untre­ ated scorpions. Cabanac and Le Guelte (1980) took these data to indicate that scorpions can generate behavioral fevers, which are mediated by PGE1, from which they inferred that the animals have a homeostatic setpoint for body temperature. The authors did not know whether scorpions expressed behavioral fever in response to bacterial infections; however, they incor­ rectly thought that arthropods lack a blood-brain barrier, which would allow hemolymph-borne pyrogens to easily influence the neurons responsible for temperature regulation. Again, the experiments were not designed to include work with other eicosanoids and controls, and we cannot draw firm infer­ ences from this work. Cabanac and Rossetti (1987) conducted analogous experiments with a freshwater snail, Limnaea auricularia. These experiments yielded negative results, indicating that the snail does not generate behavioral fevers in re­ sponse to pyrogens. Rossetti and Nagasaka (1988) also used aquatic tem­ perature gradients in similar experiments with a Japanese freshwater snail, Semisulcospira libertina. They found that the snails did not generate behav­ ioral fever in response to injections of PGE1, PGE2, or lipopolysaccharide. This work was done with a view to investigating the evolution of fever in animals, and the authors concluded that fever mechanisms do not occur in molluscs. The Mollusca comprise the second largest phylum in the animal kingdom; thus, results from two freshwater snails hardly speak for the entire phylum. As in many issues in invertebrate biology, we must await more information, upon which firmer conclusions may be constructed. An unrelated line of work provides insight into the adaptive significance of behavioral fevers in at least one invertebrate. Blandord et al. (1998) re­ ported on field trials of a biological control agent targeted for locusts and grasshoppers. The agent is a fungal insect pathogen, Metarhizium flavoviride. In trials conducted in West Africa, experimental fields were sprayed with the pathogens and control fields were not treated. The authors used thermocouples to record thoracic temperatures of fourth or fifth instars of the Senegalese grasshopper, Oedaleus senegalensis. Like many other insects, these grasshoppers rely on thermoregulatory behaviors to maintain homeo­ stasis of thoracic temperature. The preferred body temperature of uninfected grasshoppers was about 39°C. Infected individuals elevated their temperature set points to about 42°C. In this work, Blandord et al. (1998) demonstrated behavioral fevers in a natural population. The authors concluded that the ability to generate fevers in response to infections may be partly responsible for the highly variable performance of fungal insecticides. Their remarks are important to our thinking about eicosanoids in invertebrate systems, because they suggest that behavioral fevers may be quite adaptive in natural popula­ tions. While they did not worry about biochemical mechanisms underlying

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the organismal behaviors, if prostaglandins do, indeed, mediate fever reac­ tions of infections, we have another instance of important eicosanoid actions at crucial moments in life histories. Toolson et al. (1994) suggested that eicosanoids are involved in regulating the body temperature homeostatic setpoints in the cicada, Tibicen dealbatus. As seen in many insects, cicadas use various behaviors, such as seeking shade, to avoid overheating. Cicadas differ from most animals, however, because they react to dangerously high body temperatures by sweating. The mechanism involves dorsal sweat glands, which transport water to the sur­ face of the insects, where evaporation rapidly cools the animals. They re­ place the water lost to sweating by imbibing plant fluids (Toolson 1987). The role of evaporative cooling in insect thermoregulation is not well understood, and we cannot be certain how many insects express one or an­ other form of sweating. Grasshoppers, for example, may use increased tra­ cheal ventilation movements to achieve evaporative cooling (Prange 1990). Honeybees also achieve evaporative cooling, but not by sweating. Honey­ bees regurgitate fluids from the midgut, which evaporate from the surface of their heads during flight. The evaporation quickly cools their heads and tho­ raxes (Heinrich 1993). In cicadas, the sweating is stimulated when a cicada's body temperature reaches a setpoint that is somehow determined within indi­ vidual cicadas. Toolson and his colleagues suggested that the setpoint is determined by a complex mechanism, in which eicosanoids play an impor­ tant part. The sweating setpoint of individual cicadas was determined by placing individuals in a flow-through chamber. The temperature of the chamber was controlled by a computer, which also recorded the thoracic temperatures of cicadas via implanted thermocouples. The temperature in the chamber was maintained at 35°C for a low ambient temperature and at 40°C for high ambient temperature. The body temperature at which sweating began (that is, the thoracic temperature setpoint) was recorded as a departure from the linear increase in thoracic temperature as a function of time in the chamber, which was never allowed to exceed thirty minutes. Immediately after this, the cicadas were injected with ethanol (for controls), eicosanoid biosynthesis inhibitors, or fatty acids or prostaglandins. After a twenty-minute rest period at 25°C, the cicadas were returned to the experimental chamber, and a posttreatment temperature setpoint was determined. The influence of the various experimental treatments were registered as changes in the temperature setpoints. For sham-injected or ethanol-injected control cicadas, the pretreatment setpoints were not influenced by the treatments. Also, injections with 18:ln-9, a monounsaturated fatty acid unrelated to eicosanoid biosynthesis, did not alter the set points. Treatments, at 100 g/insect, with various pros­ taglandins resulted in small increases, about 0.4°C, in temperature setpoints.

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While statistically significant, this probably does not represent a biologically interesting effect. On the other hand, separate experiments with two cyclooxygenase inhibi­ tors, paeonol and aspirin, resulted in altered setpoints. The changes depended on the pretreatment setpoints and ambient temperature. At ambient tempera­ ture 35°C, but not 40°C, treatments with these compounds produced a mean of 1.2°C increase in thoracic temperature setpoints. For cicadas with rela­ tively low or high pretreatment setpoints, the cyclooxygenase inhibitors ten­ ded to moderate the extremes, slightly increasing the low setpoints and de­ creasing the high setpoints. The moderation was recorded in experiments run at both ambient temperatures, 35°C and 40°C. Similar experiments with 20:4n-6 and 20:3n-6 yielded results similar to the data just described, although the polyunsaturated fatty acids did not pro­ duce effects as large as the inhibitor effects. These results seemed contradic­ tory, because separate experiments with eicosanoid precursor fatty acids and with eicosanoid biosynthesis inhibitors produced similar patterns of influ­ ence on thoracic temperature setpoints. We interpreted these findings using a model of controlling the setpoints (Toolson et al. 1994). In this model, we considered two input pathways to the preferred body temperature integration center, thought to reside in the central nervous system. One pathway acts to increase the setpoints, while the other decreases set points. Both pathways are influenced by prostaglandins and possibly other eicosanoids. The final set point in any individual cicada reflects the relative inputs from each of the two pathways. If ambient temperature, body temperature, and various bio­ chemical parameters, including eicosanoids, simultaneously but differentially influence these neural pathways, the outcome of any given experimental ma­ nipulation with a particular cicada would not be known in advance. We concluded that eicosanoids represent one element in a complex system re­ sponsible for homeostasis of body temperature in this insect. Our work with cicadas was bolstered slightly by determining the presence of 20:4n-6 in phospholipids from adult cicadas and showing that whole cic­ adas, T. dealbatus, are competent to convert injected radioactive 20:4n-6 into prostaglandins (Stanley-Samuelson et al. 1990a). Nonetheless, for both our experiments and those of others, the results of pharmaceutical manipulations in experiments with fever and with temperature regulation have not yet pro­ duced clear, unambiguous results. The major shortcoming may be that the subject of animal body temperatures is deceptive. At one level, measuring body temperatures with small thermocouple probes seems relatively straight­ forward. Yet many factors, some internal to the animal and others external, influence instantaneous body temperatures. When temperature is investigated at the biochemical or neuronal level, it will be difficult to determine the influence of a single element on the overall physiology of thermoregulation. Eicosanoids appear to act in fever and in determining temperature setpoints

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in at least some invertebrates. Temperature is a very important aspect of animal biology, with the potential to profoundly influence cellular functions through its effects on enzymes, other proteins, and membranes. As we have seen in other areas of biology, it is not unusual to see multiple mechanisms in place as a safeguard against lethal disruption. We can look forward to more information in this area in the future.

EICOSANOIDS IN INSECT PEPTIDE HORMONE SIGNAL TRANSDUCTION In the early days of interest in prostaglandin actions in invertebrates, Yamaja Settv and Ramaiah (1982) proposed that PGE1 inhibited the release of lipid from the fat bodies of pupal silkworms, B. mori. The experiments were not directly connected to endocrine regulation of lipid mobilization, but rather were meant to explore the roles of prostaglandins in regulating metabolic events. They focused on lipids because PGE1 inhibits lipolysis in mammals. In this work, PGE1 was dissolved in saline, and 1 ^g of prostaglandin was injected into silkworm pupae in 10 μΐ volumes. Control pupae received simi­ lar saline injections. The insects were allowed to incubate for thirty minutes, hemolymph samples were collected, and the fat body was isolated and stored at — 20°C until analysis. The authors used standard spectrophotometric as­ says to determine esterase and lipase activities, and determined quantities of lipids by weighing lipid extracts. Compared to control pupae, esterase and lipase activities were reduced by about 20% and 30%. respectively. In line with these results, they found that the hemolymph lipid concentrations were reduced by about 30%. The authors concluded that PGE1 downregulates lipid mobilization in silk­ worm pupae (Yamaja Setty and Ramaiah 1982). As we have seen elsewhere, however, their conclusion suffers from a lack of convincing control experi­ ments. We do not know if the effects they measured are specific to PGE), because no other eicosanoids were considered in these experiments. Nor do we have evidence for dose-response relationships. As presented, their paper leaves us with an intriguing hypothesis that a prostaglandin modulates li­ polysis in an insect through its influence on certain enzyme activities. Wagemans. Van der Horst. and Stanley-Samuelson (preliminary data re­ ported in Stanley-Samuelson 1994a) considered the influence of PGE2 on lipid mobilization from locust fat body preparations. In these experiments, intact fat bodies were incubated in the presence of 10 pmol of adipokinetic hormone and increasing dosages of PGE2. The amount of lipid released into the incubation medium was determined at the end of the incubation periods. The quantities of lipid released declined, in a dose-dependent way, from about 1,000 mg in the presence of hormone, but no PGE2, to about 94 mg in

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the presence of hormone plus 5 X 10 ~3 M PGE2. These results were not published because they lacked additional control experiments of the sort just mentioned. Nonetheless, the results suggest that prostaglandins may modu­ late the actions of some major hormones in invertebrates. More recent work adds support to this idea. The insect hypertrehalosemic hormone is a member of the adipokinetic/ red pigment-concentrating hormone family of peptide hormones. Adipokine­ tic hormone is responsible for mobilization and release of lipids from the insect fat body during times of intense metabolic activity, such as flight (Van der Horst et al. 1993). Hypertrehalosemic hormone serves an analogous role in the formation and release of trehalose from the fat body. Trehalose, a disaccharide made of two glucose molecules, is the blood sugar of most insects. Keeley and his colleagues have been investigating the signal transduction pathways involved in the interaction of hypertrehalosemic hormone with the fat body of the cockroach, Blaberus discoidalis. The hormone exerts several actions on the fat body of this insect. Beyond stimulating the release of trehalose, the hormone is involved in stimulating the expression of some genes, as seen in increased protein biosynthesis and increased formation of mitochrondria. Of particular interest, hypertrehalosemic hormone stimulates expression of a gene for a novel cytochrome P45o, denoted CYP4C1. Keeley et al. (1996) noted that this marks the discovery of the first insect neurohor­ mone responsible for stimulating gene expression. This discovery points to the idea that this hormone exerts two different actions on fat body cells. One is the activation of the enzyme pathway in­ volved in forming and releasing trehalose, and the other is stimulating gene expression to produce new enzyme. Keeley et al. (1996) worked to deter­ mine whether these actions followed from a common signal transduction pathway, or from two independent sets of intracellular messengers. They determined that the presence of the hormone leads to increased intracellular inositol triphosphate concentrations (see Berridge 1993 for a review of inos­ itol triphosphate in signal transduction pathways). Inositol triphosphate causes the release of calcium from intracellular stores, and calcium stimu­ lates a calcium-dependent phosphorylase kinase to activate glycogen phosphorylase. This enzyme is responsible for releasing glucose residues from glycogen, thereby initiating the biosynthesis of trehalose. These events coin­ cide with the influx of extracellular calcium, after which a second calciumdependent kinase is activated. This kinase activates a transcription factor, which promotes expression of the gene for the novel cytochrome P450. The authors concluded that a common inositol triphosphate/calcium pathway is responsible for the influence of hypertrehalosemic hormone on mobilizing carbohydrate reserves and on promoting gene expression. As a part of the broader work on a common inositol triphosphate/calcium

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pathway, Keeley et al. (1996) assessed the potential for eicosanoids mediat­ ing the hormone action. Isolated fat body pieces were incubated in the pres­ ence of the eicosanoid biosynthesis inhibitor eicosatetraynoic acid and the hormone; then, the medium was analyzed to determine the release of treh­ alose from the fat body. Untreated fat body secreted about 15 g trehalose/ mg fat body dry weight, while 5 nM of the hormone stimulated trehalose secretion to about 32 g trehalose/mg fat body dry weight. At 0.1 nM, the hormone did not stimulate increased sugar secretion, nor did 100 μΜ eicosatetraynoic acid. When exposed to the hormone at 0.1 nM and the in­ hibitor together, however, the fat body released about 26 g trehalose/mg, significantly different from the controls and from the high hormone dosage. The authors conducted similar exercises with indomethacin, a cyclooxygenase inhibitor, and with BW4AC, a lipoxygenase inhibitor. Incubations in the presence of 0.1 nM hormone and 50 μΜ indomethacin, and separately in the presence of 0.1 μΜ hormone and 10 μΜ BW4AC, stimulated the fat body to release about 35 g trehalose/mg fat body dry weight. When added to fat body incubations without the hormone, neither inhibitor stimulated trehalose release. When incubated in the presence of both inhibitors with the hormone, the authors recorded a significant increase in trehalose release, about 26 g/mg. They also noted that indomethacin, but not BW4AC, dou­ bled phosphorylase activity. Neither inhibitor influenced expression of the gene for the new cytochrome P450. The authors did not articulate a firm conclusion, but rather carefully speculated that some eicosanoids may downregulate trehalose biosynthesis. Meanwhile, Steele and his colleagues have been investigating the possible actions of eicosanoids in the fat body from another cockroach, Periplaneta americana. Hypertrehalosemic hormone stimulates increased release of treh­ alose from intact fat bodies. Advancing from this basic hormone action, they first showed that the hormone stimulates increased concentrations of free fatty acids, specifically 16:0, 18:0, 18:1, and 18:2n-6, in trophocytes pre­ pared from P. americana fat body (Ali and Steele 1997a). They noted that other fat body cells, the mycetocytes and the urate cells, did not respond to the hormone. The authors offered two interpretations of these findings. On one hand, the fatty acids may be transported in hemolymph to distal tissues, where they would serve as energy sources. Alternatively, they speculated that the free fatty acids may serve as intracellular messengers. Glycogen phosphorylase, which is responsible for hydrolyzing glucose residues from glycogen, is the first step in trehalose biosynthesis. The activ­ ity of this enzyme is regulated by phosphorylation/dephosphorylation cycles that activate and deactivate the enzyme. Hypertrehalosemic hormone causes activation of this enzyme, and the activation eventually leads to increased secretion of trehalose from fat body cells. However, Steele and his col­ leagues postulated that trehalose biosynthesis must also be regulated at other

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points in the overall biosynthetic pathway, because treating fat body with cAMP or with calcium ionophore leads to increased phosphorylase activa­ tion without the attending secretion of trehalose. Hence, activation of phos­ phorylase is not the only rate-limiting step in trehalose biosynthesis. Ali and Steele (1997b) investigated this point in detail, using trophocytes prepared from disaggregated fat bodies. The cells were incubated in culture medium, then exposed to various hormone and eicosanoid biosynthesis in­ hibitor treatments. The authors determined the influence of the treatment on two parameters, the activation of phosphorylase and the release of free fatty acids. They recorded the activation of phosphorylase using a spectrophotometric assay procedure. They measured the release of fatty acids by incubat­ ing cells, then gently filtering the medium away, leaving the cells captured on a membrane. The cells were mixed in methanol, then centrifuged. The resulting supernatant represented a methanolic extract of free fatty acids. The dissolved fatty acids were methylated, then analyzed by gas chromatography. The authors investigated the influence of three eicosanoid biosynthesis inhibitors on phosphorylase activation in trophocytes. Incubations with indomethacin and nordihydroguaiaretic acid increased phosphorylase activation by about 24% and about 12%, respectively. The phospholipase A2 inhibitor 4'-bromophenacyl bromide had no influence on phosphorylase activation. In another series of experiments, batches of fat body cells were incubated with hormone and an eicosanoid biosynthesis inhibitor. The inhibitors enhanced the usual influence of the hormone on phosphorylase activation by about 6% to 8%. For example, incubations in the presence of 4'-bromophenacyl bro­ mide plus hormone increased activation by about 6% over the influence of the hormone alone. It appeared that inhibition of eicosanoid biosynthesis increased phosphorylase activation. The authors also determined the influence of the hormone and of selected inhibiitors on release of free fatty acids by the trophocytes. Incubations in the presence of indomethacin yielded significant increases in the levels of free fatty acids, including 16:0, 18:0, 18:1, and 18:2n-6. They conducted a similar series of experiments with the lipoxygenase inhibitor, nordihydrogua­ iaretic acid. This compound also stimulated increased release of free fatty acids; however, the effect was not as pronounced as it was with indometha­ cin. The nordihydroguaiaretic acid effect was seen at only one dosage, 10~4 M, and incubations in the presence of higher or lower concentrations of this inhibitor did not yield increased free fatty acids. The phospholipase A2 in­ hibitor, 4'-bromophenacyl bromide, similarly inhibited the release of free fatty acids in unstimulated trophocytes. Three more series of experiments showed that all inhibitors—indomethacin, nordihydroguaiaretic acid, and 4'-bromophenacyl bromide—did not synergize with the hormone to increase fatty acid release. To the contrary, these treatments blocked the normal hor­ mone-stimulated increases in free fatty acid concentrations.

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Ali and Steele (1997b) proposed that hypertrehalosemic hormone stimu­ lates the release of free fatty acids through an indirect action on phospholipase A2. They suggested that the hormone functions through a receptormediated mechanism to activate phospholipase A2. This idea is supported by results of another set of experiments. Melittin is a 26-amino acid bee venom peptide, which stimulates phospholipase A2 activity. The authors in­ cubated trophocvte preparations in the presence of melittin for fifteen min­ utes. then determined the release of free fatty acids. The}" recorded substan­ tial increases in 16:0. 18:0. 18:1. and 18:2n-6 following these experiments. Hence, they concluded that stimulating phospholipase A2 activity with an exogenous peptide produced results similar to the effects of hypertrehal­ osemic hormone. In another experiment, the authors prepared trophocvte membranes by centrifugation. They incubated the membrane preparations in the presence of melittin and. in separate treatments, in the presence of hypertrehalosemic hormone. The melittin treatments stimulated release of fatty acids from the membrane preparations, while the hormone treatments did not. Ali and Steele took this result to show that the hormone action obtains via a mecha­ nism different from the melittin action. Noting that the hormone stimulates the release of free fatty acids in intact cells, but not in membrane prepara­ tions. they suggested that cellular phospholipase A2 is activated by receptormediated events. Ali and Steele (1997b) then drew on the mammalian literature to sort out the influence of indomethacin and nordihydroguaiaretic acid on fatty acid release. Indomethacin inhibits cyclooxygenases more effectively than lipox­ ygenases. while nordihydroguaiaretic acid potently inhibits cyclooxygenase. lipoxygenase, and some phospholipases A2. Hence, the influence of these compounds may be expressed by inhibiting biosynthesis of prostaglandins. In this model, the hormone would stimulate phospholipase A2 activity, which would lead to release of free fatty acids. These compounds could then be converted into 20:4n-6 via the pathways described in chapter 3. The 20:4n-6 would be converted into prostaglandins, which may act via an autocoidal mechanism to influence the biosynthesis of trehalose. Using their trophocvte preparations, the authors next investigated the in­ fluence of selected free fatty acids on trehalose biosynthesis, determined by trehalose efflux (Ali and Steele 1997c). The saturated fatty acid 16:0 did not influence trehalose biosynthesis; however. 18:0. 18:1. 18:2n-6. and 20:4n-6 all similarly stimulated trehalose biosynthesis. P. americana is one of the cockroaches able to biosvnthesize 18:2n-6 de novo, from which 20:4n-6 is produced by elongation/desaturation pathways (Jurenka et al. 1987). AIi and Steele (1997c) found that hormone treatments, in the presence of indo­ methacin. doubled the rate of conversion of 18:2n-6 into 20:4n-6. Reasoning that free fatty acids released by exposure to the hormone could be converted into 20:4n-6. from which prostaglandins could be formed, they conducted

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one more experiment. Trophocytes were incubated in the presence of PGE2 and, separately, PGF2a. Whereas PGE2 did not stimulate trehalose bio­ synthesis, PGF2a did stimulate trehalose biosynthesis in a dose-dependent manner (Ali and Steele 1997c). The authors concluded that regulation of trehalose biosynthesis is more complex than earlier realized. Because inhibi­ tors of cyclooxygenase and phospholipase A2 downregulate the secretion of trehalose (Ali et al. 1998), the prostaglandins may stimulate trehalose bio­ synthesis and release from fat body cells in the American cockroach. We are left with interesting views on the roles of eicosanoids in modulat­ ing insect hormone actions. Work by the Keeley group with the fat body of the cockroach B. discoidalis suggests that eicosanoids may attenuate the in­ fluence of hypertrehalosemic hormone on trehalose mobilization. The evi­ dence from Steele's group would suggest that prostaglandins enhance the hormone effect on trehalose biosynthesis in fat body cells from another cockroach, P. americana. Again, the early work on prostaglandins and meta­ bolic events in the insect fat body suggests that prostaglandins downregulate lipid mobilization. The contradictions in this paragraph are more apparent than real. Eico­ sanoids can exert stimulatory and inhibitory influences on cellular actions in mammalian systems. Indeed, the classification of cell surface prostaglandin receptors is based in part on this point. For example, PGE2 receptors are denoted EP receptors. There are three classes of EP receptors: EP1 receptors lead to increased intracellular calcium concentrations, EP2 receptors increase intracellular cAMP concentrations, while EP3 receptors have the opposite effect (Coleman et al. 1990). In some systems, cells can express more than one class of EP receptors. Another complexity relates to receptor actions. Many receptors express differing affinities for the same eicosanoid. Where this is so, the concentrations of eicosanoids used in experimental manipula­ tions may generate misleading results. Given this brief background, it be­ comes plain that opposite, or apparently contradictory, effects of eicosanoids are to be expected. Moreover, experimental manipulations may produce concentration-driven artifacts. I suggest that the apparent contradictions in experiments on the interac­ tions between hormones and eicosanoids provide an important lesson to us: Many biological actions are simultaneously influenced by a host of signal moieties. Teasing out the actions of any particular moiety, with any hope for precision, will be a very interesting puzzle.

EICOSANOIDS IN DEVELOPMENT AND REGENERATION IN HYDROIDS Cnidarians in the class Hydrozoa make up a large assemblage of animals that exist in two forms, alternately as polyps and hydromedusae (Brusca and

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FIGURE 7-1. Hydra. Phylum Cnidaria. This drawing shows budding, which occurs in the asexual reproductive cycle. Thanks to Jon C. Bedick for the gift of this drawing.

Brusca 1990). Members of the genus Hydra (Fig. 7-1) have received a great deal of attention in studies of body pattern formation and regeneration. In recent years, the intracellular signal moieties involved in these processes have come to light. Leitz and Muller (1987) suggested that inositol triphosp­ hates and diacylglycerol act as intracellular messengers in the metamorpho­ sis of planula larvae in the hydroid Hydractinia echinata. In a similar line of work on the related genus Hydra, Muller (1989) reported that daily exposure to 1.2-dioctanoyl-5v;-glycerol caused wild-type polyps of H. magnipapillata to develop ectopic heads along the gastric column. Along the body column of the hydra, the potential to develop heads is regulated by a cellular param­ eter known as "positional value." which decreases in a gradient from the head to the foot of the polyp. The diacylglycerol was thought to act on an intracellular protein kinase C. which in turn raised the local positional value

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to a level that favors head development. When the polyps are treated with pulses of diacylglycerol, the positional value in the gastric region rises in a wave, and several ectopic heads may develop along the body column. De Petrocellis et al. (1991) reported similar findings implicating protein kinase C with another hydra, H. vulgaris. They found that two diterpenoidic diacylglycerols, verrucosins A and B, which they had isolated from the man­ tle of a marine mollusc, stimulated tentacle regeneration. In these experi­ ments, individuals were decapitated, then exposed to test compounds for twenty-four hours. Ten days later, the number of tentacles on each individual was counted, from which the average tentacle number resulting from each treatment was calculated. Again, the authors speculated that the novel di­ acylglycerols acted through protein kinase C. Additional work (De Petrocellis et al. 1993) supported this, and added complexity to the picture of signal pathways in regeneration in this animal. They found that the ver­ rucosin B effect on increased tentacle number was inhibited by protein kinase C inhibitors. The influence of verrucosin B was not enhanced in the presence of calcium, and the authors concluded that a calcium-independent protein kinase C was involved in tentacle regeneration. The additional com­ plexity emerged from experiments with the phospholipase A2 inhibitor oleyloxyethylphosphorylcholine. This compound inhibited the regeneration process, registered as reduced average numbers of tentacles. The influence of the inhibitor was reversed by adding 20:4n-6 to the preparations. The authors also tested the influence of various eicosanoid biosynthesis inhibitors on ten­ tacle regeneration, but none of them mimicked the effect of oleyloxyethylphosphorylcholine. Alternatively, they found that indomethacin strongly en­ hanced tentacle regeneration, from which they inferred that prostaglandins exert a negative control on regeneration in hydra. De Petrocellis et al. (1993) recognized that more than one signal transduction pathway is operative in hydroid regeneration. Muller et al. (1993) directly addressed the idea that eicosanoids influence positional value and formation of ectopic heads in polyps of H. magnipapillata. Small groups of polyps were prelabeled by incubating the animals in the presence of radioactive 20:4n-6. Upon stimulation with diacylglycerol, radioactive 20:4n-6 was released from complex lipids. The radioactivity did not appear in the medium, and the authors suspected that the free fatty acids remained within the polyps. In subsequent experiments, unlabeled polyps were exposed to diacylglycerol, then processed for extraction of free fatty acids and eicosanoids. These components were derivatized and analyzed by gas chromatography-mass spectrometry. The authors recorded 20:4n-6 at more than 7 g/gram fresh weight and 18:2n-6 at more than 10 g/gram fresh weight. They also detected considerable amounts of lipoxygenase prod­ ucts. Four 20:4n-6 metabolites, 5-, 8-, 12-, and 15-hydroxyeicosatetraenoic acid, were present. The four components totaled about 46 g/gram fresh

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weight, of which 12-hydroxyeicosatetraenoic comprised 39 g. Two 18:2n-6 metabolites, 9- and 13-hydroxyoctadecadienoic acid, were present, summing to about 9 g/gram fresh weight. Again, 20:4n-6 and the eicosanoid metabo­ lites were not detected in the medium. In biological experiments, the authors noted that 20:4n-6 and diacylglycerol exerted similar actions on experimental polyps. After five daily expo­ sures to 20:4n-6 (100 μΜ), the appearance of the polyps was unchanged; however, the pattern of regeneration was altered. Gastric segments were ex­ cised from whole polyps, then allowed to regenerate the lost foot. Out of 100 experiments, twelve polyps did not regenerate a foot, and nineteen formed tentacles, usually associated with the head, instead of a foot. After the sev­ enth treatment, intact polyps began to develop ectopic tentacles in the gastric region. In another series of experiments, exposure to 20:4n-6 combined with diacylglycerol yielded a synergistic acceleration of the rate of ectopic head development. These data strongly supported the idea that lipoxygenase prod­ ucts influence regeneration and positional values in the experimental animals (Muller et al. 1993). The 20:4n-6 metabolites may act through protein kinase C. Exposing the polyps to 20:4n-6 combined with the protein kinase C inhibitor chelerythrine chloride (at 600 nM) counteracted the influence of 20:4n-6 on ectopic head formation. In a related experiment, experimental polyps were incubated in the presence of 20:4n-6 plus diacylglycerol (at 10 μΜ) and the lipoxygenase inhibitor nordihydroguaiaretic acid. Compared to controls exposed to di­ acylglycerol and 20:4n-6 without the lipoxygenase inhibitor, ectopic tentacle formation was reduced from 86% to 33%. Drawing on the background information that 20:4n-6 activates some forms of protein kinase C, Muller et al. (1993) suggested that 20:4n-6 may act directly on a protein kinase C within the cells of H. magnipapillata. They also noted that the released 20:4n-6 could move into neighboring cells, and thereby expand the range of protein kinase C activation. They proposed that 20:4n-6 and diacylglycerol increase positional value, and hence the head forming potential, by activating a protein kinase C. Muller et al. (1993) also proposed that lipoxygenase products of 20:4n-6 metabolism cooperatively participate in control of body pattern. Turning back to the hydroid Hydractinia echinata, Leitz et al. (1994a) suggested that eicosanoids also act in initiating metamorphosis. They found that induction of metamorphosis resulted in release of radioactive 20:4n-6 from internal stores in larvae of the hydroid. Moreover, exposure to the lipoxygenase inhibitors nordihydroguaiaretic acid and, separately, eicosatetraynoic acid inhibited the induction of metamorphosis. Similar treatments with the cyclooxygenase inhibitors indomethacin and aspirin did not limit experimentally induced metamorphosis. Leitz et al. (1994a) inferred that li­ poxygenase products are somehow involved in the induction of metamor-

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phosis. Their case is bolstered by rigorous chemical determination of 5-, 8-, 12-, and 15-hydroxyeicosatetraenoic acids in extracts prepared from H. echinata larvae. In two independent determinations, 8-hydroxyeicosatetraenoic acid was the quantitatively major eicosanoid, present at about 7 to 17 g/gram fresh weight. In a related study, Leitz et al. (1994b) showed that cytosolic fractions of H. magnipapillata could enzymatically convert 20:4n-6 into 11-R-hydroxyeicosatetraenoic acid and 12-S-hydroxyeicosatetraenoic acid. Hence, two representatives of the Hydrozoa were shown to be compe­ tent to biosynthesize eicosanoids. Leitz et al. (1994b) considered the biological significance of the two li­ poxygenase products. First, they found that the lipoxygenase inhibitor nordihydroguaiaretic acid retarded the biosynthesis of 12-S-hydroxyeicosatetraenoic acid, but did not alter the synthesis of 11-R-hydroxyeicosatetrae­ noic acid. The authors suggested that H. magnipapillata expresses two sepa­ rate enantioselective lipoxygenases. With respect to their standard assays for ectopic head formation, they found that both enantiomers of 11-hydroxyeicosatetraenoic acid inhibited the diacylglycerol-induced ectopic head for­ mation. Alternatively, 12-S-hydroxyeicosatetraenoic acid enhanced bud for­ mation, the first step in the asexual cycle of reproduction. In a similar line of work with H. vulgaris, Di Marzo et al. (1993) noted that the polyps produced two main lipoxygenase products, 11-hydroperoxyeicosatetraenoic acid and 11-hydroxyeicosatetraenoic acid, although other hydroxyeicosatetraenoic acids were produced in minor amounts. Again, their biological assays revealed specific eicosanoid actions. First, most of the li­ poxygenase products inhibited bud formation. Second, only 11-hydrox­ yeicosatetraenoic acid elevated tentacle regeneration in decapitated polyps. These findings on two species of hydroids contribute very important in­ sights to our appreciation of eicosanoid actions in animals. The Cnidaria are among the earliest metazoan animals, with fossils dating to about 700 mil­ lion years before the present. Early members of the Cnidaria are thought to figure importantly in the evolution of multicellular life (Brusca and Brusca 1990). The idea that eicosanoids play separate and distinct roles in body pattern, regeneration, and asexual reproduction in hydroids links eicosanoid actions to the very origins of the Metazoa. It is not difficult to imagine that eicosanoids were recruited into many biological roles in the ensuing hun­ dreds of millions of years of animal evolution. The results with these two hydroids also suggest that eicosanoids are in­ volved in very fundamental aspects of cellular differentiation. We do not have more information on the roles of eicosanoids in cellular differentiation in other invertebrates; however, the research findings described in this sec­ tion open a wide door to new possibilities for the discovery of important eicosanoid actions in animals.

CHAPTER 8

Eicosanoids Mediate Ecological Interactions

ECOLOGY is the field of biological sciences concerned with the relationships between populations of organisms and their environments. Chemical ecology is a specialization within the broader field of ecology. Studies in chemical ecology take their roots in understanding that many ecological interactions between and within populations are mediated by chemicals. Chemical ecol­ ogy is a beautifully interdisciplinary line of inquiry, blending the strengths of highly sophisticated expertise in analytical and synthetic chemistry with de­ tailed knowledge of complex biological systems. Most of the literature on eicosanoids has emerged from detailed biochemi­ cal and physiological research on mammalian systems—again, particularly within the realm of the clinical significance of eicosanoids. Because of the relatively intense focus on mammals and events within mammals, the idea that eicosanoids play major roles in chemical ecology creates a comparison, and thereby allows a great broadening of our appreciation of the biological significance of eicosanoids. This chapter reviews a rapidly growing research area, namely the roles of eicosanoids in mediating ecological interactions. We will consider the roles of certain prostaglandins in predator avoidance, and the roles of prostaglandins and other eicosanoids in certain host-parasite interactions. Because this field of research is relatively new. there are only a few well-documented examples of eicosanoid actions in animal ecology. I suppose these few cases represent many others awaiting discovery. The cen­ tral point of this chapter continues the broad theme of this volume: Inquiry into the significance of eicosanoids will yield profound new information about animal biology.

EICOSANOIDS IN PREDATOR AVOIDANCE We recall that the first discovery of eicosanoids in an invertebrate, the octacoral. Plexaura homomalla, created considerable interest because the coral represented a natural, harvestable source of prostaglandins for commercial and research purposes. P. homomalla represented a source of prostaglandins because these chemicals are present in very high amounts, as much as 8% of the wet tissue weight. However, most octacoral species do not maintain these

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high levels of prostaglandins. High levels of tissue prostaglandins seem to be an adaptive feature of some coral species. Gerhart (1984) first suggested that the elevated prostaglandin levels may serve as a chemical defense against predation by coral-eating fish. He summarized the arguments supporting this idea in a later review (Gerhart 1991), from which most of the following comments are drawn. Prostaglandins induce vomiting, or emesis, in many mammalian and nonmammalian vertebrates, including humans, when delivered into the stomach by direct feeding. PGA2 or its 15-R-epimer, the prostaglandins found in some octacorals, induce vomiting in several phylogenetically disparate fish species, including the killfish Fundulus heteroclitus (Cyprinodontidae) and the yellowhead wrasse Halichoeres garnoti (Labridae). The crux of Gerhart's argument lies in the idea that fish and other vertebrate predators are capable of developing a learned aversion to emetic foods. In his model, some species of coral-eating fish might eat a portion of coral, experience a bout of vomiting, and thereafter avoid the emetic coral. Hence, the high levels of prostaglandins and their derivatives would endow the coral with a certain level of protection from predation. Gerhart (1991) tested his hypothesis in a series of field and laboratory experiments. For field experiments, he isolated PGA2 and its 15-R-epimer from P. homomalla colonies, and applied the prostaglandins to pieces of cooked fish muscle. Then he delivered three types of food pellets—those laced with PGA2, those laced with the 15-R-epimer, and those treated with solvent only—to yellowhead wrasse living at a coral reef site near Curagao, Netherlands Antilles. For purposes of data analysis, the pellets laced with either prostaglandin were pooled into a single treatment called emetic pros­ taglandins. The fish initially accepted the control and emetic pellets at identi­ cal rates. As the experiment progressed, the fish gained experience with the food pellets. After three or more prior experiences with eating each pellet type, the fish rejected all the emetic pellets and accepted all the control pellets. These results were similar to results from previous laboratory experi­ ments with yellowhead wrasse (Gerhart 1984). Gerhart inferred from these results that the fish form a strong learned aversion to the emetic food pellets. Gerhart conducted additional aversion experiments with tissues of the whip coral, Leptogorgia virgulata. In these experiments, three species of fish, the largemouth bass, Micropterus salmoides (Centrarchidae), pinfish, Lagodon rhomboides (Pomadasidae), and striped bass, Morone saxatilis (Percichthyidea), were fed pieces of coral or pellets of alginate laced with a 1% concentration of extract from the coral. These foods induced emesis in all individuals of all species, and again, the fish developed learned aversions to the coral. Gerhart put forth the reasonable idea that many fish learn to avoid emetic foods. His argument loses some of its steam, however, because L. virgulata is one of the coral species that does not maintain high levels of

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prostaglandins. This species does contain another emetic compound, the diterpenoid pukalide. Gerhart stretched his case by suggesting that this or other chemicals in the coral induced emesis by somehow causing a sharp elevation of prostaglandin levels in the stomach of predators. It remains to be deter­ mined whether this is possible. That said. Gerhart's behavioral experiments with prostaglandin-laced foods and the learned aversions to an emetic coral species amount to a sound argument: The high prostaglandin levels in some species of octocoral proba­ bly serv e as a chemical defense against predation. The idea is not without its detractors (Pawlik and Fenical 1989): however, the discussion may be more a matter of semantics than biology. Pawlik and Fenical (1989) noted that the presence of prostaglandins does not prevent fish from tasting laced artificial food pellets. Gerhart (1991) argued that the prostaglandins do not serve to reduce the palatability of foods, but rather act to create learned, lasting aversions to emetic foods. More illumination on the idea that eicosanoids act in the chemical ecology of predator-prey interac­ tions comes from a series of papers on an unrelated system, to which we now turn our attention. Gastropod molluscs include the order Opisthobranch. most members of which do not feature shells. The nudibranch. Tethys fimbria, is a Mediterra­ nean opisthobranch. As part of their investigations of marine natural prod­ ucts. Cimino et al. (1989. 1991a. b) discovered a novel class of prosta­ glandin derivatives, the prostaglandin 1.15-lactones (chapter 2). Lactone derivatives of prostaglandins E;. A;. and F2a have been determined. Two additional variations have been described. One is the lactone-11-acetate and the other is the carbon-9 or carbon-11 fatty acid esters of the lactones. In these lactones, the carbon-1 carboxylic acid functionality of the prostaglan­ din forms an oxygen bridge with carbon-15. yielding a 15-carbon closed loop with a 5-carbon aliphatic chain extending from carbon-15. Di Marzo et al. (1991» worked out the biosynthetic pathway for the pros­ taglandin 1.15-lactones. They began by injecting radioactive PGE: into the mantles of adult nudibranchs maintained in laboratory tanks. At one. two or three days after the injections, the mantles and the cerata were processed separately for the isolation of PGE; 1.15-lactone. The authors verified the purity of the radioactive lactone by chemical deri\ atization and repurification. One day after injecting the starting PGE;. they recovered lactone deriv­ atives at about 3 boiling the enzyme preparations for fifteen minutes. The activity was also inhibited by eicosatetraynoic acid, but not by indomethacin. The authors used Western blot analysis to look for the presence of lipoxygenases in the fluke preparations. They identified two fluke proteins, in the expected mo­ lecular weight ranges, that cross-reacted with antibodies to human 5- and 12-lipoxygenases. Similar exercises with antibodies to ram seminal vesicle cyclooxygenase-1. and sheep cyclooxygenase-2 yielded no evidence for cy­ clooxygenase in the S. mansoni preparations. Finally, they identified two lipoxygenase-like DNA sequences in the genomic DXA prepared from adult flukes by Southern hybridization. The results in this paper clooxygenase products. This work departs from the earlier work by Salafsky and his colleagues, showing the biosynthesis of pros­ taglandins and lipoxygenase products. With no biochemical or Western blot data supporting the presence of cyclooxygenases in S. mansoni adults. Baset

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et al. (1995) suggested that the cyclooxygenase pathways may not occur in these animals. On the other hand, they noted two quite faint bands on South­ ern blot analysis that correspond to mammalian cyclooxygenase-1 and -2 sequences. They suggested that the inability to detect prostaglandin bio­ synthesis in the adults may be related to stage specific expression of the cyclooxygenase genes. Baset et al. (1995) also considered the metabolism of 18:2n-6 by adult blood flukes. Cytosolic fractions of blood fluke homogenates were incubated with radioactive 18:2n-6, and the products were separated on thin-layer chro­ matography. The authors found a band of radioactivity corresponding with authentic 13-hydroxyoctadecadienoic acid. In their work on eicosanoid bio­ synthesis, Salafsky and his colleagues routinely used 18:2n-6 as the starting substrate. We might wonder if some of the radioactive products recovered in this work were direct oxygenation products of 18:2n-6, some of which may have appeared as cyclooxygenase products. Clarifications of this sort will emerge from continued work on this system. Having said that, I emphasize that issues such as this are technical, and can be resolved. They do not detract from the main view that parasites may influence host immune reac­ tions via alterations in eicosanoid biosynthesis.

Nematodes Nematodes are among the most successful animal groups. About 12,000 spe­ cies have been described, and many more are thought to remain unknown. Nematodes are divided into two major classes. The Adenophorea are mostly free-living animals, while most of the Secernentea are parasitic. Members of the Secernentea are variously endo- and exoparasitic. Some are parasites of plants, others of animals. Because some nematodes are responsible for very serious infections in humans and food animals, a very large body of litera­ ture is devoted to this group. Lymphatic filariasis is a parasitic disease caused by three nematode spe­ cies, Wuchereria bancrofti, Brugia malayi, and B. timori. Adults of these animals live in lymphatic vessels. The adults give rise to larval forms known as microfilariae, which are found in the blood stream. Microfilariae migrate into the peripheral circulation on a daily basis, which corresponds to the host-seeking periods of female mosquitoes. The microfilariae are ingested with blood meals by mosquitoes. They migrate through the mosquitoes, passing the midgut integument into the hemolymph, and move to the flight muscles. They eventually make their way to the salivary glands, from where they are injected into another human host during the mosquito's blood meal. Microfilariae seem to enjoy a certain level of protection from the immune reactions of their vertebrate hosts. During the course of their tenure in the

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human blood stream, microfilariae are in contact with host immunocytes, including leukocytes, platelets, and endothelial cells. Yet. immune cells rarely adhere to the parasites. Moreover, parasite-specific antigens are low in infected humans. As we have seen with other endoparasites. some eicosanoids exert attenuating actions on host immune cells. Drawing on this notion. Weller and his colleagues investigated eicosanoid biosynthesis in fil­ arial parasites. They first established the presence and ready incorporation of 20:4n-6 into the parasite lipids, and showed that the worms can biosynthesize 20:4n-6 from 18:2n-6 (Longworth et al. 1987. Liu and Weller 1989). Then. Liu et al. (1990) began direct studies on eicosanoid biosynthesis. They isolated micro­ filariae of B. malayi from infected jirds. The parasites (l06/experiment) were incubated with radioactive 20:4n-6. and the products were separated on highperformance liquid chromatography coupled to a flow-through liquid scin­ tillation counter. In some experiments, the products were separated on thinlayer chromatography. In another line of experiments, the microfilariae were incubated with no exogenous 20:4n-6. and eicosanoids were determined by radioimmunoassay. Secreted eicosanoids were determined directly in unextracted medium, and retained products were extracted from the parasites. Finally, in some experiments, the microfilariae were incubated in the pres­ ence of various eicosanoid biosynthesis inhibitiors. The researchers recorded two major peaks of radioactive eicosanoids: 6-keto-PGFIA. the stable product of PGI2. and PGE2. These products were not recorded after incubations with heat-killed microfilariae. In their second line of work, they incubated sets of IO6 microfilariae for tw enty-four hours in medium that was not supplemented with 20:4n-6. They recovered nearly 2,000 pg of 6-keto-PGFLQ. about 800 pg of PGE2. and almost 200 pg of PGD2 from the incubation medium. PGF2A and thromboxane B2 were not detected in the medium. The worms retained relatively low amounts of these products. The authors recovered only about 100 pg of 6-keto-PGFLA. about 20 pg of PGF2q. and lower amounts of the other prostanoids from the worms. As determined by the radioimmunoassay procedures, incubations in the presence of indomethacin, BW755c. and eicosatetraynoic acid resulted in over 90 percent inhibition of cyelooxygenase activity. Liu et al. (1990) concluded that the microfilariae can generate cyclooxygenase products from exogenous and endogenous 20:4n-6. and release these products into their surrounding media. They noted that the biosynthesis of PGI2 was quite meaningful, because this compound very potently inhibits platelet aggregation in mammals. Moreover, thromboxane, which enhances platelet aggregation, was not produced by the parasites. PGE2 and PGI2 are both vasodilators, and PGE2 suppresses several proinflammatory actions, in­ cluding granulocyte and macrophage functions. T lymphocyte activation, and lymphokine biosynthesis and release, in mammals.

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The formation and release of PGI2 and PGE2 may help the microfilariae manipulate the immune reactions of their hosts. Filarial-infected humans ex­ press weakened immunity, for which these parasite-derived prostaglandins may be partly responsible. Again, parasites may use eicosanoids as part of the overall evolutionary adaptation to host-parasite relationships. Liu and Weller (1990) inferred that work to gain a more detailed appreciation of eicosanoid biosynthesis and actions in parasites will yield greater insights into the chemical interactions between parasites and their host immune systems. Liu and Weller (1992) tested this idea a little more directly by assessing the ability of microfilariae to inhibit platelet aggregation. They isolated mi­ crofilariae from infected jirds following their routine protocol. Human plate­ lets were prepared from blood samples donated by healthy volunteers. The platelets were washed, and in some experiments they were pretreated with eicosanoid biosynthesis inhibitors. Microfilariae and platelets were incubated in buffer; however, to ensure mutual contact, they were pelleted by gentle centrifugation and preincubated for one hour. The pellets were resuspended, and the microfilariae were separated by centrifuging at 80 g for three min­ utes. This treatment pellets the microfilariae while leaving the platelets in suspension. Platelet aggregation was stimulated by adding thrombin, and aggregation was determined on an aggregometer. The microfilariae inhibited platelet ag­ gregation in a dose-dependent manner. Platelets exposed to parasites at a ratio of 1 parasite/IO4 platelets completely inhibited thrombin-stimulated ag­ gregation. Platelet aggregation can be stimulated by other agonists, and simi­ lar experiments in which platelet aggreagation was stimulated, separately, with collagen, 20:4n-6, or calcium ionophore A23187 showed that the para­ sites also inhibited aggregation in response to these stimulants. Aside from inhibiting aggregation, incubations in the presence of micro­ filariae influence two other platelet activities. Thromboxane A2 biosynthesis and release of radioactive serotonin were inhibited, in a dose-dependent way, by the parasites. Thromboxane generation was determined by radioim­ munoassay. To record serotonin release, the platelets were preloaded with radioactive serotonin, then incubated in the presence of varying numbers of microfilariae. It was demonstrated that the presence of microfilariae very potently reduced the ability of platelets to execute their normal immune functions. By incubating platelets and microfilariae in adjacent chambers separated by permeable membranes, Liu and Weller (1992) showed that the influence of the parasites did not require direct physical contact between the cells and the worms. This experiment demonstrated that the influence of the parasites on platelet aggregation was expressed via a soluble factor that could move across the separating membranes. In another series of experiments, the mem-

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branes were exchanged with dialysis membranes with molecular exclusion sizes of 30,000, 12,000, and 1,000. The factor moved across all these mem­ branes, and inhibited platelet aggregation in the adjacent chambers. It was thus shown that the microfilariae release one or more small molecules that influence platelets. Liu and Weller (1992) suspected that the chemicals were prostaglandins, and they tested this idea by pretreating the microfilariae with cyclooxygenase inhibitors before incubating them in adjacent chambers with plate­ lets. Pretreatments with aspirin, indomethacin, BW 755c, and eicosatetraynoic acid reduced the influence of microfilariae on platelet aggregation. Liu and Weller (1992) reached the conclusion that the parasites can inhibit host platelet aggregation, and parasite-derived prostaglandins may exert the inhibiting effect. The eicosanoid actions may be crucial elements of the para­ site life cycle. For mosquito-borne parasites, the parasites must be able to move freely through microcirculation in the periphery of the host to be avail­ able for ingestion when vectors take a blood meal. In the case of micro­ filariae, the mosquitoes are not merely carriers, as the term "vector" might suggest. The mosquitoes serve as intermediate hosts, in which the parasites pass through developmental phases of their lives. Hence, the ability to be present in unrestricted ways in host microcirculation is quite important. Eicosanoids may contribute to this important aspect of the host-parasite rela­ tionship. Given recent findings that eicosanoids mediate some insect immune reactions (chapter 5), eicosanoids may also be important in the insect phase of the parasites' life cycle. Lui and Weller also noted that their findings with B. malayi could very well apply to many other metazoan parasites. Daugschies (1995) investigated the influence of eicosanoid biosynthesis inhibitors on larval development, and the biosynthesis of eicosanoids by lar­ val stages of another nematode parasite, the nodular worm of pigs, Oesophagostonum dentatum. This parasite was maintained by routine infection of pigs at four-week intervals. Third stage larvae were produced in coproculture, and the parasites were isolated by allowing them to migrate into sterile water. The larvae were exsheathed by brief exposure to bleach, then washed. The exsheathed larvae were then maintained in vitro in a culture medium. Cultured, untreated third-stage larvae advanced to fourth-stage larvae after seven days. By contrast, larvae incubated in the presence of aspirin or indo­ methacin experienced retarded development. Of the sixteen aspirin-treated cultures, only three contained fourth-stage larvae by day 13. Similarly, incu­ bations in the presence of indomethacin completely inhibited development to the fourth stage. Daugschies (1995) interpreted these findings to support the idea that prostaglandins are involved in larval development in O. dentatum. The third-stage larvae were competent to biosynthesize and secrete eico­ sanoids into the culture medium. Levels of four cyclooxygenase products, PGE2, PGD2, PGF2a, and thromboxane B2, were determined by radioim-

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munoassay procedures, using commercial kits. The prostaglandins were ex­ tracted using solid phase extraction columns, and, without separating indi­ vidual components, were taken into the radioimmunoassay. PGD2 and PGE2 were detected in the culture medium and parasite homogenates after seven, fourteen, twenty-one, and twenty-eight days of continuous culture. PGF2a and thromboxane B2 were not detected in the homogenates, but were present in the medium throughout the twenty-eight-day culture periods. Extraction efficiencies were not reported, and the specific amounts of the prostaglandins recovered represent estimates, which ranged from about 0.1 to 2.0 pg^g homogenate protein. As seen in the other work on parasites, Daugschies (1995) speculated that the prostaglandins produced and released from 0. dentatum may be involved in modifying host defense reactions. He also rec­ ognized that the influence of the cyclooxygenase inhibitors on larval devel­ opment indicated that some eicosanoids may be involved in the developmen­ tal biology of these parasites. In a later paper, Daugschies (1996) determined the presence of leukotrienes in supernatants and homogenates of 0. dentatum in culture. The para­ sites were cultured as described above. In some experiments, the culture medium was supplemented with the leukotriene biosynthesis inhibitor diethylcarbamazine. After selected culture periods, the medium and pelleted parasites were separately processed for eicosanoid extraction and isolation of leukotrienes by solid phase extraction. Leukotriene B4 and the peptidoleukotrienes were separately determined by commercial radioimmunoassay kits. Daugschies (1996) detected both types of leukotrienes in the culture media and third-stage larval parasite homogenates after seven, fourteen, twentyone, and twenty-eight days of culture. When the data were calculated as pg leukotriene/100 larvae, the author recorded steadily increasing amounts of leukotrienes in the media and homogenates. In particular, the peptidoleukotrienes in the culture media increased from 1.3 pg/100 larvae at fourteen days to 7.2 pg/100 larvae at twenty-eight days. As we saw with the cycloox­ ygenase inhibitors, parasite cultures in the presence of diethylcarbamazine inhibited larval development from third stage to fourth stage. The inhibitory influence was reversed by placing the parasites in untreated media. Aside from its influence on development from the third to fourth stages, diethylcarbamazine also inhibited the growth of fourth stage-larvae as mea­ sured by the length of the parasites. Again, the growth retardation was re­ versed after the larvae were removed from the presence of diethylcarba­ mazine. Daugschies (1996) concluded that leukotrienes are involved in physiological events in the lives of the parasite, which can be recorded in terms of development and growth. The specific physiological actions of these eicosanoids are not yet appreciated. To gain a clearer idea of the physiological significance of eicosanoids in larvae of 0. dentatum, Daugschies and Ruttkowski (1998) assessed the influ-

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ence of eicosanoid biosynthesis inhibitors and of eicosanoids on larval mi­ gration in vitro. Third-stage larvae were harvested from their usual parasite culture system and exsheathed following standard protocols. For the migra­ tion experiments, about 100 third-stage larvae were incubated for twentyfour hours in the presence of die thy lcarbamazine. indomethacin, or aspirin. Control lan ae were incubated in the presence of the drug vehicles. The lanae were then suspended in 3.3% agar, and the resulting agar-larval sus­ pension was transferred to a dish in which a matrix made of gauze soaked in 1.3% agar had been prepared. After cooling, the gauze with the gelled agarlan al suspension was placed in a tube containing water amended with an eicosanoid biosynthesis inhibitor or vehicle. The lan ae were allowed to migrate for eighteen hours, and then the gauze with the agar-lanal gel was removed from the tube. The tube was centrifuged. and the supernatant removed. The number of lan ae that had migrated out of the agar-lan al gel was determined by counting lanae in the pellet. The nonmigrating lanae. which remained in the agar, were counted after melting the agar, then pelleting the parasites. The authors found that the parasites migrated out of the agar, with a calcu­ lated migration index of about 100. The migration index declined, in a dosedependent way. when the lanae were incubated in the presence of aspirin or indomethacin (cyclooxygenase inhibitors), or diethylcarbamazine (leukotriene biosynthesis inhibitor). For the aspirin treatments, the migration in­ dices declined from about 100 to 50 in the presence of 14 mmol aspirin, and to about 10 in the presence of 28 mmol aspirin. Higher doses virtually abol­ ished all migration. The authors reported similar results with diethylcar­ bamazine. The inhibition of migration was reversible by washing the inhibi­ tors out of the experimental systems. The authors also conducted experiments with selected eicosanoids. In these procedures, sets of lan ae were incubated in the presence of diethylcar­ bamazine or aspirin, some of which were supplemented with eicosanoids. They denoted their treatments as I + E for inhibitor plus eicosanoid and I - E for inhibitor without added eicosanoid. With diethylcarbamazine (at 25.5 mmol). they recorded the usual decline in migration index for the I-E treatments. I -I- E treatments (at 5 X 10 ~4 mmol) with leukotriene B4 and C4 reversed the effects of the inhibitor on migration. At the same dosages, leukotriene D4 also reversed the effects, but to a limited extent, while leukotriene E4 had no influence on migration. Similar experiments with aspirin and individual prostaglandins did not produce evidence for the ability of prostaglandins to reverse the effects of the inhibitor. However, lan ae incubated in the presence of aspirin plus a mixture of PGD2, PGI2, and PGF2q (at 5 X 10~4 mmol) yielded a migration index of about 100, as seen in positive control experiments. Daugschies and Runkowski (1998) point out that agar migration assays are established tools used to assess the influence of antiparasitic drugs on the

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viability of the parasites. Their discussion essentially links several important lines of evidence. First, as discussed earlier, the nematodes are competent to biosynthesize cyclooxygenase and lipoxygenase products. Second, inhibition of eicosanoid biosynthesis reversibly retarded development and growth of the larval parasites. Third, exposure to eicosanoid biosynthesis inhibitors re­ versibly arrested larval emigration from agar gels. The authors inferred from these findings that eicosanoids are important in the physiology of nematodes. Their conclusion is not overstated. Moreover, they recognized that their ma­ nipulations do not reveal specific physiological or biochemical steps that depend on eicosanoids. The excursion through larval development, rates of overall growth, and migration are taken as indices of other, probably crucial, physiological actions, the inhibition of which can be registered in terms of development, growth, and migration. Kaiser et al. (1992) suggested another point of eicosanoid-mediated inter­ action between endoparasites and their hosts. They focused on the role of endothelial cells in arterial relaxation. In their work on pathogenesis of dog heartworms, Dinofilaria immitis, they showed that the endothelium-dependent relaxation of the dog femoral artery is inhibited in dogs infected with D. immitis. They later found that exposing isolated rat aorta rings to adult heartworms also depressed the relaxation. The depressing factor was not ex­ pressed after the aorta and the parasites were co-incubated with indomethacin. The authors then posed the next question, wondering whether the indomethacin influenced prostaglandin biosynthesis in the mammalian tissue or the parasites. Their experiments required isolated aorta rings and heartworms. Rats were sacrificed, and aorta rings, with the attendant endothelial layer, were pre­ pared from the aortas. The rings were suspended in organ chambers and connected to a stationary anchor and to a force transducer. Changes in ring tension were continuously recorded via the transducer. In their protocols, the rings were relaxed by exposing them to acetylcholine, and attenuation of the relaxing treatments was recorded. Heartworms were harvested from infected dogs, then maintained in media. In their first experiments, the authors made chloroform extracts of heartworms. Aorta rings were incubated in the presence of resuspended heartworm extracts and, separately, in the presence of heartworm-free control extracts. The heartworm extracts significantly depressed relaxation in re­ sponse to increasing acetycholine doses, indicating that the active heartworm factor is chloroform-extractable. The following series of experiments showed that after pretreating heartworms with aspirin, the heartworms did not exert their usual depressant activity on aorta relaxation. On the other hand, pre­ treating the aorta rings with aspirin did not prevent the heartworm-induced depression of relaxation. The authors inferred that a prostaglandin produced and released from the heartworms depressed the relaxation response. The authors made chloroform extracts of unused heartworm medium and

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of heartworm bioassay preparations. The samples were derivatized, then an­ alyzed by gas chromatography-mass spectrometry. The authors detected two peaks, one unidentified, and the other with characteristics of PGD2. They did not obtain a complete mass spectrum of PGD2- Rather, they detected three ions known from derivatized PGD2 with chromatographic retention time of authentic PGD2. It appeared that PGD2 might be responsible for the heartworm depression of acetylcholine-driven aorta ring relaxation. Kaiser et al. (1992) investigated this possibility in two ways. In the first experiment, aorta rings were incubated in the presence of low concentrations of PGD2 (10~ I2. 10"1 and 10' 10 M). At low acetylcholine concentrations, all PGD2 treatments attenuated the normal relaxation reponse. In the second experiment, aorta rings were incubated in the presence of the standard heartworm bioassay or with PGD2 (10^12 M). As just seen, at low acetylcholine concentrations, the relaxation reactions to both treatments were identical. The authors concluded that PGD2 may act in heartworm-induced attenuation of aorta relaxation reactions. Kaiser et al. (1992) proposed on the basis of their findings that the adult heartworms secrete an eicosanoid that acts to alter the release of relaxing factors from mammalian endothelial cells. This would result in attenuated smooth muscle relaxation, which would lead to reduced blood flow rates. They speculated that this may be adaptive to the parasites by enhancing the opportunity for nutrient uptake from the blood. Given the nutrient-rich na­ ture of mammalian blood, however, it seems unlikely that this would favor the parasite. The authors also suggested that the reduced blood flow would shunt blood to cutaneous tissues to facilitate moving the microfilariae to the periphery for mosquito ingestion. Again, the advantage to the parasite seems remote. While the adaptive significance of altering vascular smooth muscle relaxation seems unclear, this stands as still another point of contact between endoparasites and their mammalian hosts. Salafsky et al. (1990) described eicosanoid biosynthesis by another nema­ tode. the human hookworm. Necator americanus. The adults of these ani­ mals attach themselves to the mucosa of the intestinal tract. The eggs of attached adults are deposited in soil along with feces. Larvae hatch and go through two molts to reach the infective third larval stage. The infective stage enters its human host by penetrating the skin, and in this regard, N. americanus is similar to S. mansoni. The larvae migrate through the skin and make their way to the lungs via blood or lymph circulation. The larvae molt into their fourth stage in the lungs, from where they migrate to the esoph­ agus. where they become established, by way of the tracheae. Salafsky et al. (1990) developed an artificial membrane made of gelatin and agar to assess larvae penetration in response to selected compounds. They found that human skin lipids significantly increased larval penetration rates. They separated human skin lipids into several fractions on thin-layer

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chromatography, and found that the free fatty acid fraction induced the high­ est larval penetration rates, nearly 80%. Drawing on their experience with S. mansoni, the authors documented eicosanoid biosynthesis from radioactive 18:2n-6. Third-stage larval parasites were incubated with the substrate; then, products were extracted and separated on high-pressure liquid chromatogra­ phy. The published radio-chromatograms indicated biosynthesis of several eicosanoids, including PGE1, PGE2, leukotrienes B4 and C4, and 15-hydroxyeicosatetraenoic acid. By analyzing the parasites and their medium sep­ arately, they found that virtually all radioactive products were secreted into the medium (although the secretory, or possibly excretory, mechanism is not known). Salafsky et al. (1990) noted several parallels in the skin penetration strate­ gies of N. americanus and S. mansoni. Both are obligate skin penetrators, stimulated by skin lipids. They both biosynthesize eicosanoids, which may be involved in the skin penetration process. Both species undergo mor­ phological changes immediately after penetration. The detailed strategies of skin penetration differ. While larvae of both species remain at the epidermaldermal junction for hours before moving into the dermis, hookworms cause a classic proinflammatory reaction in skin, while cercariae do not. The au­ thors speculated that the hookworm larvae modulate direct immune reactions directly toward themselves, and thus may gain an advantage from stimulat­ ing unrestricted inflammatory reactions. We have several points of information on eicosanoids in metazoan endoparasites. Aside from the papers cited in this chapter, several other groups have reported on the biosynthesis of eicosanoids by parasites. I believe we can safely surmise that all parasites biosynthesize eicosanoids. The eico­ sanoids may influence host defense reactions, and thereby serve an adaptive role in parasitization. The work by Daugschies and Salafsky also points to eicosanoid actions in physiology and behavior of the parasites. We will see in chapter 9 that another nematode provided the first enzyme involved in eicosanoid biosynthesis to be purified from an invertebrate source (Meyer et al. 1996). The idea that eicosanoids act in internal physiological mechanisms and are secreted into mammalian hosts, where they mediate host-parasite relationships, makes the study of eicosanoids in these invertebrates all the more interesting. We will now see that eicosanoids similarly serve multi­ functional purposes in ectoparasites.

Ticks All ticks are obligate ectoparasites, feeding on blood meals derived from their hosts. Unlike hematophagous insects, which take small blood meals in very short time frames, ticks remain on their hosts through an entire devel-

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opmental stadium, during which they continuously ingest blood. Tick sali­ vary glands are multifunctional organs, thought to act in several aspects of the lengthy ectoparasite-host relationships. As their name suggests, these glands produce a copious saliva that is injected into vertebrate hosts during blood feeding. The salivary glands also secrete the material, sometimes called cement, that ticks use to attach to their hosts' skin. More than thirty years ago, Tatchell and Moorhouse (1968) speculated that components in tick saliva could include pharmacologically active materials that may act on host tissues to facilitate tick blood feeding. A few years later, reports on the presence of prostaglandins in tick saliva began to emerge. Dickinson et al. (1976) began their work by fractionating saliva from the cattle tick, Boophilus microplus, on sephadex columns. Pros­ taglandins stimulate smooth muscle contractions, and the authors used the rat fundus (which I take to refer to the upper portions of the uterus) preparation to bioassay saliva fractions for the presence of pharmacologically active ma­ terials. The fundus was bathed in an organ bath, and connected to a smooth muscle transducer to record contractions. The authors found three fractions with smooth muscle contracting activity. They identified one fraction as pi­ locarpine, by comparison to authentic standards and by ultraviolet spectrum. Another fraction (Fraction B) eluted from the column in the same volume as authentic PGE2. Although they did not recover enough material to gener­ ate a complete log dose-response curve, abbreviated curves indicated that PGE1, PGE2, and PGF2a produced smooth muscle contracting activity simi­ lar to the activity of Fraction B. Dickinson et al. (1976) estimated about 2,000 to 8,000 ng PGE1 equivalents in 50 ml of tick saliva. They concluded that prostaglandins are present in tick saliva. To gain more data on the point, they incubated Fraction B with pros­ taglandin dehydrogenase. This enzyme is the primary enzyme responsible for metabolizing active prostaglandins into inactive 15-dehydro-prostaglandins. In mammals, about 95% of biologically active prostaglandins in blood circulation are deactivated after a single pass through the lungs due to this enzyme. This enzyme also reduced the biological activity in Fraction B by about 80%. Parallel control incubations in the absence of the enzyme did not reduce biological activity in Fraction B. Although the authors did not iden­ tify a specific compound, due to limitations in obtaining enough saliva to work with, the results of these exercises strongly supported their hypothesis that prostaglandins are present in tick saliva. Dickinson et al. (1976) speculated on the biological significance of the prostaglandins in tick saliva. On one hand, the prostaglandins may be bio­ logically active, and facilitate tick feeding by increasing the vascular per­ meability in the region of the feeding lesion. On the other, the prostaglandins may not be biologically active, and have been part of the tick hemolymph. Alternatively, they have been taken in with the blood meal, and were merely components of the excreted saliva.

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Higgs et al. (1976) independently took the work a little bit further. They collected saliva from fully engorged cattle ticks maintained in an animal culture system. The saliva was active in three biological assays for smooth muscle contracting activity: the rat stomach strip, the chick rectum, and the rat colon. In another line of work, the authors made acidic chloroform extracts of tick saliva, and fractionated the extracts on thin-layer chroma­ tography. They recovered substantial biological activity in the fraction that co-chromatographed with PGE2. They also used the bioassay to estimated quantities of PGE2 in salivary gland homogenates, recording about 5.6 ng PGE2/mg salivary gland protein. Again, Higgs et al. (1976) noted that exogenous PGE2 increases local vascular permeability. The effect is particularly interesting with PGE2 be­ cause it lasts for as long as ten hours after injection. Recognizing that the amounts of prostaglandin in the saliva are high, they suggested the pros­ taglandins could be important in starting and maintaining the feeding lesion in the tick host. In their work with another tick species, Hyalomma anatolicum excavatum, Shemesh et al. (1979) advanced the information on prostaglandins in ticks. This tick was routinely cultured in their laboratory. After the ticks detached from their hosts, their salivary glands and reproductive tracts were isolated in saline. The authors used unfed males and females and ticks that had fed for six days. The organs were cultured for twenty-four- and seventy-twohour periods in tissue culture medium. The organs and media were frozen after the incubation periods. The materials were later thawed, then processed for prostaglandin extraction. Samples were first extracted in petroleum ether to remove neutral lipids, then extracted in acidified ethyl acetate. The au­ thors used an aliquot of the extract to determine extraction efficiency, and processed the remainder through radioimmunoassays for PGE2 and PGF2a. The reported data seem to represent the total amounts of prostaglandins re­ covered in the culture media and the cultured organs, although I did not find a direct statement to this point in the paper. They recovered low amounts of prostaglandins from male and female go­ nads of unfed ticks. The fed tick yielded about 1 ng/testes of both pros­ taglandins after twenty-four hours, which approximately doubled after sev­ enty-two hours. They recovered slightly more material from the ovaries, although the data do not account for differing weights of the male and fe­ male organs. The salivary glands from unfed females yielded about 1 ng/gland of each prostaglandin after twenty-four hours, which again doubled after seventytwo hours' incubation. Salivary glands from unfed males yielded far less, about 0.035 to 0.070 ng/organ at twenty-four hours, and very little more after seventy-two hours. The situation was quite different for ticks that had fed for six days. For females, the authors recovered about 7 ng/organ of PGE2 after twenty-four hours, which increased to 26 ng/organ after seventy-two

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hours. PGF2a was also present, although in lower levels. Males yielded lower values, about 1 ng/organ. Shemesh et al. (1979) also speculated that the prostaglandins present in the tick saliva served to increase vascular permeability in the area of the tick feeding lesion. Detecting prostaglandins in the reproductive organs was a new finding, from which the authors inferred that prostaglandins also served in the physiology of reproduction in ticks. Some cattle are resistant to tick feeding, either by physically removing newly attached ticks through self-grooming or through mechanisms that re­ duce attachment and growth. In their work on cattle resistance, Kemp and Bourne (1980) developed a method to test experimentally the influence of potentially host-derived compounds on tick attachment. Cattle ticks, B. microplus, were confined by screened rings on the skin of young cattle. Se­ lected chemicals were then injected into the skin, under the confinement chambers. Numbers of attached ticks were recorded after three days. The authors found that histamine treatments significantly increased tick detach­ ments from the skin. Several other compounds, including dopamine, bradykinin, and serotonin, had no influence on tick detachments. Kemp and Bourne (1980) concluded that endogenous histamine biosynthesis by the hosts may be part of the overall resistance mechanism. More germane to our interest, the authors investigated the influence of PGE2 injections on tick detachments. These experiments showed that the prostaglandin treatment did not cause detachments. This finding lends an­ other kind of support to the idea that tick salivary prostaglandins may facili­ tate, rather than in any way inhibit, tick feeding. Ribeiro et al. (1988) used radioimmunoassays to determine the presence of two prostaglandins, PGE2 and 6-keto-PGFla (a stable product of PGI2) in the saliva of the hard tick Ixodes dammini. They surveyed saliva samples from 14 ticks, and detected broad quantitative ranges of both prostaglandins. PGE2 was present at 1 to 540 ng/ml of saliva, and 6-keto-PGFla at 35 to more than 1,500 ng/ml. Again, the authors interpreted these results in terms of hemostatic mechanisms in mammals. PGI2 may facilitate tick feeding by preventing platelet aggregation, and thus sustain blood flow around the feed­ ing wound. It could also prevent mast cell degranulation, which would mini­ mize host reaction to the ticks. The quantitative findings indicate that prostagladins are present in tick saliva in quantities that are pharmacologically significant in mammalian skin. Sauer and his colleagues have developed a very lengthy research program on the physiology of the salivary glands of the lone star tick, Amblyomma americanum (Sauer et al. 1995). As one element in a very large program, Ribeiro et al. (1992) confirmed the presence of prostaglandins in the saliva of females ticks. Saliva was collected by injecting dopamine into the ticks, then fractionated on high-performance liquid chromatography. Fractions cor-

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responding to PGE2 and PGF2a were bioassayed on the rat stomach strip for PGE2 and the rat colon for PGF2a. This work yielded positive data on the presence of large amounts of prostaglandins in the tick saliva. Again, the data indicate considerable variation (3.5 to 2,300 ng/ml), but they reported an average of 469 ng PGE2/ml saliva. The authors also derivatized the ex­ tracted prostaglandins and analyzed them on gas chromatography-mass spec­ trometry. They used selected ion monitoring, and detected ions associated with each prostaglandin. These positive findings launched a detailed research program on the presence and metabolism of 20:4n-6 in tick salivary glands. They recorded the presence of 20:4n-6, at about 8% of total fatty acids, in unfractionated lipid extracts of tick salivary glands (Shipley et al. 1993a). This work was conducted by conventional lipid extraction, class separation, and gas chromatographic procedures. Virtually all 20:4n-6 was associated with the phospholipids, and none was detected in neutral lipids or free fatty acids. Within glycerophospholipids, 20:4n-6 made up about 11% of the phosphatidylethanolamine and about 3% of phosphatidylcholine. Again, none was detected in lysophosphatidylcholine, phosphatidylinositol, or cardiolipin. As expected from the mammalian background, most of the 20:4n-6 was associated with the sn-2 position of the phospholipids. However, the asymmetry was not so striking. Almost 25% of the 20:4n-6 was esterified to the sn-1 position. Overall, compared to terrestrial insects, and other tick tis­ sues, 20:4n-6 is fairly abundant in the salivary glands. Concerned with the source of the 20:4n-6, Shipley et al. (1993b) showed that absolute amounts of 20:4n-6 increased during the lengthy course of tick feeding. They recovered about 0.04 g/gland of 20:4n-6 from the salivary glands of unfed ticks, compared to about 1.6 g/gland for fed ticks. It ap­ peared from this work that ticks were able to accumulate dietary 20:4n-6 from their blood meals into salivary gland phospholipids. This work did not, however, yield insights into possible biosynthesis of 20:4n-6 from dietary precursor fatty acids, such as 18:2n-6. Bowman et al. (1995a) investigated the possible biosynthesis of 20:4n-6 in tick salivary glands. Partially fed adult females were either injected or fed with radioactive precursors. After selected incubation periods, the salivary glands were isolated, and total lipids were extracted. Salivary gland phos­ pholipids were isolated, and fatty acid methyl esters were formed. The fatty acid methyl esters were analyzed on radio-high-performance liquid chroma­ tography. Experiments with injected radioactive acetate (2:0) produced peaks of radioactivity corresponding with 16:0, 16:1, 18:0, and 18:1. This indicates the presence of a fatty acid synthase in tick tissues. This is a multienzyme complex thought to occur in all animals (Stanley-Samuelson et al. 1988). In a similar series of experiments, separate groups of females were fed with radioactive 18:1, 18:2n-6, 18:3n-6, 20:3n-6 or 20:4n-6. The usual workup procedures followed. The resulting radio-chromatograms indicated

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that ticks are unable to metabolize Cl8 fatty acids into other components. Results with 18:1 produced a single peak of radioactivity associated with 18:1; similar results were obtained with the other four radioactive starting fatty acids. Bowman et al. (1995a) concluded that ticks lack the Δ6 desaturase required to convert 18:2n-6 into 18:3n-6, and the Δ5 desaturase involved in converting 20:3n-6 into 20:4n-6. Moreover, ticks also lack the ability to perform the elongation steps responsible for lengthening 18:3n-6 to 20:3n-6. Hence, while ticks express the ubiquitous fatty acid synthase re­ quired for biosynthesizing C16 and C18 saturated and monounsaturated components, they depend on dietary inputs to acquire all polyunsaturated fatty acids. In particular, 20:4n-6 is a dietary requirement for ticks. Sauer and his colleagues were concerned with the presence of 20:4n-6 in salivary gland phospholipids as a substrate for the biosynthesis of pros­ taglandins. They approached prostaglandin biosynthesis by tick salivary glands via several avenues. First, Bowman et al. (1995b) assessed pros­ taglandin biosynthesis by incubating microsomal preparations from isolated tick salivary glands with radioactive 20:4n-6. After twenty-minute incu­ bations, the reaction mixtures were extracted in acidified ethyl acetate and the products separated on thin-layer chromatography. The resulting radiochromatograms revealed no evidence for prostaglandin biosynthesis by iso­ lated salivary gland preparations. Bowman et al. (1995b) then developed another approach. They used par­ tially fed females to assess prostaglandin biosynthesis in living ticks. The females were fed solutions of radioactive 20:4n-6 by placing 25-μ1 glass capillary tubes over their mouthparts. The females ingested the fluid; then, the tubes were refilled with saline, and this was fed to the ticks. After feed­ ing another rinse to the females, the ticks were incubated for six to ten hours. The authors then induced the females to salivate by injecting them with dopamine. Saliva was collected for up to four hours after the treat­ ments. Prostaglandins were extracted from the saliva into acidified ethyl ace­ tate. The extracted prostaglandins were separated on thin-layer chromatogra­ phy, and radio-chromatograms were generated by scanning the plates. The published radio-chromatogram shows peaks of radioactivity associated with PGF2a, PGE2, PGD2, and PGA2AB2, and 20:4n-6. The authors concluded that ticks were, indeed, competent to biosynthesize prostaglandins, even though salivary gland homogenates did not yield positive evidence on the point. We developed another approach to the issue of prostaglandin biosynthesis (Pedibhotla et al. 1995). Adult female lone star ticks were removed from their hosts during the fast-feeding stage (which results in the complete en­ gorgement of the tick), and used to assess prostaglandin biosynthesis accord­ ing to the methods of Stanley-Samuelson and Ogg (1994). Whole ticks were homogenized, then the homogenates were centrifuged briefly to remove de­ bris. The supernatants were then centrifuged at 11,750 g for fifteen minutes.

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Ordinarily, the supernatants are taken as microsomal-enriched fractions, and these are used as sources of prostaglandin-biosynthesizing activity. We first assessed prostaglandin biosynthesis in the supernatant and pellet fractions, finding prostaglandin-biosynthesizing activity in the pellets but not the supernatants. The pellets were used as the enzyme sources. The enzyme sources were incubated with radioactive 20:4n-6 in the presence of a stan­ dard co-factor cocktail. Products were extracted in acidified ethyl acetate, amended with a mixture of chemical standards, and separated on thin-layer chromatography. Based on the idea that radioscanning was not sensitive to low levels of radioactivity (which typically has a radioactive counting effi­ ciency of about 1%), fractions corresponding to chemical standards were scraped into vials and radioactivity in each fraction was assessed by liquid scintillation counting. We recorded biosynthesis of low levels of PGE2, PGD2, PGF2ct, and PGA2/B2. Prostaglandin biosynthesis was sensitive to protein concentration (optimal at 2 mg protein/reaction), temperature (optimal at 37°C), reaction time (optimal at 2 minutes) and pH (highest biosynthesis at 6.8). Prostaglandin biosynthesis was inhibited in the presence of low indomethacin concentrations (less than 10 μΜ). We concluded that, as seen in the in vivo experiments, whole tick homogenates are able to biosynthesize prostaglandins (Pedibhotla et al. 1995). There remained, however, two points of ambiguity. First, the enzyme ac­ tivity was associated with the pellet, rather than supernatant fractions of whole tick homogenates. This is contrary to findings with other mammalian and invertebrate systems, in which the enzyme activity is typically associ­ ated with the microsomal-enriched 11,750 g supernatant fraction. We added a sonication step to our usual procedure, in which the homogenates were sonicated for ten seconds prior to the centrifugation step. With this step in the protocol, the enzyme activity was found in the 11,750 g supernatant and not in the pellet. Second, the guts of fast-feeding ticks were filled with sheep blood, which could be a source of prostaglandin biosynthesizing activity. We considered this by assessing prostaglandin biosynthesis in four preparations: whole ticks, blood-filled guts, internal tissues, and host blood. We found that about 20% of the total prostaglandin biosynthesizing activity recorded in whole tick preparations could be accounted for by the presence of host blood in the guts. Substantial enzyme activity was present in the internal tissue preparations, and we concluded that most of the activity we observed was due to enzymes endogenous to the ticks. The internal tissues included salivary glands, Malpighian tubules, ovaries, synganglion and tubular accessory glands, and these were isolated for inter­ nal tissue preparations. We used these preparations to consider the subcellu­ lar localization of the enzyme activity. In mammalian systems, the pros­ taglandin H synthase is localized in the lumen of the endoplasmic reticulum (Otto and Smith 1995), and is typically associated with the 100,000 g micro-

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somal pellet of tissue fractions. The 11,750 g superaatants of internal tissue homogenates were centrifuged at 100.000 g for one hour in an ultracentrifuge. The supernatants were taken to be the cytosolic fractions, while the pellets were taken to be the microsomal fractions of these preparations. Pros­ taglandin biosynthesis was assessed in both fractions, which yielded approx­ imately similar biosynthesizing activity. Together, these findings demonstrate that the internal tissues of adult fe­ male ticks are able to biosynthesize prostaglandins. Unlike that of the mam­ malian system, the biosynthesizing activity is not strictly associated with the microsomal fractions of the tissues. It remained to investigate prostaglandin biosynthesis in isolated salivary glands. We isolated salivary' glands from adult females in the fast-feeding stage (Pedibhotla et al. 1997). The glands were centrifuged once at low speed, then at 11,750 g for fifteen minutes. The resulting superaatants were taken as microsomal-enriched fractions, which were used as enzyme sources. The enzyme sources were incubated in the presence of radioactive 20:4n-6; then, products were extracted and separated by thin-layer chromatography. Pros­ taglandin biosynthesis by the isolated salivary gland preparations yielded four major products. PGE2. PGD2. PGF2a. and PGA2/B2. as seen in the whole tick preparations. These preparations were sensitive to protein concen­ tration. and to the presence of two cyclooxygenase inhibitors, indomethacin and naproxen. We concluded that isolated salivary glands are competent to biosynthesize prostaglandins. There still remains an important confounding issue in this work. The ana­ lytical results from Ribeiro et al. (1992) and the earlier work on prostaglan­ dins in tick saliva show relatively high levels of these compounds in saliva. The very low rates of prostaglandin biosynthesis recorded in our experi­ ments on isolated salivary glands do not in any way account for the high levels of prostaglandins found in the saliva. Pedibhotla et al. (1997) offer a couple of speculations on this point. One. this may be due to the release of 20:4n-6 from salivary gland phospholipids during the off-host period. If this is so. then the unlabeled 20:4n-6 would effectively dilute the radioactive 20:4n-6, and yield very low biosynthesis of radioactive product. Alter­ natively. the enzymes responsible for prostaglandin biosynthesis in the tick tissues may be different from their mammalian counterparts. The tick system may require unusual reaction conditions, which remain to be discovered, for optimal activity. Ribeiro (1987) and Sauer et al. (1993) summarized the major arguments on the roles of prostaglandins as immunosuppressive agents in mammalian hosts. Tick saliva attenuates host immune competence in several ways. Ho­ mogenates of salivary glands from the tick Dennacenter andersoni suppress activation of lymphocytes and the release of cytokines. PGE2 prevents mac-

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rophage activation and neutrophil activity, and promotes vasodilation. PGI2 inhibits cellular immune reactions, including mast cell degranulation. These, along with other observations on mammalian hosts, strongly support the hy­ pothesis that prostaglandins associated with tick saliva exert very important actions in tick-host ectoparasitic relationships. We have seen several instances in which it is thought that prostaglandins secreted from parasites facilitate host-parasite relationships through their ac­ tions within the host systems. Another line of work, to which we now turn, suggests that inhibition of prostaglandin biosynthesis may act in competitive relationships between insect species.

PROSTAGLANDIN BIOSYNTHESIS INHIBITORS IN INSECT DEFENSIVE SECRETIONS: DO THESE COMPOUNDS ACT IN INSECT CHEMICAL ECOLOGY? Nearly twenty years ago, Howard and his colleagues first recognized the ecological significance of insect cuticular hydrocarbons (Howard et al. 1980). As a part of an overall interest in chemical mediation of ecological interactions between and within insect species, Howard noted a similarity in the chemical structures of some compounds in insect defensive secretions and aspirin. The similarity led to the hypothesis that some of these com­ pounds inhibit prostaglandin biosynthesis. Howard et al. (1986) tested this hypothesis with two compounds in the defensive secretion of the red flour beetle, Tribolium castaneum. The potential inhibitors were 2'-hydroxy-4'-methoxyacetophenone and 2'-hydroxy-4'-methoxypropiophenone, and aspirin. The researchers used mi­ crosomal preparations made from bovine seminal vesicles and cockroach, P. americana, fat body homogenates as sources of prostaglandin biosynthetic activity. In separate experiments, the enzyme sources were incubated with radioactive 20:4n-6 with or without a putative prostaglandin biosynthesis inhibitor. Products were extracted from the reaction mixtures, and pros­ taglandins were separated on small silicic acid chromatography columns. The results from both enzyme preparations support the hypothesis. With the bovine preparations, PGE2 biosynthesis declined from about 3.2 nmol/ hour in the absence of inhibitor to 1.1 nmol/hour in the presence of both insect-derived inhibitors at 150 μΜ. In contrast, incubations in the presence of 2.5 mM aspirin resulted in a similar inhibition of the bovine preparation. Both defensive compounds also inhibited PGE2 biosynthesis by the cock­ roach preparation, from about 126 pmol/hour in positive control incubations to about 30 pmol/hour in the presence of the compounds at 2.5 mM. The authors obtained similar inhibition of PGE2 biosynthesis with aspirin.

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Howard et al. (1986) speculated that these compounds may have an unde­ termined ecological significance because beetles release their defensive se­ cretions into their environments. As an aside, this work also allows a direct comparison of the capacities for prostaglandin biosynthesis in an insect preparation and bovine seminal vesicles. Understanding that this particular mammalian preparation produces prostaglandins at the highest rates recorded so far, the data presented by Howard et al. (1986) reveal a twenty-five-fold difference in rates of pros­ taglandin biosynthesis. In general, insects produce prostaglandins at lower rates than seen in mammalian preparations. Howard and his colleagues noted that the compounds just discussed are associated with a rather specialized insect species, and the broader idea that many insect species elaborate defensive compounds remained to be tested (Jurenka et al. 1986). They approached this by purchasing selected chemi­ cals known exclusively from insect defensive secretions, but which had no known defensive functions. These are methyl anthranilate, from male ants, o-aminoacetophenone from male seed bugs; and three compounds known from beetles; methyl salicylate, 2,5-dihydroxyphenylacetic acid -γ-lactone, and salicylaldehyde. They tested the idea that these compounds inhibit pros­ taglandin biosynthesis using the enzyme sources and protocols just de­ scribed. All five compounds, as well as aspirin, effectively inhibited PGE2 biosynthesis in preparations from both animal systems. In the bovine prepa­ rations, 2.5 mM aspirin reduced PGE2 biosynthesis from about 2.9 nmol/ hour to about 1.6 nmol/hour, roughly on par with amino acetophenone and salicylaldehyde. Incubations in the presence of the other three compounds yielded less than 1 nmol/hour of PGE2. The authors tested three insect-de­ rived compounds in the insect preparations. At 2.5 mM, methyl anthranilate and methyl salicylate reduced PGE2 biosynthesis from about 556 pmol/hour to about 130 to 150 pmol/hour, not very different from the results with aspi­ rin. The -y-lactone was far more effective, reducing PGE2 biosynthesis to about 27 pmol/hour. Again, the authors speculated that these compounds would have ecological significance. Howard and Mueller (1987) determined the chemical structures of two new defensive compounds from the beetle Tribolium brevicornis. These are methyl 2.5-dihydroxy-6-methylbenzoate and methyl 2,5-dihydroxy-6-ethylbenzoate. These have not been tested for their influence on prostaglandin biosynthesis, but given their similarity in structure to other insect-derived compounds reviewed in the previous paragraphs, they speculated that these, too. would be effective inhibitors. Jurenka et al. (1989) conducted similar exercises with exocrine secretions from lace bugs, Stephanitis sp. There are hemipterans, or true bugs in the family Tingidae. The title of their paper suggests that they directly tested the

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influence of selected compounds on prostaglandin H synthase. However, this is not strictly correct, because they used their routine protocols in these ex­ periments. These protocols register prostaglandin biosynthesis, and they can­ not be certain which step in the process these chemicals interrupt. The au­ thors tested the influence of seven insect-derived compounds, and aspirin, on the bovine seminal vesicle preparation. These were 2,6-dihydroxyacetophenone, 2,4-dihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, 1-(2,6dihydroxyphenyl)dodecan-1-one, 1 -(2,4, 6-trihydroxyphenyl)dodecan-1-one, 5-hydroxy-2-nonylchromanone, and 5,7-dihydroxy-2-nonylchrome. Three of these compounds, 2,6-dihydroxyacetophenone, 2,4,6-trihydroxyacetophe­ none and l-(2,6-dihydroxyphenyl)dodecan-l-one, potently reduced PGE2 biosynthesis. The authors then tested these three compounds using the cock­ roach fat body preparation. All inhibited PGE2 biosynthesis, particularly 2,4,6-trihydroxyacetophenone and l-(2,6-dihydroxyphenyl)dodecan-l-one. Jurenka et al. (1989) noted that immature lace bugs are unusually free of predators and parasites, and they speculated that the lace bug secretions may be responsible, at least partly, for protection from various forms of attack. Their language was carefully expressed, and they refrained from suggesting that the biological actions of the secretions is exerted via their influence on prostaglandin biosynthesis. They suggested, however, that lace bugs may be a very good model to test the hypothesis that inhibition of prostaglandin biosynthesis may influence competitive interactions between and among species. These eicosanoid biosynthesis inhibitors are biosynthesized within spe­ cialized exocrine glands, which release their secretions exclusively to the exterior of the insects. The conventional wisdom of exocrine secretions holds that the inhibitors influence organisms other than the insects which produce the secretions. Drawing on the foregoing chapters, let us consider a mechanism by which exogenous inhibition of eicosanoid biosynthesis may influence ecological interactions. Eicosanoids appear to act in two major arenas of animal physiology. One is often termed a "housekeeping" arena, in which eicosanoids variously in­ fluence cellular actions in maintaining organismal homeostasis. Instan­ taneous modulation of ion transport physiology is a good example appropri­ ate to vertebrates and invertebrates. The other major arena relates to eico­ sanoid actions at crucial stages in the life history of an organism. Some of these actions, perhaps at a key point in reproduction or development, may occur only once in the life history. The roles of certain lipoxygenase prod­ ucts in oocyte maturation in starfish or barnacle hatching are examples. Other crucial points in a life history, such as cellular reactions to bacterial infections or wounding, may recur relatively frequently. Temporary inhibi­ tion of eicosanoid actions in the housekeeping arena probably would not

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result in lasting damage to most organisms. On the other hand, if eicosanoid biosynthesis were inhibited at a crucial point, say in the early phases of a bacterial infection, or perhaps during the penetration phases of a parasitization process, the effects of the inhibition could be deleterious to the organ­ ism. Under this scenario, the compounds in the defensive secretions may very well serve adaptive roles as eicosanoid biosynthesis inhibitors.

CHAPTER 9

A Research Prospectus: Approaching the Frontiers

COMPARED to our understanding of eicosanoids in invertebrates, the pub­ lished literature on the operative picture of eicosanoid actions in mammals is far ahead. To be sure, the first research on eicosanoids in mammals predates work on invertebrates by almost four decades, and a substantial head start would rather naturally put the work on mammals somewhat ahead of other work. The lengthy lead, however, is not so long as it seems. For many of these forty years, technical barriers, such as the very limited availability of chemicals, slowed all progress. It seems to me that something else is respon­ sible for the tremendous gap between progress in our understanding of eicoanoids in mammals and progress in invertebrate systems. I believe that the differences in progress lie in understanding the economic significance of eicosanoids, due to their actions in human physiology. While eicosanoids mediate many cellular actions in mammals, mediation of inflam­ matory reactions is, on an economic footing, one of the most important eicosanoid actions. Soon after the emergence of the relationship between the analgesic properties of aspirin and inhibition of prostaglandin biosynthesis, there began serious research to identify other eicosanoid biosynthesis inhibi­ tors. Following the discovery that most eicosanoids act through their influ­ ence on specific receptor sites, there ensued a great effort to understand eicosanoid receptors. Again, the significance of the work related to commer­ cial enterprises. The new information on eicosanoid receptors motivated an­ other line of research aimed at identifying novel eicosanoid receptor antago­ nists. Recognizing the strong financial linkage between eicosanoids and human medicine helps us understand why research on mammals is consid­ erably advanced relative to research on invertebrate systems. The situation may change with time, as information from invertebrate sys­ tems offers new tools with potential or actual usefulness to humans. The usefulness may spring from unexpected sources. For one example, pros­ taglandins are used to synchronize the reproductive cycles of some food animals. Could prostaglandins, or other eicosanoids, prove useful in regulat­ ing reproductive cycles in commercial invertebrate food production systems? For another example, in the previous chapter we noted the growing recogni­ tion of the significance of eicosanoids in host-parasite relationships. Do these eicosanoid systems offer the potential to design new therapeutics for use in

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protecting humans from parasitic infections? Inquiry into invertebrate eicoanoid systems is attracting increased interest, and our knowledge base is growing. Perhaps in the not-so-distant future, some of the new information will become a basis for applied, commercially motivated research. This would certainly help accelerate the pace of our progress. Meanwhile, how­ ever. some of the advances seen in mammalian systems shed a fairly bright illumination into the dimly lit corridors of research into invertebrates. These corridors will lead to very important advances. Let us briefly consider a few of these.

HOW DO EICOSANOIDS WORK?

Smith (1989) suggested a general model for the biochemical mechanisms of eicosanoid actions in mammalian cells. In his model, eicosanoids interact with G protein-linked cell surface receptors, which in turn influence intra­ cellular events. There is now a tremendous background on eicosanoid recep­ tors in mammalian systems. Coleman et al. (1990) provided a system to classify prostanoid receptors, based on information from studies of ligand binding, receptor agonists, and receptor antagonists. They recognized five receptor types, denoted DP, EP, FP, IP, and TP. These five types display the highest binding affinity for, respectively, PGD, PGE, PGE PGI, and throm­ boxane. Our understanding of receptors is greatly enhanced by the recognition of subtypes of receptors. PGE, for example, can interact with EP1, EP2, or EP3 receptors. These receptors are coupled to different G proteins, through which PGE can express seemingly contradictory actions. EP, receptors lead to in­ creased intracellular calcium concentrations, EP2 receptors are associated with increased intracellular cAMP concentrations, while interaction with EP3 receptors yields decreased intracellular cAMP concentrations. Coleman et al. (1990) expressed the long-term aim of research into eicosanoid receptors in terms of developing new medicines that act in highly selective ways to ago­ nize or antagonize specific eicosanoid receptors. While the idea that most eicosanoids act through cell surface receptors is well supported by many studies on mammalian systems, the cell surface receptor model does not apply to all eicosanoids. Specific receptors for pros­ taglandins. thromboxanes, and leukotrienes have been identified. As men­ tioned earlier, PGA and A1-PGJ. the cyclopentone prostaglandins, act through intranuclear receptors, where they can promote expression of some genes. And the hydroperoxy- and hydroxyeicosatetraenoic acids are selectively esterified into cellular phospholipids, where they may act through a "mem­ brane perturbation" model described by Spector et al. (1988). Epoxyeicosatrienoic acids also are esterified into phospholipids, where they may similarly disturb local membrane domains.

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In chapter 6, we considered the only studies of eicosanoid receptors in invertebrate systems, the gill tissue of a marine bivalve and tick salivary glands. Given the many actions eicosanoids exert in invertebrates, these two studies illuminate a very important corridor to our understanding of eico­ sanoids in invertebrates. Many subtle aspects of receptors in invertebrates await discovery. Recognition of receptor subtypes, which may express differ­ ing affinity constants for the same compound, and appreciation of the physi­ ological activation and deactivation of receptors will help us make sense of apparently contradictory results of physiological experiments. Eicosanoid re­ ceptors in invertebrate systems will eventually emerge as an established field, sure to yield important insights. The interactions between eicosanoids and their functional receptors trigger subsequent downstream events, of which we know very little in inverte­ brates. In their work on the locust rectum, Radallah et al. (1995) showed that PGE2 upregulated intracellular phospholipase C activity, which resulted in increased concentrations of inositol triphosphate. The inositol triphosphate apparently acts on L-type calcium channels, leading to increased intracellular calcium concentrations. PGE2 increased fluid resorption in the locust hindgut, which may be mediated through a phospholipase C pathway. The influence of prostaglandins and other eicosanoids on intracellular events in invertebrate systems remains to be investigated in detail.

UNDERSTANDING DEPARTURES FROM THE MAMMALIAN BACKGROUND I have often remarked that the mammalian background on eicosanoids, if taken with due skepticism, can serve as a useful guide to our work on inver­ tebrate systems. Skepticism is an important ingredient in our understanding, mostly because mammals evolved from an assemblage of invertebrates. I can imagine a future time in which points of information on the well-known mammalian eicosanoid systems will be seen as departures from the literature on the major invertebrate groups. Meanwhile, however, it may be instructive to consider some examples of how the mammalian background can be mis­ leading. Many of the reports on prostaglandin biosynthesis by various tissue homogenates prepared from insects are confounded on the issue of subcellular locations of prostaglandin biosynthesizing enzymes. Prostaglandin H syn­ thases 1 and 2 (or COX-1 and -2) are entirely associated with endomembrane fractions of mammalian cells, although each protein is found in slightly dif­ ferent locations (Otto and Smith 1995). The endomembrane fractions are typically prepared as the 100,000 g pellets of mammalian tissue homogenates. The situation is not nearly so clear in the insect preparations. Destephano and Brady (1977) found most of the prostaglandin biosynthe-

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tic activity in 12,100 g pellets prepared from male reproductive tissues, in­ cluding testes, seminal vesicles, and accessory glands, with very little activ­ ity in the corresponding supernatants. Alternatively, similar studies with fe­ male reproductive tissues showed that the enzyme activity was recovered in the 12,100 g pellets and supernatants in roughly equal distribution. This first study of prostaglandin biosynthesis in an invertebrate showed an important difference in the subcellular association of the enzyme activity. In the course of their characterization of prostaglandin biosynthesis in the housefly, M. domestica, Wakayama et al. (1986b) found most enzyme activ­ ity in the 12,000 g supernatant. After centrifuging the supernatants at 105,000 g, they found the activity distributed in the resulting pellet and supernatant fractions. Using whole-fly homogenates, they recovered 57% of the activity in the 105,000 g pellets prepared from males and 26% in the corresponding pellets from females. The enzyme activity was distributed in a different pat­ tern in combined head-thorax homogenates, with just over 80% of the activ­ ity recovered in the 105,000 g supernatants prepared from males and female. Lange (1984) reported on the transfer of prostaglandin-biosynthesizing ac­ tivity from males to females during mating in the locust, L. migratoria. In this work, she considered the distribution of the enzyme activity in centri­ fuge fractions of the opalescent glands, one of the male accessory glands. About 65% of the activity was recovered in the 11,000 g supernatants, the remainder in the pellets. After centrifuging the 11,000 g supernatants at 30,000 g, about 70% of the enzyme activity was associated with the pellet fraction. Other distributions of the prostaglandin-biosynthesizing activities are re­ ported from tissues of other insect species (Stanley-Samuelson and Loher 1986). The main thrust of these examples is to emphasize the differences in the intracellular location of the enzyme activities between various insect preparations and virtually all mammalian preparations. We can infer from these studies that the mammalian enzymes involved in prostaglandin bio­ synthesis are associated with endomembranes in ways different from the associations reported for many invertebrates. It is tempting to suggest that the enzymes in invertebrate preparations must be considerably different from their mammalian counterparts. We have virtually no information, however, on the structures or sequences of the enzymes from any invertebrate system. As it stands, it is certain that the prostaglandin biosynthesizing enzymes in various insect tissues are not associated with endomembrane fractions in the way seen in mammalian systems. Some invertebrate eicosanoid-biosynthesizing enzymes also differ from their mammalian counterparts in their sensitivities to various pharmaceutical inhibitors of eicosanoid biosynthesis. We have already seen, for example, that indomethacin very potently inhibits prostaglandin biosynthesis in mam­ mals as well as in several invertebrates, but not in reproductive tract prepara-

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tions from males of the cricket, A. domesticus (chapter 4). Again, findings of this sort indicate that eicosanoid-biosynthesizing enzymes in various inverte­ brates differ among invertebrates and from the enzymes associated with most mammals. The work of Howard and his colleagues on prostaglandin biosynthesis inhibitors associated with insect defensive secretions showed that mam­ malian preparations generally produce prostaglandins at faster rates than those seen in insect preparations (chapter 8). On the other extreme, one of the largest departures from the mammalian background is seen in some coral. These species generate very high levels of tissue prostaglandins, using an allene oxide pathway that does not involve a cyclooxygenase (chapter 2). While most mammalian lipoxygenases yield the (S) isomers of hydroxyeicosatetraenoic acids, starfish oocytes and other invertebrate systems pro­ duce the (R) isomers. Hence, the rates and stereospecific products of eico­ sanoid-biosynthesizing enzymes differ in mammals and invertebrates. The biological paradigm helps us understand these points of departure between the mammalian background and the newly acquired information on invertebrates. Invertebrates represent many more species than do mammals, as well as all other vertebrates combined; invertebrates also have been sub­ jected to the refining pressures of natural selection for many more genera­ tions than have vertebrates. Let us imagine that the discovery of eicosanoids had started with coral species that produce prostaglandins via the allene ox­ ide pathway. Drawing on Gerhart's idea that the prostaglandins in coral act as emesis-producing antipredator compounds (Gerhart 1991), information from coral would suggest that prostaglandins (which would almost certainly enjoy another common name) are produced by a pathway typically associ­ ated with plants, for ecological purposes. The eicosanoids in mammals may have subsequently been seen as the derivative systems, which they almost undoubtedly are. To the extent that speculation along these lines can be helpful, we may appreciate the differences among various eicosanoid sys­ tems in invertebrates and vertebrates as products of evolutionary forces. Seen from this perspective, the variations discussed here represent real, bio­ logical variation. This real, biological variation should be the source of our skepticism, guiding us to take information from the mammalian background with care, and to understand that each invertebrate system will present its own pattern relative to the biology of eicosanoids.

THE ENZYMES ASSOCIATED WITH EICOSANOIDS As seen throughout this volume, biosynthesis of various eicosanoids has been recorded in many invertebrate species. Aside from biochemical charac­ terizations of eicosanoid biosynthesis in fairly crude tissue homogenates or

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centrifuge fractions of homogenates, so far we have very little information on purification and characterization of the enzymes involved in invertebrate eicosanoid systems. Here, I briefly touch on two publications reporting ef­ forts to purify several enzymes. Bhagya Lakshmi and Ramaiah (1984) attempted the first purification from an invertebrate source of an enzyme involved in eicosanoid biosynthesis. They worked on the prostaglandin synthetase from testes of the silk moth, B. mori. They homogenized the testes in buffer, then centrifuged the homoge­ nates at 15,000 g for thirty minutes. They precipitated proteins in the super­ natant fraction in acetone, then resuspended the acetone-precipitated fraction in buffer. This step removed about 70% of the total protein, but yielded only a negligible increase in the specific activity of the enzyme. The resuspended fraction was passed through a phosphocellulose column, from which the au­ thors obtained a 2.5-fold increase in specific activity. Additional chroma­ tographic steps did not improve the overall purification of the protein. The authors claimed an overall threefold purification of the enzyme. This work did not, however, yield new insights into prostaglandin biosynthesis. Another group succeeded in purifying an enzyme associated with pros­ taglandin biosynthesis from adults of the nematode, Ascaridia galli (Meyer et al. 1996). In the usual scheme of PGE2 biosynthesis, 20:4n-6 is first con­ verted into PGH2. The PGH2 is isomerized into PGE2 by a PGH E-isomerase. The enzyme involved in this final step is a glutathione S-transferase, which the authors purified by classical methods. Adult worms were homogenized in buffer; then, the soluble fraction was obtained by centrifugation. The soluble fraction was frozen until further use. After thawing and rehomogenization, the samples were first fractionated on a column of sulfur-linked reduced glutathione agarose. The column was washed, and the glutathione transferases were eluted with Tris buffer amended with reduced glutathione. The eluted fraction was dialyzed, then fractionated by anion exchange on a MonoQ column. The MonoQ fractions were tested for glutathione transferase activity and for PGH isomerase activity, then fro­ zen. Aliquots of the PGH isomerase-active fractions were further separated according to hydrophobic interactions on a polyPROPYL A column. The active fractions from the hydrophobic interaction column were sorted out by reverse-phase high-performance liquid chromatography, which yielded sev­ eral subunits, all in the range of 25 to 28 kDa. The highest PGH isomerase activity was associated with subunit H, a 25.7 kDa protein. N-terminal se­ quencing showed that this protein is similar to other nematode glutathione S-transferases. Meyer et al. (1996) cast the significance of this work in terms of PGE2 biosynthesis by the parasite. Again, secreted PGE2 is thought to attenuate host defense reactions to the presence of the parasties. Other PGH E-isomerases, which also are glutathione S-transferases, have been partially purified from mammalian sources; however, the enzyme from

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this invertebrate source is the first highly specific PGH E-isomerase to be purified and partially sequenced. Of course, many enzymes involved in eicosanoid biosynthesis have been purified from mammalian sources. The success in this area has reached the point that some lipoxygenases and cyclooxygenases are now commercially available for research. Purification and more complete characterization of the invertebrate enzymes responsible for eicosanoid biosynthesis remain an open field of inquiry. Initial guidance in this area may be gleaned from published purification schemes. The obverse of eicosanoid biosynthetic enzymes may be the enzymes re­ sponsible for metabolizing some eicosanoids into biologically inactive forms (Smith et al. 1991). The biologically active prostaglandins are quickly inacti­ vated by a family of enzymes, the 15-hydroxyprostaglandin dehydrogenases. These enzymes, quite abundant in vertebrate lungs, are responsible for con­ verting active prostaglandins into their inactive, 15-keto metabolites. The PGE2 is further catabolized by Δ13 reductase to 13,14-dihydro-15-keto-PGE2. The aliphatic chains also can be shortened by ω-oxidation and β-oxidation. Leukotrienes also are quickly metabolized in mammalian systems. Human neutrophils, but not other cells, for example, catabolize leukotriene B4 via ω-oxidation. The peptidoleukotrienes undergo a series of catabolic steps. In rat liver, leukotriene C4 is cleaved to leukotriene D4, then to E4. Leukotriene E4 then goes through ω-oxidation and excretion through the urinary and alimentary tracts. The catabolism and excretion of active eicosanoids is an important ele­ ment in regulating overall concentrations of these compounds within a tissue system or organism. We have very little information on this area in inverte­ brates. Stanley-Samuelson and Loher (1985) showed that injected radioac­ tive PGE2 is rapidly cleared from hemolymph circulation in adult crickets, T. commodus. While this information is useful in understanding the phar­ macological fate of the prostaglandin, there remains a major gap in this work. The authors did not analyze the excreted radioactivity using chroma­ tography. Hence, we do not know whether the excreted radioactivity repre­ sents PGE2 in unchanged or metabolized forms. Overall, we have virtually no information on the enzymes involved in metabolizing active eicosanoids in invertebrates.

EICOSANOIDS AND NEUROBIOLOGY

Eicosanoids influence the behaviors of several invertebrates. We saw that PGE2 releases egg-laying behavior in females of T. commodus and a few other insect species (chapter 4), and eicosanoids stimulate skin penetration behaviors in some parasitic worms (chapter 8). Nanda and Ghosal (1978) reported on prostaglandins in neurosecretory cells of the cockroach, P. amer-

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9

icana. Stanley-Samuelson and Loher (1986) speculated that the target site of action for PGE2 in releasing egg-laying behavior lies in the terminal abdomi­ nal ganglion, which also houses the neural circuitry that controls the move­ ments involved in depositing eggs. Eicosanoids modulate certain behaviors in vertebrates, as well as inverte­ brates. PGF2a acts in reproductive behaviors of many vertebrate species. In goldfish, this prostaglandin acts as a postovulatory female sex pheromone (Sorensen et al. 1988). PGF2a also stimulates reproductive behaviors in fe­ male paradise fish (Villars et al. 1985). PGE2 induces female reproductive behavior in the frog Xenopus laevis (Weintraub et al. 1985). Whittier and Crews (1989) reported that PGF2a influences ovarian physiology and sexual behavior of female garter snakes. Hayaishi and his colleagues (Hayaishi 1988, Matsumura et al. 1989) articulated their hypothesis that prostaglandins regulate mammalian sleep-wake cycles. Overall, it would appear that eicosanoids, particularly prostaglandins, are intimately involved in regulating some behaviors in a wide range of vertebrates and invertebrates. In situations in which specific eicosanoids modulate the behaviors of or­ ganisms, the eicosanoids act in the mode of certain hormones that regulate behaviors. Stanley-Samuelson and Loher (1986) argued that in releasing egg-laying behavior, the action of PGE2 was consonant with classical endo­ crinology, in which the chemical is released from an identified organ, travels by way of hemolymph circulation, and interacts with specific receptors within the nervous system. In this scenario, the interaction between the eicosanoid and the nervous system is limited to the receptor protein. If this is so, then behavior-modifying eicosanoids would fall more into the realm of endocrinology than neurobiology. On the other hand, Piomelli has led a lengthy investigation into the roles of eicosanoids in the neurobiology of invertebrates. In his review of this information, Piomelli (1994) noted that nerve tissue can metabolize 20:4n-6 via all three major oxygenation pathways: cyclooxygenase, lipoxygenases, and the cytochrome P450 epoxygenases. These products can act in nerve cells in two distinct manners. In one, the eicosanoids act within cells to regulate ion channels, ion pumps, and other physiological elements. In another, the products may be released into the intracellular spaces, where they may inter­ act with G protein-linked receptors on neurons. Piomelli (1994) suggested that eicosanoids can play key roles in modulating neural and synaptic physi­ ology. He also reported a fair bit of elegant experimental work on inverte­ brates (e.g., Piomelli et al. 1987a, b, 1988). The roles of eicosanoids in neurobiology is a rapidly expanding field of inquiry, one of tremendous interest within the biological paradigm of eico­ sanoids. As seen in these few remarks, eicosanoids act in modulating behav­ iors and neurophysiology of vertebrates and invertebrates. Their actions in neurobiology, writ large, may represent important fundamental roles of eicosanoids in all animals with nervous systems.

A RESEARCH PROSPECTUS

243

THE MOLECULAR BIOLOGY OF EICOSANOIDS Various successes in chemistry are responsible for many of the early break­ throughs in our understanding of eicosanoids in mammalian systems. The chemical structures of prostaglandins, leukotrienes, and many other eico­ sanoids were determined by what at the time was (and still is) challenging analytical chemistry. The significance of 20:4n-6 as a key precursor to the biosynthesis of eicosanoids was inferred from eicosanoid structures. The ready availability of eicosanoids for research purposes is due to advances in synthetic organic chemistry. Advances in pharmaceutical chemistry yielded the many different inhibitors of various eicosanoid-biosynthesizing enzymes. Chemistry will always provide an essential foundation for studies of eicosanoids. Progress in the last ten years, however, is due more to successes in the molecular biology of eicosanoids. The late 1980s and early 1990s saw many reports on molecular structure and functions of genes for prostaglandin H synthases, for lipoxygenases, for cytochrome P450 epoxygenases, and for eicosanoid receptors. During this period, distinct forms of prostaglandin H synthases and messenger RNA were discovered, which finally led to our appreciation of what are now called COX-I and COX-2. As mentioned ear­ lier, COX-2 is an inducible form of cyclooxygenase, responsible for mediat­ ing many elements in inflammatory reactions to injury and infection. The main therapeutic gain from this discovery is the design of very specific anal­ gesics that may be taken to reduce inflammation without inhibiting the housekeeping functions of COX-1. Progress in the molecular biology of mammalian eicosanoid systems can be marked by the commercial availabil­ ity of complementary DNA for COX-2. The progress just mentioned also accounts, in part, for the tremendous gap separating progress in invertebrate systems from our understanding of mam­ malian systems. The progress in mammalian systems provides a powerful arsenal of new tools with which to approach important questions in inverte­ brates. As a single example, we can wonder if invertebrates express separate constitutive and inducible genes for prostaglandin H synthase, as seen in some, though certainly not all, mammalian tissues. Many other questions about the physiology of eicosanoids remain untouched, perhaps even un­ asked, because we have not yet aimed the molecular arsenal at invertebrates. Information on gene and amino acid sequences of proteins operational in eicosanoid actions also can be very helpful in another vein. Sequence infor­ mation would allow us to consider the relative homologies of these proteins across widely ranging phylogenetic lines. I believe we would find that some of these proteins, such as the prostaglandin receptors, would be fairly con­ served over evolutionary time. Negishi et al. (1995b) considered the homolo­ gies among several mammalian receptors, from which they reported about

244

C H A P T E R

9

20% to 30% homology. For example, the human EP2 receptor has 30% ho­ mology with the mouse EP3 receptor and 43% homology with the mouse prostaglandin D receptor. On the other hand, we have seen real differences in prostaglandin biosynthetic enzymes at the biochemical level. The subcellular localization of these enzymes appears to differ among tissues, and we saw differences in sensitivity toward various cyclooxygenase inhibitors. Varia­ tions such as this would suggest that the proteins involved in prostaglandin biosynthesis may vary considerably among animal groups. The biological significance of considering the relative homologies of these sequences extends beyond descriptive analysis. We would not learn very much in assuring ourselves that the prostaglandin H synthases from various mammals are more similar to themselves than to the counterpart sequences from representatives of far older taxa, such as the filarial worms. On the other hand, apprehending a more or less common genetic basis for the pres­ ence and biological activities of eicosanoids in invertebrates and vertebrates would add a very tangible verisimilitude to the study of eicosanoids in inver­ tebrate signal transduction systems.

AN EPILOGUE

The biological paradigm of eicosanoids can serve to integrate our under­ standing of eicosanoid actions in all animals. The paradigm extends along two major axes. On an axis of biological actions, eicosanoids influence fun­ damental cellular events in body pattern formation, regeneration, asexual reproduction, and the excursion through cell cycles. Through their actions in central and peripheral nervous systems, eicosanoids influence behaviors in vertebrates and invertebrates. Eicosanoids act in the daily housekeeping ac­ tivities required for cellular and organismal homeostasis, and they act in climactic points in life histories. Beyond their organismal actions, eico­ sanoids also play important roles in ecological relationships. On a phylogenetic axis, the presence of important biological actions of eicosanoids have been recorded in unicellular organisms, in the earliest metazoans, and in representatives of virtually all animal phyla. If we let these axes define a research space, then it is a rich space, offering the promise of a great deal of new knowledge.

Abbreviations Used in References

Acta Protozool. Agric. Biol. Chem. Adv. Insect Physiol. Adv. Lipid Res. Adv. Prostaglandin, Thromboxane and Leukotriene Res. Am. J. Physiol. Amer. Zool. Ann. Ent. Soc. Am. Ann. Rev. Biochem. Annu. Rev. Entomol. Ann. Rev. Microbiol. Annu. Rev. Pharm. Tox. Ann. Zool. Fenn. Arch. Biochem. Biophys. Arch. Insect Biochem. Physiol. Archiv. Italiano Biol. Aust. J. Exp. Biol. Med. Sci. Biochem. J. Biochem. Parasit. Biochem. Pharmacol. Biochim. Biophys. Acta Biol. Reprod. Biol. Bull. Bull. Ent. Res. Bull. Jap. Soc. Scien. Fisher. Canad. J. Zool. Cancer and Metastasis Rev. Cell Tissue Res. Chem. Ind.

Acta Protozoology Agricultural and Biological Chemistry Advances in Insect Physiology Advances in Lipid Research Advances in Prostaglandins, Thromboxane and Leukotriene Research American Journal of Physiology American Zoologist Annals of the Entomological Society of America Annual Review of Biochemistry Annual Review of Entomology Annual Review of Microbiology Annual Review of Pharmacology and Toxicology Annales Zoologici Fennici Archives of Biochemistry and Biophysics Archives of Insect Biochemistry and Physiology Archives Italiano Biologie Australian Journal of Experimental and Biological Medical Science Biochemistry Journal Biochemical Parasitology Biochemical Pharmacology Biochimica et Biophysica Acta Biology of Reproduction Biological Bulletin Bulletin of Entomological Research Bulletin of the Japanese Society of Scientific Fisheries Canadian Journal of Zoology Cancer and Metastasis Reviews Cell and Tissue Research Chemical Industry

246

A B B R E V I A T I O N S U S E D I N R E F E R E N C E S

Chem. Rev. Clinical Immunol. Immunopathol. Comp. Biochem. Physiol. Critical Rev. Neurobiol. C. R. Soc. Biol. Paris Develop. Biol. Ecol. Entomol. Ent. Exp. & Appl. Environ. Entomol. Exp. Cell Res. Exp. Parasitol. FEBS Lett. Fed. Proc. Free Radical Biol. and Med. Gamete Res. Gen. Comp. Endocrinol. Horm. Behav. Hydrobiol. Indian J. Exp. Biol. Insect Biochem. Insect Biochem. Molec. Biol. Internat. J. Invertebr. Reprod. Develop. Internat. J. Parasitol. Internat. Review Cytol. Invertebr. Reprod. Dev. Japanese J. Physiol. J. Am. Chem. Soc. J. Biol. Chem. J. Cell. Biochem. J. Chem. Ecol. J. Chromatog. J. Clin. Invest. J. Comp. Physiol. J. C. S. Chem. Comm. J. Evolutionary Biochem. Physiol.

Chemistry Reviews Clinical Immunology and Immunopathology Comparative Biochemistry and Physiology Critical Reviews in Neurobiology Comptes Rendu des Seances de la Societe de Biologie Developmental Biology Ecological Entomology Entomologica experimental!s et applicata Environmental Entomology Experimental Cell Research Experimental Parasitology FEBS Letters Federation Proceedings Free Radical Biology and Medicine Gamete Research General and Comparative Endocrinology Hormones and Behavior Hydrobiology Indian Journal of Experimental Biology Insect Biochemistry Insect Biochemistry and Molecular Biology International Journal of Invertebrate Reproduction and Development International Journal of Parasitology International Review of Cytology Invertebrate Reproduction and Development Japanese Journal of Physiology Journal of the American Chemical Society Journal of Biological Chemistry Journal of Cellular Biochemistry Journal of Chemical Ecology Journal of Chromatography Journal of Clinical Investigations Journal of Comparative Physiology Journal of the Chemical Society Chemical Communications Journal of Evolutionary Biochemical Physiology

ABBREVIATIONS USED IN REFERENCES

J. Exp. Biol. J. Exp. Mar. Biol. Ecol. J. Exp. Med. J. Exp. Parasit. J. Exp. ZooL J. Insect Behav. J. Insect Physiol. J. Invertebr. Pathol. J. Leukoc. Biol. J. Lipid Mediators Cell Signalling J. Lipid Res. J. Med. Entomol. J. Membrane Biol. J. Nutr. J. Neurosci. J. Org. Chem. J. Parasitol. J. Pharmacol. Exp. Ther. J. Physiol. Mediators of Inflamm. Marine Biol. Marine Biol. Lett. Mar. Ecol. Progr. Ser. Mol. Biochem. Parasitol. Oceanogr. Mar. Biol. Annu. Rev. Parasitol. Parasitol. Res. Parasit. Today Pest. Biochem. Physiol. Pharmac. Biochem. Behav. Pharmac. Rev. Physiol. Behav. Physiol. Entomol. Proc. Natl. Acad. Sci. USA Proc. R. Soc. Lond. Proc. Soc. Exp. Biol. Med.

247

Journal of Experimental Biology Journal of Experimental Marine Biology and Ecology Journal of Experimental Medicine Journal of Experimental Parasitology Journal of Experimental Zoology Journal of Insect Behavior Journal of Insect Physiology Journal of Invertebrate Pathology Journal of Leukocyte Biology Journal of Lipid Mediators and Cell Signalling Journal of Lipid Research Journal of Medical Entomology Journal of Membrane Biology Journal of Nutrition Journal of Neuroscience Journal of Organic Chemistry Journal of Parasitology Journal of Pharmacology and Experimental Therapeutics Journal of Physiology Mediators of Inflammation Marine Biology Marine Biology Letters Marine Ecology Progress Series Molecular and Biochemical Parasitology Oceanography and Marine Biology. An Annual Review. Parasitology Parasitology Research Parasitology Today Pesticide Biochemistry and Physiology Pharmacology and Biochemistry of Behavior Pharmacology Reviews Physiological Behavior Physiological Entomology Proceedings of the National Academy of Sciences USA Proceedings of the Royal Society of London Proceedings of the Society for Experimental Biology and Medicine

248

ABBREVIATIONS USED IN REFERENCES

Prog. Biochem. Pharmac. Prog. Chem. Fats Other Lipids Prog. Lipid Res. Rev. Aquat. Sci. Roux's Arch. Dev. Biol. Seminars Dev. Biol. Tetrahedron Lett. Trends. Biochem. Sci. Verh. dt. zool. Ges. Z. Tierpsychol. Zool. Science

Progress in Biochemical Pharmacology Progress in the Chemistry of Fats and Other Lipids Progress in Lipid Research Reviews of Aquatic Science Roux's Archives of Developmental Biology Seminars in Developmental Biology Tetrahedron Letters Trends in Biochemical Science Verhandlungsbericht der Deutschen Zoologeschen Gesellschaft Zeitschrift fur Tierpsychology Zoological Science

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Taxonomic Index

Annelida, Polychaeta Arenicola marina, 104-108 Nereis succinea, 106 Platynereis dumerilii, 106 Arthropoda, Arachnida Amblyomma americanum, 51, 168-171, 226-230 Androctonus astralis, 174 Boophilus microplus, 224-225, 226 Buthus occitanus, 174 Centruroides vittatus, 53 Dermacenter andersoni, 230-231 Dictyna sublata, 53 Grammastola sp., 53 Hyalomma anatolicum excavatum, 225226 Ixodes dammini, 226 Metaphidippus protervus, 53 Synema parvulum, 53 Arthropoda, Crustacea Balanus amphitrite, 77 Balanus balanoides, 74-79 Balanus balanus, 77 Balanus crenatus, 77 Balanus hameri, 77, 78 Balanus perforatus, 11 Cambarus bartoni bartoni, 173-174 Chthalamus montagui, 11 Eliminius modestus, 76-78 Homarus americanus, 174 Lepas anatifera, 77 Macrobrachium rosenbergii, 86-87 Penaeus duorarum, 174 Pollicipes pollicipes, 11 Procambarus paeninsulanus, 82-85 Arthropoda, Insecta Acheta domesticus, 9, 42, 143, 239. See also chapter 4 Acyrthosiphon pisum, 42 Aedes aegypti, 50, 161-165, 166 Aedes sierrensis, 50 Agrotis ipsilon, 126, 135, 150 Anopheles stephensi, 50 Anurogryllus muticus, 66 Asihs sp., 54

Blaberus discoidalis, 179-180, 183 Blattella germanica, 42, 73 Bombyxmori, 66-69, 70, 126, 131, 135, 151, 178, 240 Caliphora erythrocephala, 154 Chrysopa carnea, 42 Cicendela circumpicta, 54 Cochliomyia hommivorax, 51 Cochliomyia macellaria, 51 Culex pipiens, 39, 50 Culex tarsal is, 50 Culiseta incidens, 50 Culiseta inornata, 50 Drosophda melanogaster, 52 Ephestia sp., 37 Formica polyctena, 166-167 Galleria mellonella, 44, 48, 49, 110, 128 Gryllus assimilis, 127-128, 135, 151 Gryllus bimaculatus, 66, 73 Henosepilachna vigintioctopunctata, 69 Leucophaea maderae, 93 Locusta migratoria, 70, 178, 238 Lymantria dispar, 45, 53 Manduca sexta, 53, 91, 112, 133, 140. See a/so chapter 5 Microdon albicomatus, 54 Mkscq domestica, 42, 53, 68, 70-71, 92, 147, 238 Myrmica incompleta, 54 Myzus persicae, 42 Nilaparvata lugens, 69 Oedaleus senegalensis, 175 Periplaneta americana, 41, 42, 46, 180— 183, 231, 241 Pieris brassicae, 45 Polypedilum vanderplanki, 152 Pseudaletia unipuncta, 126, 135, 150 Sarcophaga crassipalpis, 70 Stephanitis sp., 232 Stomoxys calcitrans, 51 Teleogryllus commodus, 9, 45, 47, 48, 54, 134, 141, 241. See a/so chapter 4 Teleogryllus oceanicus, 66 Tenebrio molitor, 45, 53, 70, 164 Thermobia domestica, 71-73

274

TAXONOMIC

Arthropoda, Insecta (cont.) Tibican dealbatus, 53, 176-177 Triatoma infestans, 73-74 Tribolium brevicornis, 232 Tribolium castaneum, 231 Trichoplusia ni, 70 Zootermopsis angusticolis, 42 Zophobas atratus, 53, 124-126, 134, 150 Arthropoda, Merostomata Limulus polyphemus, 174 Cnidaria (Coelenterata) Clavularia viridis, 31, 32 Leptogorgia virgulata, 189 Plexaura homomalla, 7, 30, 75, 188 Cnidaria, Hydrozoa Hydractinia echinata, 184, 186-187 Hydra magnipapillata, 184, 185-187 Hydra vulgaris, 185 Echiniodermata, Asteroidea Asterias forbesi, 111 Asterias rubens, 97-101 Asterina pectinifera, 101 Astropecten armatus, 101 Astropecten auranciacus, 101 Astropecten irregularis, 101 Evasterias troschelii, 99, 100 Luidia ciliaris, 100 Marthasterias glacialis, 95, 97-101 Orthasterias koehleri, 99 Patiria miniata, 101 Echinodermata, Echinoidea Aracia lixula, 103-104 Arbacia punctulata, 102-104 Echinaracnius parma, 101 Paracentrotus lividus, 103 Stronglyocentrotus purpuratus, 102-104 Mollusca, Bivalvia Carunculina texasensis, 156 Ligumia subrostrata, 155, 156 Modiolus demissus, 158, 159-160 Mytilus califorianus, 92 Mytilus edulis, 111 Patinopectin yessoensis, 79-82 Mollusca, Gastropoda Hahotis rufescens, 90-92 Helisoma duryi, 87-89 Limnaea auricularia, 175 Lymnaea stagnalis, 89-90

INDEX

Semisulcospira libertina, 175 Tethys fimbria, 190 Nematoda Ascaria galli, 240 Brugia malayi, 215-218 Brugia timori, 215 Dinofilana immitis, 221-222 Necator americanus, 222-223 Oesophagostonum dentatum, 218-221 Wuchereria bancrofti, 215 Platyhelminthes, Cestoda (tapeworms) Taenia multiceps, 200-202 Taenia taeniaeformis, 197-199 Spriometra erinacei, 199 Platyhelminthes, Trematoda (flukes) Schistosoma mansom, 194,202-215,222Trichobilharzia ocellata, 212 Protozoa Phylum Ciliophora Tetrahymena, 194 Tetrahymena pyriformis, 195-196 Tetrahymena rostrata, 195 Phylum Sarcomastigophora Acanthamoeba castellanii, 194-195 Amoeba proteus, 196 Entamoeba histolytica, 194 Urochordata Styela clava, 111 Vertebrata, Agnatha Myxine glutinosa, 171 Vertebrata, Amalphibia Xenopus laevis, 242 Vertebrata, Teleost Fishes Fundulus heteroclitus, 189 Gambusia affinis, 192 Halichoeres garnoti, 189 Lagodon rhomboides, 189 Micropterus salmoides, 189 Morone saxatilis, 189 Salmo gairdneri, 28 Bacteria Aeromonas hydrophila, 173 Bacillus licheniformis, 131 Serratia marcescens. See chapter 5 Fungi Metarhizium flavoviride, 175 Planta Brassica oleracea, 45

Subject Index

accessory glands, 64-65, 70, 229, 238 acetylcholine, 221, 222 adenylate cyclase, 154, 157, 170 adipokinetic hormone, 178-179 albumen gland, 89 alimentary canal, 109, 113, 134, 147, 161, 215 allatectomized, 73 9-anthryldiazomethane, 81-82 antipyrylazo III, 102 arachidonic acid metabolism. See eicosanoid biosynthesis arterial relaxation: aorta rings, 221-222; endothelium-dependent, 221 autotomous structures, 192 behavior: egg-hatching, 75-79; egg-laying, 55-66, 68-69, 74, 79, 81, 86, 87, 92-93, 241; learned aversion, 189; mammalian skin penetration, 202-212, 213, 222, 241; swimming, cessation of, 205, 211 binding sites, prostaglandins, 160-161 biogenic amines, 111, 214 biomembranes, 43, 153, 263; proteins, mem­ brane associated, 153 carrier proteins, 153 channels, 153, 242;—L-type calcium, 168, 237 ion pumps, 29, 242 blood pressure, 171 bradykinin, 226 p-bromophenacyl ester, 57 buprofezin, 69 bursa copulatrix, 88, 89 calcium (see also chapters 6 and 7): calciumdependent kinase, 179; calcium-independent kinase C, 185; flux, 102-104; inositol triphosphate, 102, 179, 183, 184, 237; in­ tracellular, 98, 101-102, 183; ionophore, 107, 181, 217 cAMP, 181-183, 263 (see also chapter 6): blowfly salivary glands, 154, 156; cock­ roach fat body, 183; crayfish reproduc­ tion, 83-84; mosquito malpighian tubules,

162, 166; mussel gill, 156-158; prawn ovary, 86-87; starfish oocytes, 99; tick salivary glands, 169-170 cerata, 190-193 cirri, 74 chalone hypothesis, 26 cholera toxin, 170 clotting mechanisms, 199 concanavalin A, 200, 202 cuticular hydrocarbons, 152, 231 cytokines, 111, 112, 213-214, 230 defense secretions, 239. See also chapter 8 determinant, la, 201 diacylglycerol, 136, 144-145, 168, 184-187 diazomethane reaction, 76 dopamine, 168-169, 226, 228 dorsal sweat glands, 176 ectoparasites, 168, 223-224 egg flood, 93 eicosanoid biosynthesis, 153, 162-163, 169; allene oxide pathway, 30-33, 192, 239; characterization, 11, 59-60; enzyme transfer model, 58, 65-66, 89, 93; insect reproduc­ tion, 57-60; prostaglandin lactone pathway, 193; suicide inactivation of, 139-140 eicosanoids: biological paradigm, 3-4, 10, 239, 242, 244; first known action, 5; his­ tory of, 4-10; mammalian model, 11 emesis (vomiting), 189-190, 193, 239; pukalide, 190 endothelial cells, 112, 216, 221-222 eosinophils, 213 essential fatty acid deficiency, 34, 36, 38, 165, 205 esterase, 178 exocytosis, 170 fatty acid metabolism (see also phospholipid remodeling)·, desaturase, 36-37, 43, 228; elongation/desaturation pathways, 34, 3637, 45, 47-48, 58-59, 182, 204, 207, 216; fatty acid synthase, 34, 227-228; retroconversion, 48

276

SUBJECT INDEX

fatty acids, 154, 157; analysis, 38, 44-45, 163; as chemoattractants, 204; essential, 34, 37-39, 205, 206; methyl esters, 59, 64, 227; uptake by tapeworms, 199 ganglion: cerebral, 81; synganghon, 229; ter­ minal abdominal, 93, 241; visceral, 81 germinal vesicle, 95; breakdown, 96-99 gills, 81, 156-161, 171 glutamate, 93 glutathione peroxidases, 22 glutathione-S-transferase, 27, 240-241 glycogen phosphorylase, 179-180 gonadotropin, 91 gonochoristic, 94 gut epithelium, 148 heat shock proteins, 20 hindgut, 61, 150, 161, 237 histamine, 226 homeostasis, 152-153, 155, 175-177, 233 hydrogen peroxide, 90-92, 164 hypertrehalosemic hormone, 179-183 ichthyotoxicity assay, 192 immunoglobulins, 109 integument, 134, 148, 150, 152 -y-interferon, 213 interleukins, 213 3-isobutyl-l-methylxanthine, 87 jasmonic acid, 30 juvenile hormone, 72 leukocytes, 21, 27, 111, 207, 216 lipase, 178 lipopolysacchande, 175 lymphocytes, 26, 109, 152, 200-202, 213, 216, 230 lymphokine biosynthesis, 216 macrophages, 213, 216, 230-231; peritoneal, as accessory cells, 200-202 malpighian tubules, 61, 134, 153-154, 171; Aedes aegypti, 161-166; Amblyomma americanum, 229; Formica polyctena, 166, 167; Tenebrio molitor, 164; Zophobas atratus, 150 mantle, 159-161, 185. 190-192, 193 mast cell degranulation, 226, 231 mating system, 56, 66, 69

melittin, 182 1-methyladenine, 95-101 5-methyl-3-heptanone, 106 methyl sergide, 79 midgut, 109, 150, 215 miracidia, 202 morulae, 106-107 mycetocytes, 180 nephromixia, 106 nephron, 153 nerve cord, 134, 148 neurosecretory cells, 241 neutrophils, 26, 111, 112, 145, 213, 231 oocytes, 193, 239. See also chapter 4 oothecal gland, 88 opalescent glands, 70, 238 ovaries, 79-87, 225, 229 oviducal muscles, 92-93 ovotestis, 88, 193 ovulation, 84 oxylipin, 33 phagocytosis, 109, 110, 111, 117, 129-130, 132, 196 pharmaceutical effects, 60, 116 pharmaceutical influence, 67 phosphatidylcholine, 58-59, 73, 133-137, 144, 146, 163-164, 227 phosphatidylethanolamine, 58-59, 133-134, 144, 146, 163, 227 phosphatidyhnositol, 68, 73, 145, 163, 168, 227 phosphatidylserine, 144-145, 163 phosphodiesterase, 154 phospholipase C, 168, 184-187, 237 phospholipid remodeling, 145-146 phosphorylase kinase, 179 platelets, 216; aggregating activity, 197-198, 217; aggregation, inhibition of, 111, 216— 218, 226 positional value, 184, 186 prophenyloxidase, 123, 128-129, 130, 132 prostaglandin dehydrogenase, 61, 68, 224, 241 prostaglandin transporter, 14 prostate gland, 87, 89 prostomium, 106-108 protein kinase C, 168, 184-186 protein phosphorylation, 83-84, 100

S U B J E C T

receptors: eicosanoid, cell surface, 61, 129, 160, 161, 168-169, 196, 235, 236-237; eicosanoid, nuclear, 161; G protein-cou­ pled, 101, 129, 160, 168-171, 236, 242;

I N D E X

277

spermatheca, 57-59, 62-65, 70, 72, 93-94, 147 spermatophore, 57-59, 64-65, 70, 134, 141, production, 56

hormone, cell surface, 96, PGE 2 , 170, 183; prostanoid, 236; thromboxane/PGH 2 , 204;

sperm mass, 56 sperm maturation hormone, 106-108

thromboxane/PGH 2 receptor antagonist,

sterol, 52

204

synovial fluid, 12, 135

rectum, locust, 154-155, 161, 167-168 reproductive tract, 134, 147

T-cells See lymphocytes

salivary glands: blowfly, 154, 161, cement,

testectomization, 56, 59, 65 testes, 46-47, 56, 64-65, 68, 71, 81, 147,

224; mosquito, 215; tick, 51, 168-171, 224-231; tobacco hornworm, 134

225, 238, 240 thrombin, 197, 217

semen, 57, 65, 89, 92

thyroxin, 91

seminal duct, 71

transcellular metabolism, 27

seminal receptacles, 71 seminal vesicle, 30, 70-71, 87-89, 112,

trehalose See chapter 7 trophocytes, 180-183

214, 231, 232, 233, 238 serotonin, 79-81, 154, 156-157, 217.

226

urate cells, 180 urine flow rates, 171

serum albumin, 199-200

uterine muscle, 55, 92

silk glands, 148 skin, 210; interleukins, 213; keratinocytes,

vasodilators, 216, 231

212

venoms, 12, 135

somatostatin, 29 spawning, 79-81, 90-91, 94, 105

vitellogenesis, 82-84

spawning releaser, 90

water balance, 152