Innervation of the Gastrointestinal Tract (Autonomic Nervous System)

INNERVATION OF THE GASTROINTESTINAL TRACT The Autonomic Nervous System A series of books discussing all aspects of th...

Author: Simon Brookes | Marcello Costa

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INNERVATION OF THE GASTROINTESTINAL TRACT

The Autonomic Nervous System A series of books discussing all aspects of the autonomic nervous system. Edited by Geoffrey Burnstock, Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, London, UK. Volume 1 Autonomic Neuroeffector Mechanisms edited by G. Burnstock and C.H.V. Hoyle

Volume 8 Nervous Control of Blood Vessels edited by T. Bennett and S.M. Gardiner

Volume 2 Development, Regeneration and Plasticity of the Autonomic Nervous System edited by I.A. Hendry and C.E. Hill

Volume 9 Nervous Control of the Heart edited by J.T. Shepherd and S.F. Vatner

Volume 3 Nervous Control of the Urogenital Systsem edited by C.A. Maggi

Volume 10 Autonomic – Endocrine Interactions edited by K. Unsicker

Volume 4 Comparative Physiology and Evolution of the Autonomic Nervous System edited by S. Nilsson and S. Holmgren

Volume 11 Central Nervous Control of Autonomic Function edited by D. Jordan

Volume 5 Disorders of the Autonomic Nervous System edited by D. Robertson and I. Biaggioni

Volume 12 Autonomic Innervation of the skin edited by J.L. Morris and I.L. Gibbins

Volume 6 Autonomic Ganglia edited by E.M. McLachlan

Volume 13 Nervous Control of the Eye edited by G. Burnstock and A.M. Sillito

Volume 7 Autonomic Control of the Respiratory System edited by P.J. Barnes

Volume 14 Innervation of the Gastrointestinal Tract edited by S. Brookes and M. Costa

This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.


Edited by

Simon Brookes and Marcello Costa

London and New York

First published 2002 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc, 29 West 35th Street, New York, NY 10001 Taylor & Francis is an imprint of the Taylor & Francis Group

This edition published in the Taylor & Francis e-Library, 2003. © 2002 Taylor & Francis All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines.

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data A catalog record for this book has been requested

ISBN 0-203-21701-2 Master e-book ISBN

ISBN 0-203-27305-2 (Adobe eReader Format) ISBN 0–415–28377–9 (Print Edition)

Contents Preface to the Series — Historical and Conceptual Perspective of the Autonomic Nervous System Book Series

vi

Preface

xii

Contributors

xviii

1 Enteric Reflexes that Influence Motility J.C. Bornstein, J.B. Furness, W.A.A. Kunze and P.P. Bertrand 2 Motor Control of the Stomach David Grundy and Michael Schemann

1 57

3 Control of Gastric Functions by Extrinsic Sensory Neurons Peter Holzer

103

4 Neural Control of the Large Intestine Kalina Venkova, Beverley Greenwood-Van Meerveld and Jacob Krier

171

5 Neurons of the Gallbladder and Sphincter of Oddi Gary M. Mawe, Erin K. Talmage, Lee J. Jennings, Kirk Hillsley and Audra L. Kennedy

189

6 Pharmacology of the Enteric Nervous System Marcello Tonini, Fabrizio De Ponti, Gianmario Frigo and Francesca Crema

213

7 Neuroeffector Transmission in the Intestine Charles H.V. Hoyle, Pam Milner and Geoffrey Burnstock

295

8 Neural Control of Intestinal Vessels Neela Kotecha

341

9 Enteric Neuro-Immunophysiology Jackie D. Wood

363

10 Cellular Organisation of the Mammalian Enteric Nervous System Simon J.H. Brookes and Marcello Costa

393

11 Development of the Enteric Nervous System Michael D. Gershon

469

Index

527

v

Preface to the Series — Historical and Conceptual Perspective of the Autonomic Nervous System Book Series The pioneering studies of Gaskell (1886), Bayliss and Starling (1899), and Langley and Anderson (see Langley, 1921) formed the basis of the earlier and, to a large extent, current concepts of the structure and function of the autonomic nervous system; the major division of the autonomic nervous system into sympathetic, parasympathetic and enteric subdivisions still holds. The pharmacology of autonomic neuroeffector transmission was dominated by the brilliant studies of Elliott (1905), Loewi (1921), von Euler and Gaddum (1931), and Dale (1935), and for over 50 years the idea of antagonistic parasympathetic cholinergic and sympathetic adrenergic control of most organs in visceral and cardiovascular systems formed the working basis of all studies. However, major advances have been made since the early 1960s that make it necessary to revise our thinking about the mechanisms of autonomic transmission, and that have significant implications for our understanding of diseases involving the autonomic nervous system and their treatment. These advances include: (1) Recognition that the autonomic neuromuscular junction is not a “synapse” in the usual sense of the term where there is a fixed junction with both pre- and post-junctional specialization, but rather the transmitter is released from mobile varicosities in extensive terminal branching fibres at variable distances from effector cells or bundles of smooth muscle cells which are in electrical contact with each other and which have a diffuse distribution of receptors (see Hillarp, 1959; Burnstock, 1986a). (2) The discovery of non-adrenergic, non-cholinergic nerves and the later recognition of a multiplicity of neurotransmitter substances in autonomic nerves, including monoamines, purines, amino acids, a variety of different peptides and nitric oxide (Burnstock et al., 1964, 1986b, 1997; Rand, 1992; Milner and Burnstock, 1995; Lincoln et al., 1995; Zhang and Snyder, 1995; Burnstock and Milner, 1999). (3) The concept of neuromodulation, where locally released agents can alter neurotransmission either by prejunctional modulation of the amount of transmitter released or by postjunctional modulation of the time-course or intensity of action of the transmitter (Marrazzi, 1939; Brown and Gillespie, 1957; Vizi, 1979; Fuder and Muscholl, 1995; MacDermott et al., 1999). vi

PREFACE TO THE SERIES

G. Burnstock — Editor of The Autonomic Nervous System Book Series

vii

viii INNERVATION OF THE GASTROINTESTINAL TRACT

(4) The concept of cotransmission that proposes that most, if not all, nerves release more than one transmitter (Burnstock, 1976; Hökfelt, Fuxe and Pernow, 1986; Burnstock, 1990a; Burnstock and Ralevic, 1996) and the important follow-up of this concept, termed “chemical coding”, in which the combinations of neurotransmitters contained in individual neurones are established, and whose projections and central connections are identified (Furness and Costa, 1987). (5) Recognition of the importance of “sensory-motor” nerve regulation of activity in many organs, including gut, lungs, heart and ganglia, as well as in many blood vessels (Maggi, 1991; Burnstock, 1993), although the concept of antidromic impulses in sensory nerve collaterals forming part of “axon reflex” vasodilatation of skin vessels was described many years ago (Lewis, 1927). (6) Recognition that many intrinsic ganglia (e.g., those in the heart, airways and bladder) contain interactive circuits that are capable of sustaining and modulating sophisticated local activities (Saffrey et al., 1992; Ardell, 1994). Although the ability of the enteric nervous system to sustain local reflex activity independent of the central nervous system has been recognized for many years (Kosterlitz, 1968), it has been generally assumed that the intrinsic ganglia in peripheral organs consist of parasympathetic neurones that provided simple nicotinic relay stations. (7) The major subclasses of receptors to acetylcholine and noradrenaline have been recognized for many years (Dale, 1914; Ahlquist, 1948), but in recent years it has become evident that there is an astonishing variety of receptor subtypes for autonomic transmitters (see Pharmacol. Rev., 46, 1994). Their molecular properties and transduction mechanisms have been characterised (see IUPHAR Compendium of Receptor Characterisation and Classification 2000). These advances offer the possibility of more selective drug therapy. (8) Recognition of the plasticity of the autonomic nervous system, not only in the changes that occur during development and ageing, but also in the changes in expression of transmitter and receptors that occur in fully mature adults under the influence of hormones and growth factors following trauma and surgery, and in a variety of disease situations (Burnstock, 1990b; Saffrey and Burnstock, 1994; Milner et al., 1999). (9) Advances in the understanding of “vasomotor” centres in the central nervous system. For example, the traditional concept of control being exerted by discrete centres such as the vasomotor centre (Bayliss, 1923) has been supplanted by the belief that control involves the action of longitudinally arranged parallel pathways involving the forebrain, brain stem and spinal cord (Loewy and Spyer, 1990; Jänig and Häbler, 1995). In addition to these major new concepts concerning autonomic function, the discovery by Furchgott that substances released from endothelial cells play an important role in addition to autonomic nerves, in local control of blood flow, has made a significant impact on our analysis and understanding of cardiovascular function (Furchgott and Zawadski, 1980; Burnstock and Ralevic, 1994). The later identification of nitric oxide as the major endothelium-derived relaxing factor (Palmer et al., 1988; see Moncada et al., 1991) (confirming the independent suggestion by Ignarro and by Furchgott) and endothelin as an endothelium-derived constricting factor (Yanagisawa et al., 1988; see Rubanyi and Polokoff, 1994) have also had a major impact in this area.


ix

In broad terms, these new concepts shift the earlier emphasis on central control mechanisms towards greater consideration of the sophisticated local peripheral control mechanisms. Although these new concepts should have a profound influence on our considerations of the autonomic control of cardiovascular, urogenital, gastrointestinal and reproductive systems and other organs like the skin and eye in both normal and disease situations, few of the current textbooks take them into account. This is largely because revision of our understanding of all these different specialised areas in one volume by one author is a near impossibility. Thus, this Book Series of 14 volumes is designed to try to overcome this dilemma by dealing in depth with each major area in separate volumes and by calling upon the knowledge and expertise of leading figures in the field. Volume I, deals with the basic mechanisms of Autonomic Neuroeffector Mechanisms which sets the stage for later volumes devoted to autonomic nervous control of particular organ systems, including Heart, Blood Vessels, Respiratory System, Urogenital Organs, Gastrointestinal Tract, Eye Function, Autonomic Ganglia, Autonomic-Endocrine Interactions, Development, Regeneration and Plasticity and Comparative Physiology and Evolution of the Autonomic Nervous System. Abnormal as well as normal mechanisms will be covered to a variable extent in all these volumes depending on the topic and the particular wishes of the Volume Editor, but one volume edited by Robertson and Biaggioni, 1995, has been specifically devoted to Disorders of the Autonomic Nervous System (see also Mathias and Bannister, 1999). A general philosophy followed in the design of this book series has been to encourage individual expression by Volume Editors and Chapter Contributors in the presentation of the separate topics within the general framework of the series. This was demanded by the different ways that the various fields have developed historically and the differing styles of the individuals who have made the most impact in each area. Hopefully, this deliberate lack of uniformity will add to, rather than detract from, the appeal of these books. G. Burnstock Series Editor

REFERENCES Ahlquist, R.P. (1948). A study of the adrenotropic receptors. Am. J. Physiol., 153, 586–600. Ardell, J.L. (1994). Structure and function of mammalian intrinsic cardiac neurons. In Neurocardiology, edited by J.A. Armour and J.L. Ardell, pp. 95–114. Oxford: Oxford University Press. Bayliss, W.B. (1923). The Vasomotor System. Longman: London. Bayliss, W.M. and Starling, E.H. (1899). The movements and innervation of the small intestine. J. Physiol. (Lond.), 24, 99–143. Brown, G.L. and Gillespie, J.S. (1957). The output of sympathetic transmitter from the spleen of a cat. J. Physiol. (Lond.), 138, 81–102. Burnstock, G. (1976). Do some nerve cells release more than one transmitter? Neuroscience, 1, 239–248. Burnstock, G. (1986a). Autonomic neuromuscular junctions: Current developments and future directions. J. Anat., 146, 1–30. Burnstock, G. (1986b). The non-adrenergic non-cholinergic nervous system. Arch. Int. Pharmacodyn. Ther., 280(suppl.), 1–15. Burnstock, G. (1990a). Co-Transmission. The Fifth Heymans Lecture – Ghent, February 17, 1990. Arch. Int. Pharmacodyn. Ther., 304, 7–33.

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Burnstock, G. (1990b). Changes in expression of autonomic nerves in aging and disease. J. Auton. Nerv. Syst., 30, 525–534. Burnstock, G. (1993). Introduction: Changing face of autonomic and sensory nerves in the circulation. In Vascular Innervation and Receptor Mechanisms: New Perspectives, edited by L. Edvinsson and R. Uddman, pp. 1–22. San Diego: Academic Press Inc. Burnstock, G. (1997). The past present and future of purine nucleotides as signalling molecules. Neuropharmacology, 36, 1127–1139. Burnstock, G., Campbell, G., Bennett, M. and Holman, M.E. (1964). Innervation of the guinea-pig taenia coli: Are there intrinsic inhibitory nerves which are distinct from sympathetic nerves? Int. J. Neuropharmacol., 3, 163–166. Burnstock, G. and Milner, P. (1999). Structural and Chemical Organisation of the autonomic nervous system with special reference to non-adrenergic, non-cholinergic transmission. In Autonomic Failure: A Textbook of Clinical Disorders of the Autonomic Nervous System. 4th edn, edited by C.J. Mathias and R. Bannister, Oxford: Oxford University Press, pp. 63–71. Burnstock, G. and Ralevic, V. (1994). New insights into the local regulation of blood flow by perivascular nerves and endothelium. Br. J. Plast. Surg., 47, 527–543. Burnstock, G. and Ralevic, V. (1996). Cotransmission. In The Pharmacology of Smooth Muscle, edited by C.J. Garland and J. Angus, Oxford: Oxford University Press. Dale, H. (1914). The action of certain esters and ethers of choline and their reaction to muscarine. J. Pharmacol. Exp. Ther., 6, 147–190. Dale, H. (1935). Pharmacology and nerve endings. Proc. Roy. Soc. Med., 28, 319–332. Elliott, T.R. (1905). The action of adrenalin. J. Physiol. (Lond.), 32, 401–467. Fuder, H. and Muscholl, E. (1995). Heteroceptor-mediated modulation of noradrenaline and acetylcholine release from peripheral nerves. Rev. Physiol. Biochem. Physiol., 126, 265–412. Furchgott, R.F. and Zawadski, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373–376. Furness, J.B. and Costa, M. (1987). The Enteric Nervous System. Edinburgh: Churchill Livingstone. Gaskell, W.H. (1886). On the structure, distribution and function of the nerves which innervate the visceral and vascular systems. J. Physiol. (Lond.), 7, 1–80. Hillarp, N.-Å. (1959). The construction and functional organisation of the autonomic innervation apparatus. Acta Physiol. Scand., 46 (suppl. 157), 1–38. Hökfelt, T., Fuxe, K. and Pernow, B. (Eds.) (1986). Coexistence of neuronal messengers: A new principle in chemical transmission. In Progress in Brain Research, Vol. 68, Amsterdam: Elsevier. IUPHAR Compendium of Receptor Characterisation and Classification 2000, IUPHAR Media Ltd (London), UK. Jänig, W. and Häbler, H.-J. (1995). Visceral-Autonomic Integration. In Visceral Pain, Progress in Pain Research and Management, edited by G.F. Gebhart, Vol. 5, pp. 311–348. Seattle: IASP Press. Kosterlitz, H.W. (1968). The alimentary canal. In Handbook of Physiology, edited by C.F. Code, Vol. IV, pp. 2147–2172. Washington, DC: American Physiological Society. Langley, J.N. (1921). The Autonomic Nervous System, part 1. Cambridge: W. Heffer. Lewis, T. (1927). The Blood Vessels of the Human Skin and Their Responses. Shaw & Sons: London. Lincoln, J., Hoyle, C.H.V. and Burnstock, G. (1995). Transmission: Nitric oxide. In The Autonomic Nervous System, Vol. 1 (reprinted): Autonomic Neuroeffector Mechanisms, edited by G. Burnstock and C.H.V. Hoyle, pp. 509–539. The Netherlands: Harwood Academic Publishers. Loewi, O. (1921). Über humorale Übertrangbarkeit der Herznervenwirkung. XI. Mitteilung. Pflügers Arch. Gesamte Physiol., 189, 239–242. Loewy, A.D. and Spyer, K.M. (1990). Central Regulations of Autonomic Functions. New York: Oxford University Press. MacDermott, A.B., Role, L.W. and Siegelbaum, S.A. (1999). Presynaptic iootropic receptors and the control of transmitter release. Ann. Rev. Neurosci., 22, 443–485. Maggi, C.A. (1991). The pharmacology of the efferent function on sensory nerves. J. Auton. Pharmacol., 11, 173–208. Marrazzi, A.S. (1939). Electrical studies on the pharmacology of autonomic synapses. II. The action of a sympathomimetic drug (epinephrine) on sympathetic ganglia. J. Pharmacol. Exp. Ther., 65, 395–404. Mathias, C.J. and Bannister, R. (eds.) (1999). Autonomic Failure. 4th edn, Oxford: Oxford University Press. Milner, P. and Burnstock, G. (1995). Neurotransmitters in the autonomic nervous system. In Handbook of Autonomic Nervous Dysfunction, edited by A.D. Korczyn, pp. 5–32. New York: Marcel Dekker. Milner, P., Lincoln, J. and Burnstock, G. (1999). The neurochemical organisation of the autonomic nervous system. In Handbook of Clinical Neurology Vol 74(30): The autonomic nervous system – Part 1 – Normal Functions, edited by O. Appezeller, Amsterdam: Elsevier Science, pp. 87–134.


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Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109–142. Palmer, R.M.J., Rees, D.D., Ashton, D.S. and Moncada, S. (1988). Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem. Biophys. Res. Commun., 153, 1251–1256. Rand, M.J. (1992). Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, non-cholinergic neuroeffector transmission. Clin. Exp. Pharmacol. Physiol., 19, 147–169. Rubanyi, G.M. and Polokoff, M.A. (1994). Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev., 46, 328–415. Saffrey, M.J. and Burnstock, G. (1994). Growth factors and the development and plasticity of the enteric nervous system. J. Auton. Nerv. Syst., 49, 183–196. Saffrey, M.J., Hassall, C.J.S., Allen, T.G.J. and Burnstock, G. (1992). Ganglia within the gut, heart, urinary bladder and airways: studies in tissue culture. Int. Rev. Cytol., 136, 93–144. Vizi, E.S. (1979). Prejunctional modulation of neurochemical transmission. Prog. Neurobiol., 12, 181–290. von Euler, U.S. and Gaddum, J.H. (1931). An unidentified depressor substance in certain tissue extracts. J. Physiol., 72, 74–87. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411–415. Zhang, J. and Snyder, S.H. (1995). Nitric oxide in the nervous system. Annu. Rev. Pharmacol. Toxicol., 35, 213–233.

Preface Eating is an extremely hazardous way to make a living, biologically speaking. The strategy of ingesting foreign organisms, breaking them down and reusing the spare parts is fraught with risks. For one thing, the organism concerned is likely to resist being broken down for scrap and may, in fact, have designs on the body parts of the diner. For another, the chemicals required to digest food are equally capable of breaking down the structure of the gut wall and the rest of the consumer’s body as well. It seems that minimising these risks, while meeting the imperative to absorb nutrients, has influenced many of the design principles of the gastrointestinal tract. This long tube is composed of a variety of tissue types and is the largest internal organ of the body. Its main function is to digest food and absorb the released nutrients. It is subdivided into functionally distinct regions, so that ingested material is processed serially in a way that serves to minimise the risks of self-digestion and attack by ingested pathogens. After rapid propulsion through the thorax via the oesophagus, food is subjected to mechanical and chemical breakdown in the harshly acidic environment of the stomach. This tends to reduce the viability of (inadvertently) swallowed micro-organisms. If toxic material is detected, propulsion can be reversed and the ingested material can be rapidly expelled by vomiting. During its sojourn in the stomach, sphincteric muscles at either end of the organ prevent spillage of the corrosive acidic content into the susceptible neighbouring regions of the duodenum and oesophagus. Gastric contents are then gradually aliquoted into the duodenum at a carefully controlled rate, adapted to the calorific and nutrient content of the meal. In the duodenum, the acidity of the contents is neutralised and they are mixed with digestive enzymes, bile and watery secretions, to allow the complex chemistry of digestion to get to work. The resulting chyme is mixed, moved to and fro and gradually propelled along the small intestine, at a rate adapted to the requirements for absorption of nutrients. A few hours after the meal, the small intestine is basically empty, the residual matter having been expelled into the colon. Here it may reside for several days, as water and ions are reabsorbed, playing a potentially vital role in the water balance of the organism. Eventually, the near-solid material is expelled from the body. In mechanical terms, this is a decidedly non-trivial event. The prolonged retention of contents in the colon makes it inevitable that large numbers of bacteria will be present, posing an enormous potential danger to the organism. If large numbers of micro-organisms colonise the small intestine, the barrier between the vasculature and the outside world is rapidly compromised and xii

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massive septicaemia results. Parasites in food may survive the digestive process and then attack the wall of the gut. The consuming organism defends itself against these perils with specialised motor patterns, which are triggered to remove such noxious threats with great rapidity from the body. The multiplicity of functions required of the digestive tract makes it a fascinating subject for biological research by a range of disciplines, including immunology, microbiology, biochemistry, oncology etc. However it provides an especially appealing subject for neuroscientists. Neuronal circuitry is intimately involved in controlling many of the functions of the gut. It controls the activity of the smooth muscle in the gut wall, which mixes and propels the contents. Between meals, specialised patterns of motor activity periodically empty sloughed-off epithelial cells and accumulated mucous, thus preventing the build-up of a culture medium for the ever-threatening bacteria. Other highly propulsive motor patterns can be triggered by toxic chemicals, bacterial infection, parasitic infestation or identified allergens. While we consider the symptoms associated with these motor patterns (diarrhoea and pain) to be unpleasant, there is little doubt that these specialised mechanisms are of profound adaptive significance to our survival. The feature of the innervation of the gastrointestinal tract that is most appealing to neuroscientists is the presence of extensive networks of neural circuits embedded in its walls – the enteric nervous system. Containing approximately the same number of nerve cells as the spinal cord, it is connected with the central nervous system via visceral afferents and sympathetic and parasympathetic efferents of the autonomic nervous system. The presence of an entire, specialised nervous system within this peripheral organ system provides an opportunity for detailed study, without having to contend with the central nervous system. Since the studies of Bayliss and Starling (1899), Langley and Magnus (1905) and Trendelenburg (1917), it has been apparent that the enteric nervous system is capable of displaying a great deal of autonomy in controlling gastrointestinal function. Thus the enteric nervous system is studied by gastroenterologists seeking to understand how gut function is controlled, and also by neuroscientists endeavouring to understand how a relatively simple, autonomous mammalian neural circuit is organised. Obviously these two motivations are not mutually exclusive and many students of the enteric nervous system have foot in both camps. The complexity of the enteric nervous system began to be recognised with the discovery of the two major neural plexuses, the submucous plexus by Meissner (1857) and the myenteric plexus by Auerbach (1862). Langley (1921) placed the intrinsic enteric innervation in a class of its own after he recognised that many of the neural functions depend on the circuits entirely intrinsic to the digestive tract, rather than being solely determined by extrinsic neural inputs. Despite this early insight, it took many more years before the enteric nervous system was recognised by gastroenterologists as a relatively independent component of the autonomic nervous system. Combined studies by neuroanatomists, pharmacologists and physiologists eventually demonstrated that the study of the enteric nervous system was not only relevant to gastroenterology, but essential for its progress. During much of the twentieth century, the rate of progress was relatively slow in this field, which seemed to have fallen into a gap between neuroscience and gastroenterology. During this period, much emphasis was placed on the myogenic control of gastrointestinal motility, with little direct investigation of the roles of enteric neurons. It is only recently

xiv INNERVATION OF THE GASTROINTESTINAL TRACT

that the cellular basis of the spontaneous electrical activity of gut smooth muscle has begun to be understood in detail. It has been discovered that the mysterious cells, first described by Cajal as “interstitial cells” are involved both in generating myogenic activity and in mediating the input of enteric motor neurons to the smooth muscle (Sander, 1996; Huizinga et al., 1997). This breakthrough promises eventually to bridge the historical divide between the two schools of thought (myogenic versus neurogenic) about the control of gut motility. In this volume of the series on the autonomic nervous system, the innervation of the gut by the enteric nervous system, and its interface with the extrinsic innervation, is examined from a number of different perspectives. It will become apparent that all of these different aspects of nervous control can be related directly to how gut function is adapted to minimise risk, while maximising digestive efficiency. For example, the detailed analysis of enteric neural circuits controlling reflex motor activity of the intestine is elegantly summarised in the chapter by Joel Bornstein and colleagues. They provide an insightful synthesis of data gathered over many years by their own laboratory and by others around the world, as to how simple circuits may propel the gut contents, preventing excessive distension and perhaps contribute to the expulsion of pathogens. As described above, the stomach plays a very different role in the process of dealing with food, as it is not involved in digestion or absorption but is rather adapted for storage, mechanical breakdown and aliquoting of contents. Not surprisingly, the neuronal control of gastric motility has many different features from those of the small intestine. This is summarised in the extensive review of David Grundy and Michael Schemann who have contributed widely to the study of both the extrinsic and intrinsic innervation of the stomach. A particularly important role for extrinsic sensory nerves in protecting the stomach from a range of damaging agents has been identified in the last decade. Peter Holzer has been at the centre of this field and has written an authoritative review, summarising the role and mechanisms of action of these nerves in gastric protection. At the other end of the gastrointestinal tract, the colon also plays a significant role as an organ of storage, but it has to deal with very different material. In particular, much of the activity of the colon is modulated by extrinsic neuronal inputs from both the sympathetic and parasympathetic divisions of the autonomic nervous system. For many years the contributions of the extrinsic innervation have been rather over-simplified. This is corrected in the chapter of Kalina Venkova, Beverely Greenwood-Van Meerveld and the late Jack Krier, who carried out so many foundation studies on the extrinsic innervation of the distal gut. The last specialised region to receive attention in this volume is the gallbladder and sphincter of Oddi. Gary Mawe’s group have established themselves as leading investigators of this often overlooked accessory to the gut. Given the amount of trouble that it causes clinically, the biliary system and its motility are medically important subjects for study. The major differences between the organisation of the enteric nervous system of the gallbladder and sphincter of Oddi and that of the extensively studied small intestine are highlighted in this chapter. Since the pioneering study of Paton (1955), the small intestine, particularly of the guinea-pig, has been a favoured preparation for pharmacologists around the world. A vast literature has developed about the effects of drugs acting on a huge range of receptors found in the enteric nervous system and smooth muscle of this preparation. Marcello

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Tonini and his colleagues have summarised an enormous body of work to provide an accessible, yet comprehensive summary of this field. It should be pointed out that the future ability of gastroenterologists to provide treatment for disorders of the gut will rely on the development of new drugs. Understanding the pharmacology of the enteric nervous system is a crucial step in this undertaking. This analysis is greatly extended by the review of Charles Hoyle, Pam Milner and Geoffrey Burnstock on the nature of neuroeffector transmission in the gut. A wide-ranging summary of the transmitters, receptors and pharmacology of neuromuscular transmission is provided in their contribution. Frequently, enteric neurobiologists forget about the secretory and absorptive functions of the gastrointestinal tract, which are so central to its role in digestion. Charles Hoyle and colleagues also give us a wonderful account of the major pathways, largely arising from the submucous plexus, which control epithelial function. Of course, the secretory and absorptive capability of the epithelium is closely coupled with maintenance of an adequate blood supply, as are the requirements for mucosal protection described in Peter Holzer’s review. Neela Kotecha’s chapter summarises what is known about the enteric and extrinsic control of the gut vasculature, providing a valuable foundation for appreciating how different aspects of neuronal control are integrated. Lay people and funding agencies are often particularly interested in what scientists can tell them about the nature of medical disorders. This is as true for the innervation of the gut as it is for any other organ system. Many of the disorders of the gastrointestinal tract are related to the interactions between the immune system and the nerve cells and fibres in the gut wall. Jackie Wood and his many colleagues, over a long period, have been pioneers in this field. In his chapter, Professor Wood provides a compelling account of the central role of mast cells and mediators from leukocytes in modifying and sometimes determining the motor patterns and secretion of the small and large intestine during pathogen challenge. In the chapter by Simon Brookes and Marcello Costa, the cellular organisation of the enteric nervous system and some of the changes associated with gut disorders have been summarised. This raises the question of how the enteric nervous system develops to the normal adult form. This field has made enormous progress in the last decade and at the forefront of the advance has been the New York-based group of Michael Gershon. In a scholarly review, Professor Gershon describes some of the major influences on the colonisation and phenotypic development of enteric neurones. He discusses evidence for the involvement of a number of powerful growth factors and their receptors. In addition, he examines how defects in them may contribute to the aganglionosis of the distal colon, which is one of the most common developmental disorders of the enteric nervous system. The investigations summarised in this volume are beginning to reveal a large repertoire of neural mechanisms present in the digestive tract. It is not possible for a single book to encompass all knowledge about such a large and rapidly growing field. However, the chapters presented here give a good indication of the state of current knowledge. By reading between the lines, the astute reader will also be able to identify some of the current gaps in our knowledge. For example, it should not be surprising that there have been only few attempts to link cellular mechanisms to the functions of the entire organ, either in vitro or in vivo. In only a few cases have investigators been able to establish the circumstances under which a particular neural mechanism actually operates in normal living

xvi INNERVATION OF THE GASTROINTESTINAL TRACT

conditions. The gap between cellular physiology and organ physiology is still very wide. It should be clear from the reviews presented here that we now know a lot about what the neural system in the gut can do, but we know much less about how it actually operates under normal conditions. We know even less about how it changes, at the cellular level, to deal with different diets, with pathogens and in disease states. This represents a major challenge for the next generation of investigators. It can be expected that addressing these questions will be invaluable in the rational design of new therapies for disorders of the gastrointestinal tract. Simon Brookes and Marcello Costa

REFERENCES Auerbach, L. (1862). Über einen plexus myentericus, einen bisher unbekannten ganglionervösen Apparat im Darmkanal der Wirbelthiere. Breslau, Morgenstern, E. p. 13. Bayliss, W.M. and Starling, E.H. (1899). The movements and innervation of the small intestine. Journal of Physiology (London), 24, 99–143. Huizinga, J.D., Thuneberg, L., Vanderwinden, J.M. and Rumessen, J.J. (1997). Interstitial cells of Cajal as targets for pharmacological intervention in gastrointestinal motor disorders. Trends in Pharmacological Sciences, 18, 393–403. Langley, J.N. and Magnus, R. (1905). Some observations of the movements of the intestine before and after degenerative section of the mesenteric nerves. Journal of Physiology (London), 33, 34–51. Meissner, G. (1857). Über die Nerven der Darmwand. Zeitschrift für Ration Medezin, 8, 364–366. Paton, W.D.M. (1955). The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes. Journal of Physiology (London), 127, 40P–41P. Sanders, K.M. (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology, 111, 492–515. Trendelenburg, P. (1917). Physiologische und Pharmakologische Versuche über die Dunndarmperistaltik. Naunyn Schmiedebergs Archiv für Experimentelle Pathologie und Pharmakologie, 81, 55–129.

Marcello Costa and Simon Brookes — Editors of this volume

Contributors P.P. Bertrand Department of Physiology University of Melbourne Victoria 3010 Australia J.C. Bornstein Department of Physiology University of Melbourne Victoria 3010 Australia Simon J.H. Brookes Department of Human Physiology and Centre for Neuroscience Flinders University GPO Box 2100, Adelaide South Australia 5001 Geoffrey Burnstock Autonomic Neuroscience Institute Royal Free and University College Medical School Royal Free Campus Rowland Hill Street London NW3 2PF UK Marcello Costa Department of Human Physiology and Centre for Neuroscience

Flinders University GPO Box 2100, Adelaide South Australia 5001 Francesca Crema Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Fabrizio De Ponti Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Gianmario Frigo Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy xviii

CONTRIBUTORS

J.B. Furness Department of Anatomy and Cell Biology University of Melbourne Victoria 3010 Australia Michael D. Gershon Department of Anatomy and Cell Biology Columbia University College of Physicians and Surgeons 630 W 168th Street New York NY 10032 USA Beverley Greenwood-Van Meerveld Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA David Grundy Department of Biomedical Science The University of Sheffield The Alfred Denny Building Western Bank Sheffield S10 2TN UK Kirk Hillsley Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Peter Holzer Department of Experimental and Clinical Pharmacology University of Graz, Universitätsplatz 4 A-8010 Graz Austria

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Charles H.V. Hoyle Department of Anatomy and Developmental Biology University College London Gower Street London WC1E 6BT UK Lee J. Jennings Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Audra L. Kennedy Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Neela Kotecha Department of Physiology Monash University Clayton Victoria-3800 Australia Jacob Krier Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA Wolfgang A.A. Kunze Department of Anatomy and Cell Biology University of Melbourne Victoria 3010 Australia

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Gary M. Mawe Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA Pam Milner Autonomic Neuroscience Institute Royal Free and University College Medical School Royal Free Campus Rowland Hill Street London NW3 2PF UK Michael Schemann Department of Physiology School of Veterinary Medicine Bischofsholer Damm 15/102 D-30173 Hannover Germany Erin K. Talmage Department of Anatomy and Neurobiology C423 Health Science Complex The University of Vermont Burlington VT 05405 USA

Marcello Tonini Department of Internal Medicine and Therapeutics Section of Clinical and Experimental Pharmacology University of Pavia Piazza Botta 10 I-27100 Pavia Italy Kalina Venkova Oklahoma Foundation for Digestive Research Basic Science Labs V.A. Medical Center Oklahoma City OK 73104 USA Jackie D. Wood Department of Physiology College of Medicine The Ohio State University 300 Hamilton Hall 1645 Neil Avenue Columbus Ohio 43210 USA

1 Enteric Reflexes that Influence Motility J.C. Bornstein1, J.B. Furness2, W.A.A. Kunze2 and P.P. Bertrand1 1

Department of Physiology and Department of Anatomy and Cell Biology, University of Melbourne, Victoria 3010, Australia

2

The gut exhibits a variety of movements that depend on the region studied and the timing and composition of the last meal. Primary control of these movements is exerted by the enteric nervous system. Except in the oesophagus and the sphincters, external inputs modulate enteric neural activity, rather than directly controlling muscle movements. In isolated intestine, localised mechanical and chemical stimuli excite enteric neurons to produce stereotypic reflexes in the smooth muscle. The reflex circuits form the basis of the circuitry responsible for more complex motor patterns evoked by more generalised stimuli. The enteric neural circuitry responsible for stereotyped reflexes in the guinea-pig ileum has been studied in detail and the neurons involved have been identified. These include at least three types of intrinsic primary afferent neurons (IPANs), a population of ascending interneurons, three populations of descending interneurons, excitatory and inhibitory motor neurons innervating the circular muscle and longitudinal muscle motor neurons. Synaptic transmission in the ascending excitatory reflex pathway is primarily via fast excitatory synaptic potentials (EPSPs) mediated by acetylcholine acting at nicotinic receptors; slow EPSPs mediated via NK3 tachykinin receptors may also be involved. Acetylcholine is less important in descending pathways. Transmission from interneurons to inhibitory motor neurons depends on ATP acting at P2X receptors and tachykinins play a significant, but not exclusive, role in transmission from IPANs to descending interneurons. Transmitters at other synapses in this pathway remain to be identified. These properties of simple reflexes will be important for understanding more complex behaviour. KEY WORDS: myenteric plexus; intestinal reflexes; synaptic transmission; intrinsic primary afferent neurons; enteric neural circuits; neurotransmitters.

INTRODUCTION The gastrointestinal (GI) tract breaks down food into absorbable nutrients by mechanisms that are almost entirely without conscious control. While chewing, swallowing and defaecation are consciously initiated; processes such as digestion, secretion and absorption, and the motility of the GI tract are essentially automatic. Both motility and secretion are under the direct control of the nervous system. This is manifested at several different levels. The enteric nervous system (ENS) is contained 1

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entirely within the wall of the GI tract and can mediate many gastrointestinal functions without input from the central nervous system. Nevertheless, under normal circumstances several levels of external control can be identified. Emotions such as fear have well known gastrointestinal effects without the involvement of a visceral stimulus. In addition, many visceral stimuli alter gastrointestinal behaviour via pathways that include neurons of the central nervous system (e.g. vago-vagal reflexes). However, some stimuli applied to the gut modify gastrointestinal functions via neural pathways that bypass the central nervous system, but include the prevertebral sympathetic ganglia. Furthermore, a variety of hormonal mechanisms can also regulate intestinal function. For example, the interdigestive motor patterns of the stomach and both acid secretion and gastric emptying after a meal are modified by the chemical composition of the chyme in the small intestine (Lin et al., 1990a,b; Spencer et al., 1990; Orloff et al., 1992). The latter two effects are depressed when all neural pathways into the stomach are interrupted, but they can still be observed. This implies that both the extrinsic nerves and hormonal influences are involved in the control of gastric function by the contents of the small intestine. Behaviours evoked by stimulation of the GI tract, that are independent of the endocrine system, fall into the general class of entero-enteric reflexes. Many, notably the vago-vagal reflexes referred to above (for review see Grundy and Scratcherd, 1989), depend on the integrity of connections between the GI tract and the central nervous system. Others depend only on intact connections between the GI tract and the prevertebral sympathetic ganglia (for review see Szurszewski and King, 1989). A characteristic feature of the extrinsic nervous supply to the GI tract is that efferent nerves provide terminals largely to the intrinsic ganglionated plexuses rather than directly to the muscle, except in the sphincters and in the striated muscle of the oesophagus (Furness et al., 1999). Thus, the extrinsic reflex pathways coordinate the activities of different regions of the GI tract largely by modifying the activity of enteric neurons, rather than via direct effects upon the muscle, the enteric neurons representing a final common level of control of gastrointestinal function. This chapter focuses on the control of motility. In particular, we discuss recent data about the neural circuitry responsible for motility reflexes and the way this circuitry is organised to produce the motor programs responsible for complex patterns of movement. MOTILITY REFLEXES AND MOTOR PROGRAMS The neural circuits that control real motor behaviours anywhere in the body are complex and are generally studied by the use of reduced preparations in which the number of factors affecting a motor response is kept to a minimum. In the extreme case, such reduced preparations give rise to simple reflexes in which each element of the circuit can be readily identified. At the next level, the neural circuits that produce more complex behaviours can be considered to act as motor pattern generators, producing coordinated and often complex interactions between different muscles to give rise to stereotyped behaviour. To take an example from another system, in the simple tendon jerk reflex, primary afferent neurons from muscle spindles directly excite motor neurons. However, when the spinal cord is isolated from central input, appropriate sensory stimuli trigger the entire suite of coordinated movements required for normal walking. The difference between the simple reflexes of reduced preparations and the more complex, but still stereotyped, motor

ENTERIC REFLEXES THAT INFLUENCE MOTILITY

3

patterns is that the simple reflexes always have the same form when initiated by a specific stimulus, while a single organ can generate several distinct motor patterns. In systems where pattern generators have been studied, including the GI tract, the neurons in simpler reflex circuits are an integral part of the pattern generator circuits. GI tract reflexes vary in complexity as they do in all other parts of the nervous system. Even if those reflexes involving the central nervous system or the prevertebral ganglia are excluded, enteric reflexes range from local constrictions resulting from pinches applied to the serosal surface to coordinated waves of activity sweeping along the intestine. The former may be analogous to the spinal monosynaptic reflex, while the latter are due to activation of complex motor patterns controlled by circuitry analogous to the motor pattern generators of the central nervous system. This chapter explores the circuitry underlying simple reflexes and the reflex motor patterns seen in the gut, with an emphasis on the ileum of the guinea-pig, about which we have the most detailed information.

PHYSIOLOGICAL STUDIES OF ENTERIC REFLEXES The first studies of intestinal reflexes were performed towards the end of the nineteenth century when it was shown that various stimuli applied to the intestine trigger movement of intestinal content or secretion across the intestinal wall (Nothnagel, 1882; Mall, 1896; Bayliss and Starling, 1899, 1900a,b). By 1899, it was clear that a substantial part of the mechanisms that control propulsion of intestinal contents must be confined to the intestinal wall. Bayliss and Starling (1899) showed that a bolus inserted into a region of intestine triggered a complex motor pattern which caused the bolus to pass anally along the intestine even when all connections to the central nervous system had been severed. This study identified two reflexes which appear to underlie much of the normal propulsive behaviour of the intestine; the ascending excitatory reflex, in which the circular muscle contracts oral to the point of stimulation, and the descending inhibitory reflex, in which the circular muscle relaxes anal (or aboral) to it. Bayliss and Starling believed that any stimulus that evoked one of these reflexes would evoke the other as well and they termed this the “Law of the Intestine”. Other workers, including Langley and Magnus (1905), quickly confirmed that propulsion of material along the intestine did not depend on the central nervous system. However, the motility patterns of the intestine in the presence, and absence, of food in vivo are more complex than the propulsive response to an inserted stimulus in vitro. STUDIES OF MOTOR PATTERNS IN VIVO Using radiometric methods, Cannon identified several distinct types of propulsive behaviour in the presence of food in vivo (Cannon, 1902, 1906, 1912). These can be broadly described as segmentation, peristalsis and peristaltic rushes. Segmentation seems to be a mixing behaviour and involves rhythmic stationary contractions that repeatedly divide and redivide a mass of intestinal content. In contrast, peristalsis and peristaltic rushes each propel the intestinal content in an anal direction, with the difference between them being a matter of degree, duration and velocity. He also identified propulsion of content in an oral direction, antiperistalsis.

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Radiometric methods require the presence of a material opaque to X-rays in the lumen of the GI tract and have been extended by the use of radioactive labels within the food so that its movement can be monitored with scintigraphy. Such studies, in combination with the multisite recording methods discussed below, have provided valuable information about the effects of varying the composition of a meal on the motility of the intestine. An important observation is that the rate of movement of a meal through the stomach and along the intestine is critically dependent on its composition. Chemically inert meals move along the intestine more rapidly than meals with significant nutritive content, with the relative proportions of fat, carbohydrate and protein being significant determining factors (Eeckhout et al., 1984; Schemann and Ehrlein, 1986c; Buhner et al., 1988; Buhner and Ehrlein, 1989; Siegle, Schmid and Ehrlein, 1990; Schmid and Ehrlein, 1993). Even the pattern of contractions seen within the intestine varies according to the chemical composition of the meal. This is illustrated by a study by Schemann and Ehrlein (1986c) who found that meals containing nutrients induced a larger proportion of stationary contractions, attributed to segmentation, than did non-nutritive meals. The rate of propagation of propulsive contractions (peristalsis) was unchanged, but the length over which they propagated was substantially reduced by increased nutrient density. Thus, segmentation predominates when nutrient is present in lumen, while peristalsis predominates when the chyme is nutrient free (Schemann and Ehrlein, 1982, 1986b,c; Siegle and Ehrlein, 1988; Buhner and Ehrlein, 1989; Schmid and Ehrlein, 1993; Huge, Weber and Ehrlein, 1995). In many species, different patterns of activity occur in fed and fasted states. Studies using a variety of widely spaced recording devices – strain gauges, extracellular electrodes, multichannel pressure sensing catheters – have shown that when an animal is fasted a complex wave of activity propagates repeatedly, but slowly, along the intestine from the stomach to the caecum. The behaviour is variously known as the migrating myoelectric complex (MMC), the migrating motor complex or the interdigestive cycle (for a recent review of this, and other motility patterns in vivo, see Hasler (1999)). The MMC essentially consists of an anally propagating wave of strong excitation of the intestinal muscle that alternates with a longer period of very low activity or possibly inhibition of the muscle (Figure 1.1). These two phases (Phase III and Phase I, respectively) are separated by periods of irregular muscular activity (Phases II and IV). Interestingly, while MMCs in the small intestine have never been convincingly recorded in isolated tissue, they are seen in extrinsically denervated GI tract in vivo. Thus, the neural circuit generating MMCs must be contained within the intestinal wall, i.e. part of the enteric nervous system (Sarna et al., 1981). In many animals, including humans, MMCs disappear immediately after the animal eats and the activity of the intestine changes to the fed, or post-prandial state described above. MMCs are widely identified with the gastric antrum, the duodenum, jejunum and ileum. However, migrating motor patterns, which have been termed MMCs, have also been observed in the colon, but not in the caecum (Sarna, Prasad and Lang, 1988). The colonic MMCs are independent of those in the small bowel. Another motor pattern seen predominantly in the fasted state is the giant migrating contraction (GMC): a large contraction that is much briefer than phase III of the MMC, propagates rapidly and continues for a substantial distance (Sarna, Prasad and Lang, 1988; Sarna, 1991a,b; Otterson and Sarna, 1994; Sethi and Sarna, 1995). GMCs are seen in the ileum, the caecum and the


J1 J2 J3 J4 J5 J6 J7 J8 J9 J10 J11 J12

III

I

II

5

III

24 min

80 mmHg

Figure 1.1 Multichannel manometric recordings taken from 12 sites (J1–J12) within the jejunum of a fasting human. Each recording site was separated from its neighbours by 2 cm. The different phases of the migrating motor complex are indicated for the upper trace as Phase I (I), Phase II (II) and Phase III (III), in this set of recordings Phase IV was not readily discernable. Recordings provided by Prof. Sushil Sarna.

colon and are clearly distinct from MMCs, but are similar to the peristaltic rushes seen in the fed state. In the fed state, the distal ileum has a characteristic pattern of motility not seen elsewhere in the GI tract (Fich et al., 1990). When a meal arrives in the terminal ileum of the dog, a series of high frequency contractions of the circular muscle is initiated. These contractions occur more frequently than the slow waves that are usually thought to control the frequency of contractions within the intestinal muscle. They are seen only when the material entering the terminal ileum contains nutrient; i.e. they are not seen during an isotonic saline infusion. As yet their function is unknown. Thus, in vivo studies have demonstrated the presence of several distinct motor patterns whose presence and persistence depends on the region being studied, the content of that region and of other regions. The relationship between some of these behaviours and the intestinal microcircuitry mediating simple reflexes will be discussed in the last section of this chapter. REFLEXES AND MOTOR PATTERNS IN ISOLATED SYSTEMS The work of Mall (1896) and then Bayliss and Starling (1899; 1900a,b) may fairly be described as the first substantial studies of motor patterns in isolated segments of intestine and hence in reduced systems. The results were essentially similar to those seen over the next 2 decades by other workers in studies performed on segments of intestine in vitro (Magnus, 1904; Langley and Magnus, 1905; Trendelenburg, 1917). Stimulation of the intestinal segment by irritating the mucosa or by distension with either a solid or liquid bolus evoked a stereotyped propulsive movement of the intestine that tended to move the stimulus in an oral to anal direction. The movement involved a shortening of the longitudinal muscle, followed by a contraction of the circular muscle oral to the stimulus and,

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in some cases, a relaxation of the circular muscle anal to the stimulus. This last component of the response was not seen by all subsequent workers (e.g. Bolzer, 1949b), perhaps because the smooth muscle must first generate some active tension for a relaxation to be detected. Bolzer (1949a,b) recognised that peristalsis evoked by stroking the mucosa or by distension was a more complex phenomenon than a simple reflex. He found that contractions evoked oral to a solid bolus propagate anally along the intestine at a rate faster than the movement of the bolus itself. The contraction passed over the bolus implying that movement of the stimulus is not the sole mechanism leading to anal propagation of the contractions in the circular muscle. He speculated that the consistency of the bolus may be an important determinant of the final pattern of the propulsive behaviour. The idea that differing stimuli evoke different patterns of contractile behaviour has been supported by studies of intestinal segments isolated from the central nervous system in vivo and in vitro by Hukuhara and colleagues in the late 1950s and throughout the 1960s (Hukuhara, Yamagami and Nakayama, 1958; Hukuhara and Miyake, 1959; Hukuhara, Nakayama and Sumi, 1959; Hukuhara et al., 1959; Hukuhara, Nakayama and Nanba, 1960a,b, 1961; Hukuhara, Sumi and Kotani, 1961; Hukuhara and Fukuda, 1965; Hukuhara, Neya and Tsuchiya, 1969). In these studies, stretching the intestinal wall inhibited ongoing contractions oral to, and anal to, the point of stimulation in the dog and the rabbit small intestine, but mechanical or acid stimulation of the mucosa evoked contraction on the oral side and inhibition on the anal side. The response to a specific stimulus also depended on the species tested, with guinea-pigs showing only oral excitation as a result of chemical or mechanical stimulation of the mucosa. In this species, stretch evoked contractions on both the oral and the anal side, unlike the responses seen in dogs and rabbits. Anal excitation evoked by stretch has been seen only intermittently in subsequent studies of guinea-pig intestine (see below). Hukuhara’s studies demonstrated several important points about the motor behaviour of the intestine. First, removal of the myenteric plexus interrupted transmission of reflex activity along the intestine, but disruption of the submucous plexus did not. Thus, the myenteric plexus probably contains the interneurons and the cell bodies of motor neurons mediating these reflexes. Second, reflex responses in the circular muscle to chemical and mechanical stimulation of the mucosa were essentially identical. Third, mucosal stimulation does not always evoke responses similar to those evoked by stretch. Thus, the nature of the motor program activated by a stimulus may depend on the overall state of the tissue at the time the stimulus is applied. Finally, they showed that chemical stimuli (acetylcholine, histamine, pilocarpine or BaCl2) applied to the serosa could evoke stereotyped motor responses in the intestine. The variety of stimuli employed by Hukuhara and his colleagues, raises the issue of the modes of stimulation appropriate for studies of motor reflexes. Recently, three distinct sensory modalities have been shown to excite stereotyped intestinal reflexes consisting of excitation oral and inhibition anal to the point of stimulation in isolated segments of small intestine or distal colon in vitro. Distension, mechanical stimulation of the mucosa and acid applied to the mucosa, all evoke such reflexes (Smith and Furness, 1988; Smith, Bornstein and Furness, 1990, 1991, 1992; Smith et al., 1992; Yuan et al., 1991; Grider, 1994; Grider and Jin, 1994; Foxx-Orenstein, Kuemmerle and Grider, 1996; Smith and McCarron, 1998).


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Despite the dependence of propulsion and local motor patterns on the chemical composition of the chyme (see above), mechanical stimuli are nearly always used in studies of enteric motility reflexes, perhaps because the onset and offset of the stimulus can be well controlled by the investigator. Distension, or a localised stretch applied to the intestinal wall, has been the stimulus of choice in the vast majority of studies, either in vivo or in vitro. This does not affect chemoreceptors and so distension is a relatively pure stimulus. However, exclusive use of any stimulus modality to analyse propulsive behaviours carries the inherent assumption that the neural circuitry activated by that stimulus includes all the elements that might be excited by other stimuli. Detailed electrophysiological analysis of the functional classes of neurons excited by different sensory modalities in the guinea-pig small intestine indicates that these different stimuli excite common populations of circular muscle motor neurons (Smith, Bornstein and Furness, 1992). However, as discussed below, many of the primary afferent neurons excited by the different sensory modalities that trigger intestinal reflexes are functionally distinct, indicating that this assumption does not hold. The other underlying assumption of exclusive use of distension to initiate reflexes is that the motor program excited by this stimulus provides the basic pattern underlying all motility in the GI tract. Although the basic reflexes excited by the three distinct sensory modalities appear similar, the results outlined earlier in this chapter indicate that chemical stimuli may initiate motor patterns distinctly different from those evoked by distension. Thus, as “physiological stimuli” used in most studies of enteric reflexes are constrained to a single sensory modality or a mix of mechanical stimuli (see below), they may excite only a proportion of the behaviours of which the stimulated region is capable. When an isolated segment of intestine is distended by a static increase in the pressure of isotonic saline contained within the lumen, it exhibits rhythmic contractions of the circular muscle (e.g. Trendelenburg, 1917; Tonini et al., 1981; Buchheit and Buhl, 1991). These contractions propagate anally, propelling the saline in the same direction and can empty the entire segment. Prior to each circular contraction, the longitudinal muscle contracts in what is known as the “preparatory phase” of peristalsis. In a variant of this method, a length of intestine is steadily distended with increasing amounts of physiological saline until a propulsive reflex is generated (Bülbring and Lin, 1958; Kottegoda, 1969; Waterman, Costa and Tonini, 1992, 1994a). The behaviour evoked consists of a ring of contraction of the circular muscle, which typically starts at the oral end of the intestinal segment and propagates anally (Hennig et al., 1999; SchulzeDelrieu, 1999). The contraction usually occludes the lumen and is preceded by a relaxation of the circular muscle, so that the saline within the segment is propelled anally and expelled from its anal end. This method has the advantage of allowing a threshold for the initiation of the behaviour to be determined along with both the pressure changes and the transport of liquid through the intestinal segment. Such studies have shown how complex the propulsion of material along the intestine can be. For example, intraluminal acid reduces the threshold pressure needed to trigger propulsion, but leads to a reduced flow at any given stimulus pressure once threshold is exceeded (Hukuhara, Nakayama and Sumi, 1959). Similarly, blockade of inhibitory motor neurons reduces the threshold pressure for initiation of the contraction, but can lead to a complete cessation of propulsion (Waterman and Costa, 1994, 1994b). Moreover, while these methods produce responses that are similar to the behaviours seen in vivo, there are several possible differences. For example,

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the normal content of the intestine is not as chemically inert as physiological saline and does not steadily, or evenly, distend long segments of intestine. A simpler behaviour can also be evoked by gradual filling of a segment of the GI tract. This is reflex accommodation which involves a relaxation of the circular muscle at the site of distension when the pressure inside the stomach (Azpiroz and Malagelada, 1990; Hennig et al., 1997), the small intestine (Waterman, Costa and Tonini, 1994a) or the large intestine (Davison and Pearson, 1979; Ciccocioppo et al., 1994) exceeds a threshold value. This threshold is typically below that required to evoke a propulsive reflex (Waterman, Costa and Tonini, 1994a). Thus, reflex accommodation may be part of the preparatory phase of the propulsive behaviour evoked in the intestine by a fluid distension, as well as a separate behaviour in its own right. Saline distensions have also been used to study motor behaviours in the stomach (Hennig et al., 1997) and colon (Ciccocioppo et al., 1994; Smith and Robertson, 1998). In the latter, however, artificial faecal pellets, which represent a more physiologically realistic stimulus, are often used (Costa and Furness, 1976; Foxx-Orenstein and Grider, 1996; Kadowaki, Wade and Gershon, 1996; Foxx-Orenstein, Jin and Grider, 1998). The pellet is inserted into the oral end of an isolated segment of colon and the time taken to expel it from the anal end is measured. This time is consistent between trials and provides a reliable measure of the propulsive behaviour of the colon. The propulsive mechanism is very similar to that in the small intestine with pellets being propelled anally by waves of contraction within the circular muscle that are preceded by relaxation of the same muscle layer. An elegant refinement of this method has been the use of a mobile balloon attached to a device for measuring rate of transit as the artificial pellet (Crema, Frigo and Lecchini, 1970; Frigo and Lecchini, 1970; Tonini et al., 1989; Ciccocioppo et al., 1994; Onori et al., 2000). This allows the size of the balloon to be varied between trials and the pressure generated to be determined along with the speed of propulsion. These studies have shown that, in the rabbit colon, the dependence of propulsion on inhibitory neuromuscular transmission is greater for larger pellets. Another type of experiment uses a confined stimulus, which is held stationary, to evoke reflexes within the intestine. Responses are measured as changes in the contractility of the smooth muscle layers (Holzer, 1989; Tonini and Costa, 1990; Holzer, Schluet and Maggi, 1993; Maggi et al., 1994; Spencer, Walsh and Smith, 1999, 2000), as electrical responses in the muscle (Hirst, Holman and McKirdy, 1975; Smith and Furness, 1988; Smith, Bornstein and Furness 1990, 1991; Smith et al., 1992; Yuan et al., 1991) or as changes in the activity of enteric neurons measured either extracellularly or intracellularly (Hirst and McKirdy, 1974; Hirst, Holman and McKirdy, 1975; Bornstein et al., 1991a; Smith, Bornstein and Furness, 1992). Local distension with a balloon evokes an excitatory reflex oral to the point of stimulation (Holzer, 1989; Smith, Bornstein and Furness, 1990, 1992; Tonini and Costa, 1990; Holzer, Schluet and Maggi, 1993; Maggi et al., 1994), but this reflex typically propagates a few centimetres orally rather than anally (Smith, Bornstein and Furness, 1990, 1992). Such stimuli (and also mechanical and chemical stimuli applied to the mucosa) may excite local reflexes which do not propagate at all. However, recent studies by Spencer, Walsh and Smith (1999, 2000) found that both radial distension and mechanical stimulation of the mucosa evoked anally propagating contractions of both longitudinal and circular muscle in the guinea-pig ileum.


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In contrast, electrophysiological studies have rarely shown any evidence that the excitatory reflex contractions evoked by stationary distensions propagate anally in the small intestine. Similarly, stimulation of the mucosa either mechanically or with acid evokes an orally propagating excitatory reflex (Smith and Furness, 1988; Smith, Bornstein and Furness, 1991), which has yet to be shown to propagate anally. Thus, the anal propagation of contraction seen in the propulsion experiments cannot be explained by a simple combination of reflexes evoked by stationary stimuli applied to a confined region of intestine. This will be discussed in the last section of this chapter.

NEURAL CIRCUITS MEDIATING REFLEXES EVOKED BY STATIONARY STIMULI IN VITRO The basic circuit excited by a stationary stimulus is now clearly established for the small intestine of the guinea-pig (Figure 1.2). This is based on the responses of enteric neurons and the intestinal muscle and correlated electrophysiological, immunohistochemical and morphological studies of individual enteric neurons. In combination, these experiments

Figure 1.2 Diagram showing the neural circuitry underlying simple reflexes within the guinea-pig small intestine. This circuit, which contains seven types of neurons, has been inferred from many experimental sources. At each point along the intestine there is a network of IPANs with outputs to all other neuronal subtypes other than SOM descending interneurons. The interneurons form chains along the intestine and also make specific connections to motor neurons (ascending interneurons to excitatory motor neurons, descending interneurons to inhibitory motor neurons), which also receive input from local IPANs. The identities of specific neuronal subtypes are shown below the main circuit and are used in subsequent figures.

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have allowed virtually all component neurons of the reflex pathways to be identified both structurally and physiologically. A stationary stimulus excites one or more populations of intrinsic primary afferent neurons (IPANs), which project circumferentially to excite local motor neurons and separate populations of orally and anally directed interneurons. Orally directed (ascending) interneurons activate both excitatory motor neurons and other ascending interneurons, while the anally directed (descending) interneurons excite other descending interneurons and inhibitory motor neurons. A significant subset of the IPANs project anally to contact inhibitory motor neurons (Johnson et al., 1996; Johnson, Bornstein and Boucher, 1998; Bian, Bertrand and Bornstein, 2000). These studies are discussed in detail in the remainder of this section. CLASSIFICATION OF ENTERIC NEURONS There have been four primary methods of classifying enteric neurons prior to identification of their functions: morphological, neurochemical, pharmacological and electrophysiological. Morphological studies date back to the work of Dogiel (1899) in the nineteenth century. This work provided the basic description of the major morphological types of cell bodies seen in the many subsequent studies (e.g. Stach, 1979, 1989; Scheuermann and Stach, 1983; Brehmer and Stach, 1998; Brehmer et al., 1998). In the small intestine of the guinea-pig, two broad classes of neurons can be identified using morphological techniques: large multipolar neurons are usually called Dogiel type II neurons, and several poulations of monoaxonal neurons (Figure 1.3) (Furness, Bornstein and Trussell, 1988).

Figure 1.3 Representative silhouettes of myenteric neurons from the guinea-pig duodenum that had been injected with neurobiotin during electrophysiological recordings (Clerc et al., 1998a). These shapes are typical of the shapes of myenteric neurons in the duodenum, ileum, proximal colon and distal colon.


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A subset of the monaxonal neurons has short lamellar dendrites; these are Dogiel type I neurons. Other subsets of the monoaxonal neurons include a population with long filamentous dendrites and a population of small neurons (Furness, Bornstein and Trussell, 1988). Small neurons with short filamentous dendrites have been identified in the guineapig duodenum (Clerc et al., 1998a,b) and in the proximal colon (Messenger, Bornstein and Furness, 1994) and distal colon of this species (Lomax et al., 1999). These small filamentous neurons have similar projection patterns to the small neurons of the ileum and may represent the same functional class. The immunohistochemical studies that have led to classification of enteric neurons according to their neurochemistry and projections are described in detail elsewhere in this volume (see Chapter 11 of this volume). Electrophysiological identification of enteric neurons began in the early 1970s with studies using extracellular recording methods (for review see Wood, 1975). These were rapidly overtaken by classification schemes based on intracellular recordings that began a little later. The first substantial intracellular study of enteric neurons published was that of Nishi and North (1973) who divided neurons into type 1 and type 2 on the basis of their firing properties, specifically whether they adapted rapidly or slowly to an imposed depolarization. In work published a few months later, Hirst, Holman and Spence (1974) divided myenteric neurons of the duodenum into two groups on the basis of their synaptic inputs and the after-potentials following their action potentials. S-neurons exhibited prominent fast excitatory synaptic potentials (EPSPs) in response to electrical stimulation of synaptic inputs and had only a brief (2 s Dogiel II

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Figure 5.1 Active electrical properties of guinea-pig gallbladder neurons. A. Four consecutive overlapping traces showing the response of a neuron to prolonged depolarizing current pulses (250 ms; 0.2 nA). As is typical of these cells, this neuron generated only one action potential at the onset of the current pulse. B. Response of a neuron to a brief current pulse illustrating a typical AHP, which is composed of an early and a late phase that are sensitive to tetraethyl ammonium and apamin, respectively. C. Elimination of the late phase of the AHP by apamin (100 nM) converts the neuron from an adaptive to a non-adaptive state, thus allowing for the generation of repetitive action potentials. (From Mawe et al., 1997).

guinea-pig gallbladder neurons since suppression of this component of the AHP with apamin causes the cells to fire action potentials repetitively throughout the duration of a depolarizing current pulse (Figure 5.1C; Mawe, 1990). The neurons of the North American opossum gallbladder are classified into two groups (Bauer et al., 1991), adaptive neurons which respond to intracellular current pulses with a short burst of action potentials, and rapidly adaptive neurons that respond to current pulses with a single action potential. The adaptive cells are more numerous, comprising about 70% of the population. Action potentials of neurons in these ganglia are TTX-sensitive, and are followed by a brief AHP lasting about 30 ms. Synaptic inputs to guinea-pig gallbladder neurons Synaptic input to gallbladder neurons, in response to stimulation of interganglionic fibre bundles, has been studied in the guinea-pig and the opossum (Mawe, 1990; Bauer et al.,

NEURONS OF THE GALLBLADDER AND SPHINCTER OF ODDI

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1991; Hillsley, Jennings and Mawe, 1998). Both fast and slow excitatory postsynaptic potentials (EPSPs) can be elicited, but inhibitory synaptic potentials have not been reported. Fast EPSPs, which can be activated by low frequency (0.5 Hz) stimulation of interganglionic fibre tracts, are sensitive to hexamethonium and are reversibly eliminated when the tissue is bathed in a Ca2+-free/high Mg2+ Krebs solution. Although interganglionic communication in the form of nicotinic EPSPs is likely to exist in these ganglia, the major source of nicotinic input to gallbladder neurons is from vagal preganglionic nerve fibres (see section on Vagal efferent fibres provide the driving force for gallbladder neurons; Mawe, Gokin and Wells, 1994). Slow EPSPs can be elicited by high frequency fibre tract stimulation (10–20 Hz) in about 30% of the neurons in guinea-pig (Mawe, 1990), and 20% of the neurons in opossum (Bauer et al., 1991). In the guinea-pig, the most thoroughly studied species, these slow synaptic events are Ca2+-dependent, and are associated with a decrease in input resistance. The slow EPSP is likely to involve an activation of a non-selective cation conductance since its amplitude decreases as cells are depolarized and increases when the membrane is hyperpolarized, with an estimated reversal potential near 0 mV. Recent evidence suggests that tachykinins and calcitonin gene-related peptide (CGRP) released from extrinsic afferent fibres mediate slow EPSPs in gallbladder ganglia (see section on Extrinsic sensory fibres; Mawe, 1995).

REGULATORY INPUTS TO GALLBLADDER GANGLIA Vagal efferent fibres provide the driving force for gallbladder neurons As described above, all gallbladder neurons exhibit nicotinic fast EPSPs in response to stimulation of interganglionic fibre tracts (Mawe, 1990; Bauer et al., 1991; Hillsley, Jennings and Mawe, 1998). Since gallbladder ganglia are developmentally associated with enteric ganglia, and since synaptic inputs to enteric ganglia are mainly intrinsic (Furness and Costa, 1987; Wood, 1989; Costa et al., 1992), it could not be presumed that nicotinic inputs to gallbladder neurons are from preganglionic neurons. However, it does appear that all gallbladder neurons do receive nicotinic input from the vagal preganglionic fibres. Several lines of evidence verify a significant vagal input to gallbladder ganglia. Stimulation of the vagus nerves causes gallbladder contraction, or, in the presence of atropine, relaxation (Ryan, 1987; Dodds, Hogan and Geenen, 1989; Dahlstrand, 1990). Neurons in the dorsal motor nucleus of the vagus have been retrogradely labelled from the gallbladder (Mawe, 1990). Also, nerve fibres that are immunoreactive for the biosynthetic enzyme of acetylcholine, choline acetyltransferase (ChAT), are present in ganglia, surrounding gallbladder neurons, and in the paravascular plexus, indicating that extrinsic cholinergic nerve fibres enter the gallbladder. Data from a study involving stimulation of extrinsic inputs to the gallbladder in normal and vagotomized guinea-pigs indicate that all gallbladder neurons receive vagal input, and that the vagal input is the major source of fast synaptic input to gallbladder neurons (Mawe, Gokin and Wells, 1994). Vagotomy results in the complete elimination of extrinsic nicotinic inputs, which are normally received by all gallbladder neurons. Therefore, unlike the ganglion cells in the bowel, all gallbladder neurons receive direct input

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from the central nervous system. Following vagotomy, fast EPSPs could be elicited in some neurons by stimulation of interganglionic fibre tracts, indicating that interganglionic neural projections may exist amongst gallbladder ganglia. Sympathetic postganglionic fibres In addition to receiving input from vagal preganglionic fibres, intramural ganglia are the target of sympathetic postganglionic inputs to the gallbladder. The ganglionated plexus of the gallbladder is rich in nerve fibres that express catecholamine histofluorescence (Cai and Gabella, 1983a, 1984; Mawe and Gershon, 1989), as well as immunoreactivities for tyrosine hydroxylase and dopamine β-hydroxylase (Mawe and Gershon, 1989). It is likely that these nerve fibres arise in the celiac ganglia because celiac neurons are labelled following injection of retrograde axonal tracers in the wall of the gallbladder (Mawe and Gershon, 1989). We have established that noradrenaline can be released from catecholaminergic nerve fibres and that it acts to attenuate the release of acetylcholine from vagal terminals in gallbladder ganglia (Mawe, 1993; Mawe, Gokin and Wells, 1994). Exogenously applied noradrenaline decreases the amplitude of fast EPSPs in a concentration-dependent manner by acting on α2-adrenoceptors. The action of noradrenaline is mimicked by the α2-adrenoceptor agonist, clonidine, and is antagonized by the α2-adrenoceptor antagonist, yohimbine. When endogenous catecholamines are released from sympathetic nerves by tyramine application or by electrical stimulation of the vascular plexus, a yohimbine-sensitive decrease in fast synaptic activity is observed. Since it has been demonstrated that the major source of fast synaptic input to gallbladder neurons is vagal preganglionic fibres, and that fast EPSPs elicited by cystic nerve stimulation are sensitive to noradrenaline (Mawe, Gokin and Wells, 1994), it is likely that vagal input to the gallbladder can be attenuated by sympathetic activity. Therefore, the decrease in gallbladder tone, that can be elicited by stimulation of the splanchnic nerves (Pallin and Skoglund, 1964; Persson, 1971, 1972, 1973; Yamasato and Nakayama, 1990), may be the result of a presynaptic inhibitory effect of sympathetic nerves on the vagal terminals in gallbladder ganglia. Yamasato and Nakayama (1990) reported that subthreshold stimulation of the celiac nerve in the dog, which had no effect on gallbladder motility, markedly decreased the responsiveness of the gallbladder to vagal stimulation. It is possible that this inhibitory influence of sympathetic input on the vagal tone facilitates gallbladder filling. Furthermore, administration of sympathomimetics for treatment of low blood pressure could have the side effect of decreasing gallbladder motility by decreasing vagal output to gallbladder ganglia, thus leading to gallbladder stasis. Extrinsic sensory fibres The concept that sensory neurons can release neuroactive compounds from their peripheral processes, and initiate local reflex activity, has now been established in several organ systems, including the gallbladder. In the gallbladder, nerve fibres that are immunoreactive for substance P (SP) and CGRP are abundant in the ganglionated and vascular plexuses (Goehler, Sternini and Brecha, 1988; Maggi et al., 1989; Mawe and Gershon,


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1989). Furthermore, application of capsaicin to gallbladder strips causes contraction and the release of SP and CGRP (Maggi et al., 1989). Since gallbladder neurons do not express CGRP-immunoreactivity (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1992, 1996), the SP/CGRP-immunoreactive nerve fibres must be extrinsic and are likely to arise from thoracic sensory ganglion cells which project to the gallbladder (Mawe and Gershon, 1989). Application of tachykinins, CGRP or capsaicin to guinea-pig gallbladder neurons causes a depolarization that is similar to the slow EPSP recorded in these preparations (Mawe, 1995; Gokin, Jennings and Mawe, 1996). Both tachykinins and CGRP depolarize gallbladder neurons by activating a non-selective cation conductance, and increase the excitability of these cells. The tachykinin-mediated depolarization of gallbladder neurons has been studied with receptor-specific agonists and antagonists (Mawe, 1995). Naturally occurring tachykinins depolarize gallbladder neurons with a rank order potency that is characteristic of neurokinin-3 (NK3) receptors (neurokinin B > neurokinin A > SP). Consistent with this finding, the NK3 receptor agonist, senktide, was more potent than any of the naturally occurring tachykinins in eliciting a depolarization. Once the involvement of NK3 receptors was established, [Trp7,β-Ala8 ]-NKA(4–10), which acts as an antagonist at NK3 receptors, was used in the preparation. This compound shifted the concentration-effect curve for SP to the right, and depressed both capsaicin-induced depolarizations and stimulus-evoked slow EPSPs. Therefore, sensory fibres are likely to contribute to slow EPSPs in gallbladder ganglia by releasing tachykinins, and probably CGRP, in an axon reflex response. It is possible that such a response may contribute to gallbladder inflammation since release of neuroactive peptides from extrinsic sensory fibres is one of the initial steps in toxin A-induced inflammation in the bowel (Mantyh et al., 1996). Hormonal cholecystokinin Cholecystokinin (CCK) has long been recognized for its ability to cause gallbladder contractions (Ivy and Oldberg, 1928). However, the issue of whether CCK elicits its physiological effect through a direct action on gallbladder smooth muscle or by facilitating the neural output to the organ has been debated. Over the past 10 years, studies from several different laboratories provide strong support for the concept that, physiologically, CCK acts through neural mechanisms to cause gallbladder emptying. Studies of in vivo preparations have provided support for the view that the principle physiological effect of CCK in the gallbladder involves a neural mechanism (Grossman, 1975; Behar and Biancani, 1980, 1987; Takahashi et al., 1982; Gullo et al., 1984; Fisher, Rock and Malmud, 1985; Marzio et al., 1985; Strah et al., 1985, 1986; Pozo, Salida and Madrid, 1989; Hanyu et al., 1990b; Takahashi, May and Owyang, 1991). These studies demonstrate that gallbladder contractions elicited by feeding, or by intravascular injections of physiological post-feeding concentrations of CCK, were disrupted by atropine, hexamethonium, TTX, or vagal blockade. Similar results have been reported in an in vitro preparation by Brotschi, Pattavino and Williams, (1990). Furthermore, when applied to gallbladder muscle strips, CCK causes the release of ACh (Yau and Youther, 1984; Yamamura et al., 1986; Rakovska, Milenov and Bocheva, 1989; Galligan and Bertrand,

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1994). Taken together, these data indicate that it is likely that feeding and CCK-induced physiological contraction of the gallbladder occurs through a neural mechanism rather than a direct action of CCK on smooth muscle. It is clear that CCK receptors do exist on gallbladder smooth muscle cells, but these receptors may not normally play a role in gallbladder emptying. It is plausible that the neural effect that was described was a result of actions of CCK on vagal afferent fibres and/or vagal terminals within gallbladder ganglia since hexamethonium and vagal blockade disrupt the meal- and CCK-induced gallbladder contraction (Hanyu et al., 1990b; Takahashi, May and Owyang, 1991). Electrophysiological studies of gallbladder whole mount preparations support this view. Direct studies of the actions of CCK in gallbladder ganglia have been conducted in the guinea-pig (Mawe, 1991; Mawe, Gokin and Wells, 1994) and in the opossum (Bauer et al., 1991) using intracellular electrophysiological recording techniques. In both of these species, CCK has a profound presynaptic facilitory effect on ganglionic transmission, but does not have a direct effect on the gallbladder neurons. Upon application of CCK, the amplitude of cholinergic fast EPSPs is increased, usually converting subthreshold EPSPs to suprathreshold EPSPs (Bauer et al., 1991; Mawe, 1991). CCK increases the quantal content three-fold without altering quantal size, but does not alter the sensitivity of these neurons to exogenously applied acetylcholine, indicating that CCK acts through a presynaptic mechanism (Mawe, 1991). Most importantly, it has been shown that CCK is quite potent in its ability to promote the release of acetylcholine. The concentration-effect relationship for CCK in gallbladder ganglia peaks at 1.0 nM, and has a half-maximal effective concentration (EC50) of 33 pM; the EC50 for the direct contractile effect of CCK on gallbladder muscle is 10 nM. In the presence of 10 pM CCK, which is within the range of postprandial serum levels of CCK (Takahashi, May and Owyang, 1991), the peptide increases synaptic currents by about 20%. The nerve terminals that are sensitive to CCK are from the vagus nerve since synaptic responses to cystic nerve stimulation are sensitive to CCK, and since these inputs are eliminated following vagotomy (Mawe, Gokin and Wells, 1994). CHEMICAL CODING OF GALLBLADDER NEURONS Despite the cumulative knowledge that has been gained from motility and ganglion electrophysiological studies, it is not entirely clear what compounds are actually released from gallbladder neurons to modulate smooth muscle function. It is recognized, from muscle strip studies involving electric field stimulation, that excitatory transmitters are released to promote gallbladder emptying, and it is possible that inhibitory transmitters are released to promote gallbladder filling. Two major theories have been proposed to explain how the gallbladder fills. One theory suggests that the gallbladder undergoes a “passive” filling between meals (Ryan, 1987; Shaffer, 1991), and the other theory suggests that the gallbladder actively expands to draw hepatic bile into its lumen, much as a bellows draws air in as it is expanded (Lanzini, Jazrawi and Northfield, 1987; Jazrawi et al., 1995). In order for the gallbladder to act as a bellows, a potent inhibitory output from the ganglia of the gallbladder would be necessary to induce an active relaxation. An important step in determining how gallbladder neurons could influence gallbladder function was to identify the neurochemical phenotypes of gallbladder neurons. We and


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others have studied the chemical coding of gallbladder neurons in several species, including guinea-pig, human, dog and opossum, and have identified sets of neuroactive compounds that are co-expressed. Characteristic trends, as well as interspecies differences, have emerged from these investigations. For example, all gallbladder neurons are likely to be cholinergic since all detectable neurons express immunoreactivity for the essential biosynthetic enzyme for acetylcholine, choline acetyltransferase (Talmage et al., 1996). Furthermore, gallbladder neurons can express immunoreactivities for tachykinins, vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS) and either neuropeptide Y (NPY) (guinea-pig and human) or galanin (dog and opossum) (Keast, Furness and Costa, 1985; Talmage et al., 1992, 1996; De Giorgio et al., 1995). Species specific patterns of neuropeptide co-expression have been identified in neurons of the human, canine and guinea-pig gallbladder (Talmage et al., 1992, 1996; De Giorgio et al., 1995). These are schematically represented in Figure 5.2. In the human, the vast majority of neurons express VIP and NPY, and most of these neurons express SP as well (De Giorgio et al., 1995; Talmage et al., 1996). In the dog, where NPY appears to be replaced by galanin, most neurons express VIP and galanin, and most of these express SP as well (Talmage et al., 1996). In the guinea-pig, most neurons express SP and NPY, and a distinct subpopulation of neurons expresses VIP (Talmage et al., 1992). It is clear that gallbladder ganglia comprise neurons that express the enzymatic machinery to synthesize nitric oxide. In all species that have been studied, including the human (Talmage and Mawe, 1993; De Giorgio et al., 1995), monkey (De Giorgio et al., 1994), dog (Talmage and Mawe, 1993), opossum (Talmage and Mawe, 1993), guinea-pig (Talmage and Mawe, 1993; Grozdanovic et al., 1994; Siou, Belai and Burnstock, 1994), gerbil (Talmage and Mawe, 1993) and mouse (Grozdanovic, Baumgarten and Bruning, 1992), neurons in the gallbladder express NOS and/or NADPH-diaphorase (NADPH-DA) activity. In guinea-pig there is a direct correlation between NOS-immunoreactivity and NADPH-DA activity in gallbladder ganglia (Mawe et al., 1997). As mentioned above, all gallbladder neurons express ChAT; coexpression of ChAT and NOS is also observed in these ganglia (Figure 5.3). The physiological role of neurally released nitric oxide in the gallbladder, if there is one, is unclear at this time. However, nitric oxide can relax the gallbladder (Mourelle et al., 1993a; McKirdy, McKirdy and Johnson, 1994) and may modulate the CGRP-induced relaxation of the gallbladder (Kline and Pang, 1994).

Figure 5.2 Chemical coding in neurons in ganglia of the human, dog and guinea-pig gallbladder. Note the coexpression of neuroactive compounds that have excitatory (+) and inhibitory (–) effects on gallbladder smooth muscle. ACh, acetylcholine; VIP, vasoactive intestinal peptide; NPY, neuropeptide Y; SP, substance P; NOS, nitric oxide synthase. (Modified from Talmage et al., 1996).

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Figure 5.3 Immunoreactivities for ChAT and NOS in ganglia of the guinea-pig gallbladder and sphincter of Oddi. Immunoreactivities in these double stained preparations illustrate the co-expression of ChAT and NOS in a subset of gallbladder neurons, and the exclusive expression of ChAT or NOS by sphincter of Oddi neurons. Note that in the micrographs of the SO ganglion, the NOS-immunoreactive neurons are located in the spaces that are devoid of ChAT immunoreactivity.

Unlike the ganglia of the bowel, ganglia of the gallbladder lack CGRP-immunoreactive neurons. Immunohistochemical studies in the human (De Giorgio et al., 1995; Talmage et al., 1996), dog (Talmage et al., 1996), pig (Sand, Tainio and Nordback, 1993), guineapig (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1992), Australian possum (Padbury, 1990), Monodelphis domesticus opossum (Talmage et al., 1996), and toad (Davies and Campbell, 1994) failed to identify neurons in the gallbladder wall that expressed immunoreactivity for CGRP. However, CGRP-immunoreactive nerve fibres have been described in the gallbladders of all of these species, where they are most abundant in the ganglionated plexus and in association with blood vessels, primarily in the paravascular plexus. In the human, dog, opossum, Australian possum, guinea-pig, and toad gallbladders these fibres have been shown to co-express SP-immunoreactivity, and probably originate in sensory ganglia (Goehler, Sternini and Brecha, 1988; Mawe and Gershon, 1989; Talmage et al., 1996). It is quite possible that CGRP can be released from these fibres as part of an axon reflex circuit (see section on Extrinsic sensory fibres of gallbladder ganglia), and exert influences on neurons, muscle and/or epithelial cells. CGRP causes a relaxation of gallbladder muscle (Kline and Pang, 1992) that is due to an activation of an ATP-dependent K+ conductance (Zhang et al., 1994). As described above, in guinea-pig gallbladder ganglia, CGRP causes a depolarization of neurons that is quite similar to the slow excitatory postsynaptic potential that occurs in these ganglia (Gokin, Jennings and Mawe, 1996).


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Together with the knowledge that all of the gallbladder neurons are cholinergic, these patterns of expression of neuroactive compounds are somewhat perplexing; as they suggest the coexpression of excitatory and inhibitory transmitters in single neurons of all of these species. In muscle strip assays, acetylcholine (Ryan, 1987), SP (Meldrum, Bojarski and Calam, 1987; Dahlstrand et al., 1988; Guo et al., 1989; Maggi et al., 1989) and NPY (Lillemoe, Webb and Pitt, 1988) have been shown to increase gallbladder tone, whereas VIP (Ryan and Cohen, 1977; Ryan and Ryave, 1978; Dahlstrand, Dahlström and Ahlman, 1989) can relax the precontracted gallbladder. The ostensibly opposing outputs from individual gallbladder neurons that express excitatory and inhibitory compounds could be explained by the following theoretical scenarios: (1) excitatory and inhibitory neuroactive compounds are separately released from a given neuron in response to distinct inputs; (2) compounds with opposing actions are released onto the same target sequentially, with one acting as a physiological antagonist of the other; or (3) both sets of compounds are co-released, but act on different targets. For example, if acetylcholine, SP and VIP were released together, acetylcholine and SP may act on the muscle to elicit a contraction, while VIP may act on epithelial cells. Neuroactive compounds may also act on adjacent nerve terminals to modulate further release. Further studies will be required to test these models.

SPHINCTER OF ODDI GANGLIA To date, many studies that have involved the direct study of SO ganglia have involved the guinea-pig model. Therefore, most of the following information is related to ganglia of the guinea-pig SO region. This is important to note because considerable interspecies variation exists with regard to the structure and function of the SO (see Mawe, 1992). In the guinea-pig, this area includes the terminal portion of the common bile duct (choledochal sphincter), an ampulla, and a terminal papilla (Dahlstrand et al., 1989; Hirose and Ito, 1991; Mawe, 1992). The studies that are reviewed here relate to neurons located throughout the anatomically defined regions of the ampulla and the choledochal sphincter. No regional differences have been observed in the electrical or morphological characteristics of neurons in these areas. STRUCTURAL AND ELECTRICAL PROPERTIES OF SPHINCTER OF ODDI NEURONS Morphological properties of sphincter of Oddi ganglia and neurons The ganglionated plexus of the guinea-pig SO includes a network of ganglia and interganglionic fibre bundles that is similar in appearance to the myenteric plexus of the small intestine. When stretched and pinned out flat, the SO region encompasses an area of approximately 20–25 mm2 containing an average of about 60 ganglia, and a total of about 2000 neurons (Wells and Mawe, 1993). The structure of individual SO neurons has been determined by injecting neurobiotin into individual neurons, from intracellular recording electrodes, and labelling the neurobiotin

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with fluorophore- or horseradish peroxidase-conjugated avidin (Wells and Mawe, 1993). Guinea-pig SO neurons conform to the morphological classification scheme described by Dogiel for myenteric neurons (see Furness and Costa, 1987). The neurons of the SO that were classified as both tonic and phasic cells (see section on Electrophysiological properties of gallbladder neurons), which together represent the majority of SO neurons, have the shape of Dogiel type I cells. These neurons typically have a single long process and several very short processes emanating from the soma. In most cells, the short processes were flattened and “club-shaped”, and the remainder of the neurons had several thin processes that were approximately the length of the somal diameter. The long process of most cells, thought to be the axon, usually projects into adjacent ganglia. These processes frequently end as expansion bulbs, indicating that they may have been severed during the dissection. Processes of some cells were followed to strands of circular muscle that had not been removed during the tissue preparation. A small proportion of the neurobiotin-filled SO neurons, which are classified electrophysiologically as AH cells, are shaped like Dogiel type II cells. These neurons have several long branched processes that terminated both within the ganglionated plexus as well as within the muscularis. Calbindin-immunoreactive neurons in SO ganglia have a shape similar to that of the AH cells. Electrical properties of sphincter of Oddi neurons Unlike the guinea-pig gallbladder, where only one electrically classified type of neuron exists, three classes of neurons have been identified in the guinea-pig SO, on the basis of their active and passive electrical properties as determined by intracellular voltage recordings (Table 5.1, Figure 5.4; Wells and Mawe, 1993). The two most prevalent cell types, which together comprise about 95% of the total neuronal population, are called tonic and phasic cells. Tonic cells are very excitable; they generate spontaneous action potentials and elicit action potentials continuously throughout a prolonged suprathreshold depolarizing current pulse. The tonic cells of the SO are comparable to the S/type 1 cells of the small intestine (Wood, 1989) and rectum (Tamura and Wood, 1989), and the gastric Type I (Schemann and Wood, 1989) and colonic type 1 cells (Wade and Wood, 1988). Phasic cells have passive membrane characteristics that are similar to tonic cells, but differ in their active properties. Phasic cells do not exhibit spontaneous activity, and when directly

Figure 5.4 Typical responses of sphincter of Oddi tonic and phasic cells to a prolonged depolarizing current pulse. Tonic cells, which often exhibit spontaneous activity, generate action potentials throughout the duration of a depolarizing current pulse. Phasic cells, which are not spontaneously active, generate action potentials only at the onset of the current pulse, regardless of its amplitude or duration.


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stimulated, they adapt rapidly, typically firing only 1–3 action potentials at the onset of a prolonged depolarizing current pulse. This type of neuron has not been reported in the duodenum or small intestine; however neurons with similar properties have been described in the stomach (gastric type II; Schemann and Wood, 1989; Tack and Wood, 1992) and the distal colon (colonic type 4; Wade and Wood, 1988). The remainder of SO neurons, comprising about 5% of the population, are called AH cells. These cells have properties identical to those of the Type 2/AH cells of the bowel (Wood, 1989). Action potentials in these cells, which are TTX-insensitive, have a broadened shoulder during the repolarization, and the AHP of these cells lasts several seconds. Synaptic inputs to sphincter of Oddi neurons When interganglionic fibre bundles are stimulated, three different types of synaptic potentials can be elicited: fast EPSPs, slow EPSPs and inhibitory postsynaptic potentials (IPSPs). The fast excitatory input, which can be activated by low frequency stimulation, is present in most tonic and phasic cells, and some AH cells. These events are reversibly eliminated when the tissue is bathed in a Ca2+-free/high Mg2+ Krebs solution, and they are sensitive to hexamethonium. Potential sources of fast synaptic input to SO neurons include other SO neurons, neurons of the duodenum, and vagal preganglionic fibres. In some SO neurons, membrane depolarizations and hyperpolarizations can be activated by trains of high-frequency (10–20 Hz, 0.5 ms pulse) fibre tract stimulation (Wells and Mawe, 1993). These responses are eliminated by exposure to Ca2+-free Krebs, and therefore are presumed to be slow EPSPs and IPSPs. The sources and ionic mechanisms of slow excitatory inputs have not been evaluated, but SO neurons could receive modulatory input from other SO neurons, duodenal neurons, and/or extrinsic sensory fibres. Inhibitory synaptic inputs to SO neurons probably involves the activation of a K+ conductance, and these inputs are provided by sympathetic postganglionic input from the coeliac ganglion (see below). REGULATORY INPUTS TO SPHINCTER OF ODDI GANGLIA As indicated above, not as much is known about the neural control of the SO relative to other regions of the gut, including the gallbladder. Of the potential physiological modulators of SO ganglion function, two that have been studied directly in SO ganglia are sympathetic postganglionic inputs and CCK. Sympathetic inputs to sphincter of Oddi ganglia The action of sympathetic input to this region appears to increase SO tone. Activation of the sympathetic input to the feline SO, in vivo, by stimulating the splanchnic nerves or postganglionic nerve bundles, increases the tone of the sphincter musculature (Persson, 1972, 1973), whereas in non-sphincter regions of the gut, sympathetic input results in decreased motility (see Furness and Costa, 1987). Although the muscularis of the SO does contain catecholamine-histofluorescent nerve fibres, the area of most dense catecholaminergic innervation lies within the intrinsic

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ganglionated neuronal plexus (Baumgarten and Lange, 1969; Mori, Azuma and Fujiwara, 1971; Cai and Gabella, 1983b). In the ganglionated plexus of the SO, immunoreactivities for tyrosine hydroxylase and dopamine β-hydroxylase are abundant, and coexistent in nerve fibres, but these synthetic enzymes are not expressed by SO neurons (Wells and Mawe, 1994). The actions of noradrenaline and endogenous catecholamine release have been studied in the guinea-pig SO (Wells and Mawe, 1994). Exogenous noradrenaline reversibly decreases the amplitude of fast EPSPs evoked by stimulation of interganglionic fibre tracts. Since noradrenaline does not alter the responsiveness of the neurons to acetylcholine, the action of noradrenaline in SO ganglia is thought to be presynaptic. The α2-adrenoceptor agonist, UK14304, mimics the noradrenaline-induced effect, and this effect is attenuated by the α2-adrenoceptor antagonist, Idazoxan. Agonists of α1- and β-adrenoceptor have no detectable effect in SO ganglia. Results of experiments involving application of exogenous noradrenaline are supported by the finding that release of endogenous catecholamines, by tyramine, causes an idazoxan-sensitive decrease in the fast EPSP. In SO neurons that exhibit IPSPs, noradrenaline causes hyperpolarization of the membrane potential. The IPSP and the noradrenaline-induced hyperpolarization have reversal potentials near the K+ equilibrium potential, and are inhibited by α2-adrenoceptor antagonists. Further evidence for the concept that sympathetic nerves mediate IPSPs is that desipramine, a catecholamine reuptake inhibitor, reversibly increases the amplitude of the IPSP. The findings from morphological and electrophysiological studies indicate that sympathetic nerves target SO ganglia, and that within these ganglia, noradrenaline can act both pre- and postsynaptically as an inhibitory neurotransmitter. Therefore, ganglia of the SO can be added to the growing list of enteric and parasympathetic ganglia that receive interganglionic input from sympathetic postganglionic nerves. Although noradrenaline has actions in SO ganglia that are similar to actions in other gastrointestinal ganglia, the net effect of sympathetic input to the sphincter is contraction rather than relaxation. The activation of divergent net effects through common mechanisms supports the view that precise circuitry exists within ganglia in various regions of the gut. Sympathetic pre- and postsynaptic activity is likely to have the net effect of decreasing the release of inhibitory substances and/or increasing the release of excitatory neuroactive compounds. Since the effects of noradrenaline are inhibitory it is likely that it decreases the release of inhibitory compounds. It is not clear whether the IPSPs occur selectively on inhibitory neurons of the SO, but this type of circuitry could contribute to the increased SO tone that results from sympathetic nerve stimulation. Actions of CCK on sphincter of Oddi neurons At the level of the SO, the stimulatory effect of CCK on bile flow appears to involve a neural mechanism. Several studies, in various species, have shown that the actions of CCK on the SO can be attenuated by muscarinic blockade with atropine and/or complete neural blockade with TTX (Behar and Biancani, 1987; Vogalis, Bywater and Taylor, 1989; Hanyu et al., 1990a). Furthermore, CCK-induced release of neuroactive compounds such as acetylcholine (Harada, Katsuragi and Furukawa, 1986), VIP (Wiley, O’Dorisio


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and Owyang, 1988; Dahlstrand et al., 1990) and nitric oxide (Pauletzki et al., 1993) have been reported. The actions of CCK on guinea-pig SO neurons have been investigated by application of CCK while recording from SO neurons with intracellular recording electrodes (Gokin, Hillsley and Mawe, 1997). CCK has a direct, excitatory effect on tonic and phasic cells in SO ganglia. The depolarization that is elicited by CCK likely involves the activation of a non-selective cation conductance since it reverses near 0 mV, and it is significantly reduced in a low Na+ solution. The CCK-induced depolarization of tonic and phasic cells involves the CCK-A receptor subtype; it is significantly reduced by the CCK-A antagonist, PD 140,548 N-methyl-D-glucamine, but not by the CCK-B receptor antagonist, PD 135,158 N-methyl-D-glucamine. Although SO neurons express CCK receptors, and CCK can act on these receptors to depolarize SO neurons, it is disputable whether hormonal CCK could act physiologically at this site to alter SO tone. This is because of the discrepancy between the sensitivity of SO neurons for CCK and the peak concentrations of CCK in the serum following a meal. The lowest concentration of CCK that results in a detectable response in SO neurons is 1 nM, and the EC50 in these neurons is 10 nM. Since peak serum concentrations of CCK following a meal are in the low picomolar range, hormonal CCK would be delivered at subthreshold concentrations. It is possible that CCK could have a paracrine effect on SO neurons due to the close proximity of the SO to the duodenum, where CCK is released. Alternatively, CCK may alter SO tone through vagal and/or duodenum-SO neural circuits (see section NEURAL INTERACTION BETWEEN THE GUT AND THE SPHINCTER OF ODDI). CHEMICAL CODING OF SPHINCTER OF ODDI NEURONS To identify potential chemical coding in sphincter of Oddi neurons, immunohistochemistry and histochemistry have been employed to test for the presence of putative neurotransmitters and related synthetic enzymes. In the guinea-pig SO, the pattern of chemical coding is less ambiguous than that found in the gallbladder; there is a distinction between the expression of excitatory and inhibitory compounds in SO neurons (Figure 5.3). In the guinea-pig SO, about 70% of the neurons are immunoreactive for ChAT, indicating that they are likely to synthesize acetylcholine and most of these neurons are also immunoreactive for SP and enkephalin (Wells, Talmage and Mawe, 1995; Talmage et al., 1997). Since acetylcholine, SP and opiates increase SO tone (Azuma and Fujiwara, 1973; Kudoh et al., 1981, Behar and Biancani, 1984; Dahlstrand et al., 1985, 1988), and since nerve fibres that are immunoreactive for SP and ChAT are abundant in the muscularis of the SO (Talmage et al., 1997), it is likely that the SP/enkephalin/ChAT-immunoreactive population includes excitatory motor neurons. A distinct population of guinea-pig SO neurons express NOS, and some of the NOS-positive neurons express VIP, whereas others express NPY. The NOS/VIP and NOS/NPY neurons are likely to be inhibitory neurons because both nitric oxide (Allescher et al., 1992; Kaufman et al., 1993; Mourelle et al., 1993b; Pauletzki et al., 1993) and VIP (Behar and Biancani, 1980; Wiley, O’Dorisio and Owyang, 1988; Dahlstrand et al., 1990) have been shown to decrease SO tone. To determine whether a correlation exists between the electrical and chemical coding properties of SO neurons, we conducted a study combining intracellular recording and dye

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injection, along with chemical coding. Results of this study clearly demonstrated that there is no relationship between the electrical properties of SO neurons (Tonic versus Phasic) and their chemical coding patterns (cholinergic versus nitrergic) (Hillsley and Mawe, 1998).

REFLEX INTERACTIONS BETWEEN ENTERIC AND BILIARY NEURONS Since the beginning of the twentieth century, when Langman, Bayliss and Starling, and Trendelenberg were evaluating the neural activity of the bowel, it has been recognized that peristaltic reflex responses could be observed in the absence of the central nervous system. Evidence also exists for reflex interactions between the duodenum and the gallbladder and sphincter of Oddi, and between the gallbladder and sphincter of Oddi. Although these gutSO-gallbladder interconnections have not been evaluated as extensively as the intrinsic circuits of the gut, it is clear that the circuitry exists for reflex interactions between duodenal and biliary tract neurons.

NEURAL INTERACTIONS BETWEEN THE GUT AND THE GALLBLADDER In 1933, DuBois and Kistler found that stimulation of the duodenal ampulla causes the gallbladder to contract. This contractile response to ampulla stimulation was eliminated by transection of the bile duct, but gallbladder responses to vagal stimulation and stimulation of the cut end of the bile duct persisted. On the basis of these findings, DuBois and Kistler proposed that a direct neural connection exists between the gut and the gallbladder, and that these axons pass along the cystic duct. Morphological evidence from retrograde labelling experiments in the guinea-pig and Australian possum, show that the gallbladder receives extrinsic neural projections from several sources, and support the concept of a direct gut-gallbladder projection (Mawe and Gershon, 1989; Padbury et al., 1993). Sources of input to the gallbladder include the dorsal motor nucleus of the vagus nerve, nodose ganglia, coeliac ganglia, and thoracic dorsal root ganglia. In addition, when retrograde tracers were injected into the wall of the gallbladder, neurons of the duodenal myenteric plexus and the ganglia of the SO were labelled. These data indicate that, in addition to being regulated in conventional reflex responses that involve central nervous system processing, the circuitry exists for the gallbladder to receive direct inputs from the bowel. The evidence described above indicates that gut neurons project to the gallbladder where they exert an excitatory influence. However, the physiological relevance, if there is any, of such a pathway, and the precise origins and targets of the gut-gallbladder projection have not been resolved. According to the DuBois and Kistler study, activation of this circuit has an excitatory influence on gallbladder motor activity; however it is not known whether the target of this input is on gallbladder ganglia, on the smooth muscle cells, or at both of these locations. When retrograde tracers were injected into the gallbladder they could have been taken up by axon terminals in any layer of the organ.


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It is doubtful that the gut–gallbladder projection contributes to the fast EPSPs that have been studied in gallbladder ganglia. Following vagotomy, nicotinic input to gallbladder neurons, in response to stimulation of the nerve bundles that pass along the cystic duct, are completely eliminated (Mawe, Gokin and Wells, 1994). However, if this pathway is similar to the projection from enteric neurons to the pancreas (Kirchgessner and Pintar, 1991), there may be a serotonergic projection from the gut to the ganglia of the gallbladder. Nerve fibres that are immunoreactive for serotonin do exist in the ganglia of the gallbladder (Mawe and Gershon, 1989), and when serotonin is applied to gallbladder neurons, it causes a prolonged depolarization that is associated with an increase in the excitability of gallbladder neurons (Mawe, 1990). One potential physiological role of an excitatory neural pathway from the gut to the gallbladder would be to activate the gallbladder contractions that occur in coordination with the migrating myoelectric complex. During the interdigestive period, the gallbladder undergoes periods of increased intraluminal pressure, in phase with the migrating myoelectric complex, that are accompanied by a delivery of bile from the gallbladder to the lumen of the duodenum (Itoh et al., 1983; Ryan, 1987). It is thought that the migrating myoelectric complex serves a “housekeeping function”. According to this model, increased motor activity would advance undigested food from the proximal bowel toward the large intestines, and the associated delivery of bile into the intestinal lumen would facilitate the overall digestive process. NEURAL INTERACTIONS BETWEEN THE GUT AND THE SPHINCTER OF ODDI Duodenal distension and electrical field stimulation causes changes in SO tone in the dog, cat, and Australian possum (Wyatt, 1967; Thune, Jivegård and Svanvik, 1989; Saccone et al., 1994). In the possum, these responses are abolished both by crushing the duodenum between the site of stimulation and the SO and by TTX, but not by vagotomy. This indicates that neural projections from duodenal neurons to the SO are responsible for this response. Also in the Australian possum, Padbury and colleagues (1993) have demonstrated that injections of retrograde tracers into the SO resulted in retrograde labelling of duodenal myenteric neurons. In the guinea-pig, SO ganglia are richly innervated by calbindin-immunoreactive nerve fibres, but these ganglia contain few, if any, calbindin-immunoreactive neurons (Wells and Mawe, 1993). Since a high proportion of neurons in the nearby myenteric ganglia of the duodenum are calbindin-positive, the duodenum-myenteric projection neurons represent a likely source of calbindin-positive fibres in SO ganglia. This suggestion is supported by findings from studies demonstrating that the calbindin-immunoreactive neurons project from the myenteric plexus of the duodenum to SO ganglia (Kennedy and Mawe, 1998). Evidence from retrograde labelling studies validates the existence of a neural projection between the duodenum and the SO in the guinea-pig and Australian possum. Injection of DiI into the wall of the SO, in the Australian possum, in vivo, results in retrograde labelling of neurons in the duodenal myenteric plexus (Padbury et al., 1993). In vitro retrograde label studies in the guinea-pig have demonstrated that specific subpopulations of guineapig duodenal myenteric neurons project to the ganglionated plexus of the SO in guinea-pig (Kennedy and Mawe, 1998). An average of 110 neurons project from the duodenum to the

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SO in the guinea-pig, and these neurons express ChAT-, but not NOS- or calretininimmunoreactivity. Importantly, many (~20%) of these projection neurons are calbindinpositive. This is a critical detail because calbindin is a marker for intrinsic primary afferent neurons in the guinea-pig enteric nervous system, and calbindin-positive neurons in the myenteric plexus send projections to the mucosa. The duodenum-SO projection neurons are depolarized by CCK, indicating that they are capable of responding to CCK released from the mucosa. Because calbindin-IR myenteric neurons send a projection to the mucosa, and postprandial release of CCK also occurs in the mucosa, it is possible that the duodenum-SO neural circuit is activated by postprandial release of mucosal CCK. To examine the nature of the synaptic signals received by SO neurons from neurons in the duodenum, membrane potential recordings were obtained from SO neurons with intracellular microelectrodes while duodenal nerve bundles and villi were stimulated (Kennedy and Mawe, 1999). Focal stimulation of interganglionic fibre bundles in the myenteric plexus of the duodenum, or duodenal mucosal villi, elicits nicotinic fast synaptic potentials in SO neurons. In these studies, antidromic action potentials were also detected in SO neurons indicating that SO neurons project to the duodenum. This reciprocal projection was verified morphologically by retrograde labelling studies demonstrating that SO neurons are labelled when DiI is applied to the duodenal myenteric plexus, in vitro. Thus, it is clear that duodenal neurons provide input to SO neurons and, at least in the guinea-pig, this input is in the form of nicotinic fast EPSPs through a reflex that can be activated at the duodenal mucosa. This circuit could play a role in any or all of the SOs functions. For example, it is possible that the neurons that project to the SO are activated, directly or through other neurons, by the release of CCK. According to this model, CCKinduced changes in SO tone would involve a local neural circuit rather than a direct action of hormonal CCK on SO neurons. This is consistent with the finding, described above, that SO neurons are not sensitive to the levels of CCK that exist in the serum. Another possible role of the duodenum-SO circuit could be to cause contraction of the SO in coordination with contractions of the duodenum. This would help prevent the reflux of lumenal contents into the biliary and pancreatic ducts. Finally, the gut-SO circuit could be involved in coordinating the relaxation of the SO in concert with the gallbladder contractions and delivery of bile that occur in phase with the migrating myoelectric complex. NEURAL INTERACTIONS BETWEEN THE GALLBLADDER AND THE SPHINCTER OF ODDI In addition to reflex interactions between the gut and the biliary tract, there exists a neural reflex circuit that links the gallbladder to the SO. In dogs, cats and humans, distension or electrical stimulation of the gallbladder results in a decreased motility, or flow resistance, in the SO (Wyatt, 1967; Thune, Thorness and Svanvik, 1986; Thune, Jivegård and Svanvik, 1989; Thune et al., 1991). In the cat, this response was eliminated by TTX or application of local anaesthetics to the bile ducts (Thune, Thorness and Svanvik, 1986). The mechanisms for the reflex between the gallbladder and the SO are unknown. Authors of these reports have suggested that the reflex may involve a direct neural link between the gallbladder and the SO; however, these studies have not ruled out the possibility of a vagal reflex mechanism.


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CONCLUDING REMARKS The neurons of the gallbladder have characteristics that are more similar to parasympathetic neurons than those of enteric neurons. To release their neuroactive compounds onto the effector tissues of the organ, gallbladder neurons must be stimulated, and the major source of excitatory input to these cells is vagal preganglionic fibres. Modulatory inputs that can up- or down-regulate the efficacy of this nicotinic ganglionic transmission include CCK and noradrenaline, which have presynaptic excitatory and inhibitory effects on vagal terminals, respectively. Furthermore, sensory fibres can release SP and CGRP in gallbladder ganglia to depolarize and increase the excitability of gallbladder neurons. The concept of how gallbladder neurons, which express acetylcholine plus an array of excitatory and inhibitory neuromodulators, conduct unambiguous signals to the smooth muscle and epithelial cells of the gallbladder needs to be resolved in future studies. The neurons of the SO represent a more heterogeneous population than those of the gallbladder. Electrically, SO ganglia comprise excitable neurons with spontaneous activity, less excitable neurons that do not exhibit spontaneous activity, and a small contingent of AH cells. Neurochemically, SO ganglia include neurons that appear to be either excitatory or inhibitory; the excitatory neurons express ChAT, SP and enkephalin, and the inhibitory neurons express NOS and VIP or NPY. Although the extrinsic inputs to SO ganglia have not been extensively studied as of yet, mounting evidence suggests that significant regulatory inputs to SO ganglia originate in the myenteric plexus of the duodenum. Future studies will focus on the neurochemical mediators of duodenal-SO interactions and the physiological activators of this pathway.

ACKNOWLEDGEMENTS The studies performed in the Mawe laboratory have been supported by NIH grants DK 45410 and NS 26995. We thank the alumni and active members of the Green Mountain Gallbag Company, including Wendy Pouliot, Ellen Cornbrooks, David Wells, Lei Zhang and Alex Gokin for their contributions to many of the studies that have been described here.

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6 Pharmacology of the Enteric Nervous System Marcello Tonini, Fabrizio De Ponti, Gianmario Frigo and Francesca Crema Department of Internal Medicine and Therapeutics, Section of Clinical and Experimental Pharmacology, University of Pavia, Piazza Botta 10, I-27100 Pavia, Italy The intestine is capable of complex behaviours which allow mixing, propulsion, digestion and absorption of food. The mechanism whereby the intrinsic neurons of the enteric nervous system and the circular smooth muscle coordinate their activities to produce propagated contractions have been the subject of intensive investigations for almost a century. Since the intestine contains a large number of neuroactive substances and an equally large number of receptors for a variety of chemicals, preparations of longitudinal muscle with attached myenteric plexus have been widely used by pharmacologists and therefore have provided detailed information about the effect of drugs on motor neurons of the gastrointestinal tract. The complexity of the chemical coding of enteric neurons mirrors the complexity of the receptors involved in neurogenic intestinal functions. This chapter is intended to review the findings that associate any pharmacologically induced change in enteric nerve activity with the appropriate receptor type or subtype recognized by means of selective agonists and/or antagonists. KEY WORDS: enteric nervous system; chemical coding; cholinergic transmission; adrenergic transmission; NANC transmission; serotonergic transmission; tachykinins.

INTRODUCTION An explosive growth has recently characterized the pharmacology of the enteric nervous system, thanks to the improved anatomical, electrophysiological and functional knowledge of the various classes of neurons intrinsic to the intestinal wall and of their chemical coding (Costa and Brookes, 1994; Furness et al., 1994; Costa et al., 1996). This gave impetus to the search for highly selective agonists and antagonists for the pharmacological characterization of the receptor subtypes involved in fast and slow transmission between neurons, in the excitatory and inhibitory transmission to smooth muscle cells, and in the modulation of transmitter release by presynaptic or prejunctional mechanisms (Starke, Göthert and Kilbinger, 1989; Fuder and Muscholl, 1995). 213

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In spite of the fact that enteric neural pathways are involved in the regulation of several important functions, such as absorption/secretion, maintenance of vascular tone and motility, those involved in the regulation of gastrointestinal motility (i.e. the myenteric plexus) have been investigated much more extensively. This led to an outstanding development of the pharmacology of receptors mediating the effects of neurotransmitters on intestinal motility, which will be one of the main topics of the present chapter. Myenteric neurons can be divided into several functional classes that include sensory neurons, ascending and descending interneurons, ascending and descending (both short and long) motor neurons to the circular muscle, and excitatory motor neurons to the longitudinal muscle coat (Costa et al., 1996). All these neurons contain a wide array of neuroactive substances, which can be co-stored in and co-released from the same neuron upon appropriate stimulation. They include acetylcholine (ACh), tachykinins and opioid peptides (usually enkephalins) in the ascending pathways, and ACh, serotonin (5-hydroxytryptamine: 5-HT), somatostatin, vasoactive intestinal peptide (VIP), nitric oxide (NO) and dynorphin in descending interneurons, while descending motor neurons may contain VIP, NO, pituitary adenylate cyclase-activating peptide, gastrin-releasing peptide, dynorphin/ enkephalin and, as a result of pharmacological evidence, ATP. Some other substances are present in extrinsic pathways innervating the intestine (e.g. noradrenaline) and in mucosal endocrine cells (e.g. 5-HT) and mast cells. The complexity of the chemical coding of enteric neurons mirrors the complexity of the receptors (in terms of their subtypes, distribution, density, ionic and transduction mechanisms) involved in neurogenic intestinal functions. The presence of some of these receptors in the gut has been documented only from a pharmacological point of view and their functional significance is debated. In fact, it is a general rule that, for any given endogenous substance, a number of receptor subtypes exists even in the same tissue, leading either to similar or contrasting effects. Furthermore, it is now well documented that disease states can lead to alterations in the level of expression of a given receptor subtype. This concept of inducible receptors (Donaldson, Hanley and Villablanca, 1997) can of course profoundly affect drug action in vivo. In some cases, compounds behaving as antagonists on receptors activated by endogenous ligands offer a widely used pharmacological tool to assess the contribution of a given neurotransmitter in mediating a biological response. Indeed, if a given receptor is under tonic control by an endogenous transmitter, administration of an antagonist will elicit effects that are opposite to those of the endogenous ligand. This is based on the assumption that antagonists, according to the classical definition, lack efficacy. However, evidence has recently begun to accrue that, in the case of G protein-coupled receptors, some antagonists not only bind to the receptor, but also induce a conformational change that favours uncoupling of the receptor from its G protein (Schütz and Freissmuth, 1992; Kenakin, 1994). This implies that, in some in vitro systems with constitutive receptor activity, antagonists may show a property termed negative efficacy (or inverse agonism). Antagonists may thus be classified into compounds with no intrinsic activity and those with negative intrinsic activity (i.e. those reducing constitutive receptor activity, such as timolol or ICI 118551; Chidiac et al., 1994; Samama et al., 1994). These considerations cast some doubts on data obtained with receptor antagonists simply with the assumption that they prevent agonists binding. However, it should be noted that, while constitutive

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activity of the unliganded receptor is a well-established phenomenon in reconstituted systems with purified components, its physiological relevance is still unknown (Schütz and Freissmuth, 1992; Milligan, Bond and Lee, 1995) and, at the present state of knowledge, the effect observed with antagonists in vivo can be tentatively ascribed to antagonism of the endogenous ligand, unless evidence for inverse agonism is obtained. With these basic concepts in mind, this chapter is mainly intended to review those findings that associate any pharmacologically induced change in enteric nerve activity with the appropriate receptor type or subtype recognized by means of selective agonists and/or antagonists. Because of the complexity of the subject, this chapter should have had the size of a book. We hope the reader will forgive us if substantial information had to be neglected due to space limitations. Whenever possible, reference to extensive reviews on the subject has been provided.

PHARMACOLOGY OF CHOLINERGIC TRANSMISSION MUSCARINIC RECEPTORS Acetylcholine is the major transmitter in the enteric nervous system (Furness and Costa, 1987). Inhibition of muscarinic receptors by hyoscine was found to inhibit peristalsis (Tonini et al., 1981), although a weaker hyoscine- or atropine-resistant peristalsis may develop over time (Tonini et al., 1981; Barthó et al., 1982b; Schwörer and Kilbinger, 1988). One of the most popular intestinal preparations used to demonstrate ACh release from cholinergic nerve endings is the longitudinal muscle with attached myenteric plexus (LM-MP) from the guinea-pig small intestine (Paton and Zar, 1968). ACh is released from unstimulated LM-MP preparations and in greater amounts in response to electrical field stimulation. The postjunctional target of neuronally released ACh is a muscarinic receptor associated with membrane phosphoinositide breakdown, which causes the effector cell to contract (Eglen et al., 1994). Recently, this receptor has been recognized as the M3 receptor subtype using the M3-selective antagonist hexahydrosiladifenidol (HHSiD) in both the longitudinal (Giraldo et al., 1988) and circular muscle (Dietrich and Kilbinger, 1995). The prejunctional target of neuronally released ACh, as well as of applied muscarinic agonists, are inhibitory muscarinic autoreceptors, which depress, through a negative feed-back mechanism, the release of ACh itself. Indeed, muscarinic receptor antagonists were found to increase the electrically stimulated ACh release (Starke, Göthert and Kilbinger, 1989; Vizi et al., 1989). The evidence that muscarinic autoreceptors were antagonized with low affinity by the M1-selective antagonist pirenzepine (Kilbinger et al., 1984) and the M2-selective antagonist AF-DX 116 (Dammann et al., 1989) and with high affinity by the M3-selective antagonists HHSiD (Fuder, Kilbinger and Müller, 1985) and 4-DAMP (Kilbinger et al., 1984) suggests that these prejunctional sites belong to the M3 subtype. However, since HHSiD and 4-DAMP possess high affinity also for the M4 receptor, the participation of this site in negative feed-back mechanisms cannot be ruled out (Eglen and Watson, 1996). Furthermore, there is also evidence that cholinergic ganglionic transmission is regulated by presynaptic inhibitory muscarinic autoreceptors characterized so far as M2 subtypes (North, Slack and Surprenant, 1985).

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In contrast with the longitudinal muscle, prejunctional autoreceptors that inhibit ACh release in the circular muscle of the guinea-pig ileum belong to the M1 subtype (Dietrich and Kilbinger, 1995). This is of particular relevance, since M1 receptors located on the cell bodies of myenteric S neurons (i.e. interneurons and motor neurons) cause a slow membrane depolarization (North and Tokimasa, 1982; Morita, North and Tokimasa, 1982) and stimulate resting ACh output (Kilbinger and Nafziger, 1985; Vizi et al., 1989) through a mechanism inhibited by pirenzepine. Functionally, M1 receptors on myenteric (inter) neurons are activated during the ascending excitatory reflex in response to localized gut wall distension. In fact, in a partitioned organ bath, administration of pirenzepine at the site of distension inhibited the reflex contraction recorded in the oral compartment. Conversely, administration of pirenzepine at the site of reflex recording was ineffective (Tonini and Costa, 1990). Previous experiments by Schwörer and Kilbinger (1988) had indicated that M1 receptors also play a role in peristalsis since nanomolar concentrations of pirenzepine were found to enhance peristalsis, an effect apparently in contrast with that observed in the ascending excitatory reflex (Tonini and Costa, 1990). Pirenzepine-induced facilitation of peristalsis was explained in terms of antagonism of M1 receptors activating an inhibitory pathway. Based on recent findings, however, this effect can be easily explained by the removal of a tonic M1 receptor mediated inhibition of ACh release from the circular muscle (Dietrich and Kilbinger, 1995). The existence of muscarinic heteroceptors, i.e. modulating release of neurotransmitters other than ACh (e.g. noradrenaline), is also documented (Alberts and Stjärne, 1982; Manber and Gershon, 1979; Yokotani and Osumi, 1993; Marino et al., 1997). NICOTINIC RECEPTORS Nicotinic receptors are ligand-gated ion channels which are present on certain subclasses of myenteric neurons, where they cause rapid membrane depolarization (fast excitatory post-synaptic potentials, fast EPSPs) leading to fast communication between neurons. Stimulation of these receptors evokes release of numerous transmitters including ACh (Yau, Dorsett and Parr, 1989), NO (Jin and Grider, 1992), somatostatin (Grider, 1989) and L-glutamate (Wiley, Lu and Owyang, 1991). A polysynaptic chain of cholinergic ascending interneurons are involved in the distension-evoked ascending excitatory reflex of the ileal circular muscle. The ACh released from these neurons acts on nicotinic receptors on interneurons and motor neurons. Inhibition of these receptors by hexamethonium added to the oral, intermediate or anal compartment of a partitioned organ bath, markedly reduced the amplitude of the reflex contraction (Tonini and Costa, 1990), indicating that fast ganglionic nicotinic transmission exerts a crucial role in such responses. In contrast, probably because ACh is not handled by all classes of descending interneurons, the descending inhibitory reflex is reportedly less sensitive to nicotinic receptor blockade (Smith and Furness, 1988; Smith, Bornstein and Furness, 1990; Smith and Robertson, 1998). Nicotinic ganglionic transmission is also crucial for normal peristalsis, since hexamethonium abolishes propulsion both in the ileum and colon (Fontaine, Van Nueten and Janssen, 1973; Barthó, Holzer and Lembeck, 1987; Barthó et al., 1989; Kadowaki, Wade and Gershon, 1996; Tonini et al., 1996). In the ileum, weaker peristalsis can be obtained


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using greater distending volumes, providing naloxone is present to block the inhibitory effects of endogenous opioids (Barthó, Holzer and Lembeck, 1987; Barthó et al., 1989). Thus, non-nicotinic (i.e. hexamethonium-resistant) ganglionic transmission also occurs during peristalsis. This form of transmission is inhibited by endogenous opioids. Typically, nicotinic receptors are localized to the somatodendritic region of excitatory interneurons and motor neurons. Recently, however, prejunctional and presynaptic nicotinic receptors have been described in myenteric plexus neurons of the guinea-pig small intestine, where they mediate the release of excitatory non-cholinergic (tachykinergic) transmitters (Galligan, 1999; Schneider and Galligan, 2000; Schneider, Perrone and Galligan, 2000).

PHARMACOLOGY OF NON-CHOLINERGIC EXCITATORY (TACHYKINERGIC) TRANSMISSION Tachykinins represent a family of peptides which have been isolated from mammals (substance P, neurokinin A, neurokinin B) and amphibians (physalaemin, phyllomedusin, uperolein, eledoisin, kassinin) and whose role in intestinal motility has been reviewed recently (Holzer and Holzer-Petsche, 1997a,b). In the gut, although tachykinins may be present in the enterochromaffin and immune cells of the mucosa, most of them are found in intrinsic enteric neurons and extrinsic primary afferent nerve fibres (Holzer and HolzerPetsche, 1997b). In the myenteric plexus of the guinea-pig small intestine, substance P (SP) immunoreactivity has been found in excitatory motor neurons projecting to the circular (CM) and longitudinal muscle (LM) layers, ascending interneurons and intrinsic primary afferent neurons (Llewellyn-Smith et al., 1988; Brookes and Costa, 1990; Brookes, Steele and Costa, 1991a,b, 1992; Brookes et al., 1992; Costa et al., 1996). SP and neurokinin A (NKA) have been found in synaptic vesicles from enteric neurons where they are co-released with ACh, whereas neurokinin B (NKB) is absent (Deacon et al., 1987; McDonald et al., 1988; Regoli, Bondon and Fouchère, 1994; Lippi et al., 1998).

TACHYKININ RECEPTORS Three distinct receptors mediate the biological effects of endogenous tachykinins in the gastrointestinal tract. They are known as tachykinin NK1, NK2 and NK3 receptors, which have some preferential (though sometimes negligible) affinity for SP, NKA and NKB, respectively (Maggi, 1995). Since NKB is not expressed in the gastrointestinal tract, NK3 receptors are agonized by SP and NKA, which may act as full agonists at these sites (Maggi, 1995). Nevertheless, the pharmacological characterization of tachykinin receptors was made possible by the development of potent and selective peptide-derived agonists, e.g. NK1 receptor: SP methyl ester, [Sar9]-SP sulphone; NK2 receptor: [β-Ala8]-NKA(4–10), [Nleu10]-NKA(4–10); NK3 receptor: senktide, [MePhe7]-NKB, and peptide (FK-888; MEN11420; PD-161182) or non-peptide antagonists (SR-140333; SR-48968; SB-142801) (Holzer and Holzer-Petsche, 1997a).

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Effects of tachykinin receptor stimulation on neuromuscular preparations The NK1 and NK2 subtypes are located on smooth muscle cells, where they mediate contraction (Holzer and Lembeck, 1980; Costa et al., 1985; Maggi et al., 1990a, 1994a) and on neurons. In the guinea-pig ileum and colon LM, the direct contractile response to SP is mediated by the NK1 subtype (Lee et al., 1982; Briejer et al., 1993a), while in the CM both NK1 and NK2 receptors are involved (Maggi et al., 1990a, 1994a). In the colon, NK1 and NK2 receptors produce a non-adrenergic non-cholinergic (atropine-resistant) excitation of CM to nerve stimulation, which show a remarkable specialization. NK1 receptors mediate a “fast” excitatory transmission, which depend on the activation of voltage-sensitive calcium channels, whereas NK2 receptors mediate a “slow” excitatory transmission that is largely independent from calcium channels (Maggi, Zagorodnyuk and Giuliani, 1994b). Species-related pharmacological differences detected by variable affinities of competitive antagonists have emerged for both NK1 and NK2 receptors (Maggi, 1995). NK1 receptors are expressed on both ascending and descending neuronal pathways and on the interstitial cells of Cajal (Sternini et al., 1995; Legat et al., 1996; Portbury et al., 1996b; Johnson, Bornstein and Burcher, 1998; Lomax, Bertrand and Furness, 1998; Lecci et al., 1999; Bian et al., 2000). In CM strips from the guinea-pig small intestine, contractions mediated by NK1 receptors are atropine-resistant, but partially sensitive to tetrodotoxin (TTX) (Maggi et al., 1990a; Bian et al., 2000), suggesting that NK1 receptors are present, at least in part, on excitatory neurons, where they stimulate release of a non-cholinergic transmitter. In the same preparation, use of GR-73632, a selective NK1 receptor agonist, produced a TTX-sensitive decrease of spontaneous contractility, due to release of NO from inhibitory motor neurons (Lecci et al., 1999). NK1 receptor-mediated, TTX-sensitive relaxations due to release of NO have been also demonstrated in the colonic circular muscle (Bian et al., 2000). Lastly, prejunctional inhibitory NK1 receptors operating a feedback inhibition of ACh release have been described in LM-MP preparations (Kilbinger et al., 1986; Loeffler et al., 1994). Neuronal NK2 receptors have been described in descending pathways only (Portbury et al., 1996a; Zagorodnyuk and Maggi, 1995). In the guinea-pig colon CM strips, NKA and the NK2 receptor-selective synthetic agonist [β-Ala8]-NKA(4–10), in addition to causing a direct excitation, were found to activate inhibitory non-adrenergic non-cholinergic motor neurons with consequent release of NO and an apamin-sensitive inhibitory transmitter (Zagorodnyuk and Maggi, 1995). NK3 receptors are predominantly neuronal in origin. They are located on intrinsic primary afferent neurons (Mann et al., 1997a), and on ascending and descending pathways (Maggi et al., 1990a, 1993, 1994c; Legat et al., 1996; Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998; Jenkinson et al., 1999). Intrinsic sensory neurons are connected to each other via synapses communicating with NK3 receptors, which therefore contribute to the reinforcement of perceptive signal (Furness et al., 1998). In ascending pathways, activation of NK3 receptors by senktide or NKB promotes release of tachykinins (SP and NKA) and ACh from the myenteric plexus of the guinea-pig ileum (Guard and Watson, 1987; Yau et al., 1992). NK3 receptors located on descending pathway induce release of NO and inhibition of circular muscle activity in both the ileum and colon (Maggi et al., 1993; Maggi, Zagorodnyuk and Giuliani, 1994c).


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Electrophysiological effects of tachykinins on neurons Substance P depolarizes 85% of AH neurons and 76% of S neurons in the myenteric plexus when applied electrophoretically. The depolarization is associated with an increase in input resistance due to inactivation of a resting potassium conductance (Katayama, North and Williams, 1979). Slow EPSPs in S and AH neurons evoked by repeated pulse stimuli are due to the release of a non-cholinergic neurotransmitter and can be abolished by desensitization to SP (Katayama and North, 1978; Johnson et al., 1981). Thus, tachykinins are likely to mediate some slow EPSPs in myenteric neurons. On pharmacological grounds, it might be predicted that NK3 (and NK1), but not NK2, receptors are involved (Hanani et al., 1988; Willard, 1990; Bertrand and Galligan, 1994, 1995). Role of endogenous tachykinins Electrically induced, nerve-mediated, non-cholinergic contractions of the CM are reduced in amplitude by antagonists at the NK1 and NK2 receptor subtypes (Costa et al., 1985; Barthó et al., 1992). This indicates the participation in the contractile response of both SP and NKA, which are co-released upon electrical stimulation (Lippi et al., 1998). The ascending reflex contraction of the CM produced by radial distension of the gut wall is abolished by hyoscine at low distensions, but partly reduced at higher distension volumes. Under the latter conditions, the reflex contraction is reduced by tachykinin NK1 and NK2 receptor antagonists (Costa et al., 1985; Holzer, 1989), and in particular, by those acting at the NK2 receptor subtype (Barthó et al., 1992; Maggi et al., 1994d). Exposure of guinea-pig ileum CM to SP produces depolarization and action potentials (Niel, Bywater and Taylor, 1983a). The depolarization is associated with decreased membrane resistance. Noncholinergic fast excitatory junction potentials in the intestinal CM are abolished by desensitization to SP and by SP receptor antagonists acting at NK1 and NK2 receptors (Niel, Bywater and Taylor, 1983a; Taylor and Bywater, 1986; Serio et al., 1998). Taken together, these results indicate that endogenous tachykinins mediate non-cholinergic excitatory transmission to the CM. This transmission is mainly mediated by the NK2 receptor subtype, although NK1 receptors participate in this event. There is now convincing evidence that tachykinins play a role in peristalsis. Figure 6.1 illustrates the distribution of tachykinin receptors on effector cells and intrinsic neuronal pathways (i.e. myenteric plexus), which mediate propulsive activity in the gastrointestinal tract. SP is released into venous effluent in response to distension of the small intestine during peristalsis. This release is blocked by TTX and enhanced by the nicotinic receptor agonist dimethylphenylpiperizine (DMPP) (Donnerer et al., 1984; Donnerer, Holzer and Lembeck, 1984). SP stimulates peristalsis when the intestine is distended by a subthreshold volume (Holzer and Lembeck, 1979) and increases the frequency of emptying (Barthó et al., 1982a). Furthermore, atropine-resistant peristalsis is greatly inhibited by tachykinin receptor antagonists (Barthó et al., 1982b), whereas hexamethonium-resistant peristalsis is abolished by spantide (Barthó et al., 1989). Analysis of the receptors by which tachykinins influence peristalsis has shown that activation of NK1 receptors inhibits, while activation of NK2 and NK3 facilitates propulsion (Holzer, Schluet and Maggi, 1995). The NK1 receptor-mediated depression of peristalsis involves descending motor pathways utilizing

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Figure 6.1 Scheme illustrating the neuronal pathways regulating standing reflexes and peristalsis in the gastrointestinal tract. Radial distension of the gut wall by an intraluminal bolus excites ascending and descending pathways leading to excitatory and inhibitory reflexes. All three tachykinin receptor subtypes (NK1,2,3) are expressed on neurons where they mediate excitatory (+) or inhibitory effects (–) on propulsion. Tachykinin receptors located on smooth muscle cells mainly belong to the NK1 and NK2 subtypes and are excitatory in nature.

NO as a transmitter (Portbury et al., 1996b; Lecci et al., 1999), since the inhibitory effect was prevented by a nitric oxide synthase (NOS) inibitor (Holzer, 1997). On the other hand, blockade by SR-140333 of NK1 receptors markedly reduced the amplitude of inhibitory junction potentials evoked in the circular muscle by activation of descending pathways during motility reflexes. This clearly indicates that endogenous tachykinins acting via NK1 receptors partly mediate transmission to inhibitory motor neurons (Johnson, Bornstein and Burcher, 1998). Use of antagonists at NK1 (SR-140333), NK2 (SR-48968) and NK3 (SR-142801) receptors made it possible to establish that endogenous tachykinins acting via NK1 and NK2 receptors contribute to maintain intestinal peristalsis when cholinergic ganglionic and neuromuscular transmission via muscarinic receptors is suppressed. SR-142801 lacked a major influence on peristalsis, indicating that NK3 receptors play little role in peristalsis generation (Holzer et al., 1998). Conversely, NK3 receptors have been found to play a substantial role in transmission from intrinsic sensory neurons and from ascending (and descending) interneurons to excitatory motor neurons during motility reflexes (Johnson et al., 1996; Johnson, Bornstein and Burcher, 1998). It is interesting to point out that distension-evoked standing reflexes of the circular muscle subserve but cannot be identified with motor propulsive events (Tonini et al., 1996). In the guinea-pig isolated colon, NK1 and NK2 receptors contribute to peristalsis (FoxxOrenstein and Grider, 1996). In another study, blockade of NK1, NK2 and NK3 receptors inhibited propulsion. NK1 and NK2 receptors showed a synergistic interaction with muscarinic receptors, an effect observed also in the ileum (Holzer and Maggi, 1994), whereas inhibition produced by NK3 receptor blockade was additive with the decelerating effect


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caused by nicotinic receptor blockade. Simultaneous blockade of all the three receptors produced a 50% inhibition of the velocity of propulsion indicating that endogenous tachykinins acting at NK1, NK2 and NK3 receptors play an important role in maintaining propulsion (Tonini et al., 2001). In the rabbit isolated distal colon, NK2 and NK3 receptors located on descending pathways decelerate propulsion by activating a NO-dependent pathway. Both receptors, however, may also accelerate propulsion via distinct mechanisms. NK2 receptors mediate a facilitatory effect by a postjunctional synergistic interaction with muscarinic receptors, while NK3 receptors located on ascending pathways facilitate propulsion through a synergistic interaction with nicotinic receptors (Onori et al., 2000, 2001).

PHARMACOLOGY OF ADRENERGIC TRANSMISSION Noradrenaline is well established as the transmitter of postganglionic sympathetic neurons supplying the gastrointestinal tract, since it meets all the criteria for identification of a substance as a neurotransmitter (Furness and Costa, 1987). However, sympathetic neurotransmission may not be exclusively noradrenergic, as indicated, for instance, by the fact that adrenoceptor antagonists and reserpine pretreatment do not inhibit neurogenic vasoconstriction in submucosal arterioles, at least in the guinea-pig (Surprenant, 1994). In this species, noradrenergic axons supplying intestinal arterioles and submucous ganglia also contain neuropeptide Y and somatostatin, respectively, while sympathetic neurons reaching the myenteric plexus and the circular muscle coat contain neither peptide (Furness and Costa, 1987). The chemical coding of motility-inhibiting adrenergic neurons is different in the rat, a species where these neurons contain neuropeptide Y (McConalogue and Furness, 1994). Noradrenergic axons in the gut are most numerous in the myenteric and submucous ganglia and around arterioles, but there is also evidence of a sparse noradrenergic supply to the circular muscle and to the mucosa (Gabella, 1979). Concerning the sympathetic fibers reaching directly the muscle layer, in general the adrenergic innervation of the longitudinal muscle is less abundant than that of the circular layer (Gabella, 1979). Histochemical studies show that the noradrenergic innervation is particularly rich in the muscle coat of the so-called sphincteric regions such as the gastroesophageal junction, the pyloric region and the anal canal (Gabella, 1979; Furness and Costa, 1987). With few exceptions, stimulation of the sympathetic supply to sphincteric muscle is excitatory (Furness and Costa, 1974) and is thought to be due to a direct effect of noradrenaline on smooth muscle α-adrenoceptors. Electrophysiological studies have shown that noradrenergic fibers supplying the myenteric ganglia cause presynaptic inhibition of cholinergic transmission (Hirst and McKirdy, 1974a), while stimulation of noradrenergic nerves at frequencies of up to 50 Hz causes no changes in the membrane properties of myenteric neurons (Hirst and McKirdy, 1974b), although exogenous α2- and β1-adrenoceptor agonists can respectively hyperpolarize (Morita and North, 1981; Surprenant and North, 1985; Galligan and North, 1991; Wells and Mawe, 1994) or depolarize (Schemann, 1991; Tack and Wood, 1992) some neurons. Exogenous noradrenaline was shown to act presynaptically at different gut levels

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inhibiting fast EPSPs (Nishi and North, 1973; Schemann, 1991; Tack and Wood, 1992; Mawe, 1993). The most direct evidence supporting the concept of a tonic sympathetic inhibition on gut motility would be provided by electrophysiological studies showing a tonic discharge of sympathetic efferent neurons supplying the myenteric plexus and/or smooth muscle cells. Unfortunately, while release of noradrenaline from sympathetic terminals accounts for the inhibitory post-synaptic potential (IPSP) through activation of postsynaptic α2-adrenoceptors in submucosal neurons, little information is available on IPSPs in myenteric neurons, where IPSPs are seldom recorded, and there are only a few data on the possible transmitter(s) involved (Surprenant, 1994). Although noradrenaline can hyperpolarize myenteric neurons, hyperpolarization of myenteric neurons in response to sympathetic activity has never been shown (McConalogue and Furness, 1994), while, in the case of secretomotor neurons, neuronal hyperpolarization seems to be the main mode of action of the sympathetic input (North and Surprenant, 1985). In a recent study, designed to test whether sphincter of Oddi ganglia are a target of sympathetic input (Wells and Mawe, 1994), noradrenaline, when applied to neurons exhibiting IPSPs (which were, however, a minority), induced membrane hyperpolarization. Interestingly, α2-adrenoceptor blockade inhibited, whereas application of desipramine increased the amplitude of the IPSPs, an observation consistent with the hypothesis put forward by the authors that noradrenaline may mediate IPSPs at least in this preparation. ADRENOCEPTOR SUBTYPES At present, adrenoceptors are classified into three major classes: α1, α2 and β, each further divided into several subtypes (Bylund et al., 1994; Alexander and Peters, 2000), but only fragmentary information is available on the distribution of adrenoceptor subtypes in the enteric nervous system (Table 6.1). On a pharmacological basis, α1-adrenoceptors are defined as receptors selectively stimulated by methoxamine, cirazoline or phenylephrine and competitively blocked by low concentrations of prazosin, WB-4101 or corynanthine, while α2-adrenoceptors are characterized by a high sensitivity to agonists such as clonidine, xylazine, UK14 304 and antagonists such as rauwolscine, yohimbine and idazoxan (Ruffolo and Hieble, 1994). Concerning β-adrenoceptors, although many experimental data can be explained in terms of the existence of β1- and β2-adrenoceptors, a β-adrenoceptor with “atypical” pharmacological characteristics was described in some tissues such as the adipose tissue, gastrointestinal tract and skeletal muscle (Arch et al., 1984; Bond and Clarke, 1987; Croci et al., 1988; Bianchetti and Manara, 1990; Molenaar et al., 1991) and is now classified as a β3-adrenoceptor (Alexander and Peters, 2000). This β-adrenoceptor displays atypically low pA2 values for conventional antagonists such as propranolol, ICI 118551 and CGP 20712A and high sensitivity to agonists such as BRL 37344 and SR 58611A (Arch and Kaumann, 1993). Among conventional β-adrenoceptor antagonists, alprenolol is one of the compounds displaying the highest affinity (Blue et al., 1990), although its pA2 value for β3-adrenoceptors is rather low (Arch and Kaumann, 1993). Molecular biological approaches have confirmed the existence of at least three human genes encoding different β-adrenoceptors corresponding to the β1-, β2- and β3-subtypes (Frielle et al., 1987;

Phenylephrine, methoxamine, cirazoline

Prazosin, corynanthine, WB4101

Gq/11

Selective agonists

Selective antagonists

Predominant effectors

CGP20712A, atenolol Gs

Gi/o

Xamoterol, prenalterol

Smooth muscle relaxation

Smooth muscle, enteric neurons (?)

β1-adrenoceptors

Yohimbine, idazoxan

Clonidine, UK14304

Presynaptic inhibition, smooth muscle contraction

Adrenergic neurons (autoreceptors), myenteric neurons (heteroceptors), smooth muscle

α2-adrenoceptors1

Gs

ICI118551

Terbutaline, salbutamol, ritodrine

Smooth muscle relaxation, facilitation of transmitter release2

Smooth muscle, peripheral noradrenergic neurons (?)

β2-adrenoceptors

Gs

SR59230A

BRL37344, SR58611A, CL316243

Smooth muscle relaxation

Smooth muscle

β3-adrenoceptors

α1- and α2-adrenoceptors are further divided into the following subtypes, respectively: α1A, α1B, α1D and α2A, α2B, α2C (Alexander and Peter, 2000), but only fragmentary information is available at present on their distribution in the gut; 2 This effect has been reported in peripheral organs other than the gastrointestinal tract (Langer, 1981; Brunn et al., 1994).

Smooth muscle contraction or relaxation, neuronal depolarization (gastric neurons)

Functional response

1

Smooth muscle, gastric neurons

Distribution in the gut

α1-adrenoceptors1

TABLE 6.1 Synopsis of adrenoceptors in the gut

PHARMACOLOGY OF THE ENTERIC NERVOUS SYSTEM 223

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Kobilka et al., 1987; Emorine et al., 1989). More recently, the development of selective β3-antagonists has provided a new tool for β3-adrenoceptor identification in functional tests (Manara et al., 1995; De Ponti et al., 1996; De Ponti, 1997). ROLE OF PREJUNCTIONAL AND POSTJUNCTIONAL ADRENOCEPTORS Adrenoceptors of both α- and β-subtype have been recognized at different levels of the gastrointestinal tract where they are involved in the regulation of motility and secretion. In particular, α1-adrenoceptors are located postsynaptically on smooth muscle cells and, to a lesser extent, on intrinsic neurons, while α2-adrenoceptors may be present both pre- and postsynaptically (Table 6.1). At a presynaptic level, α2-adrenoceptors may act either as autoreceptors inhibiting noradrenaline release from adrenergic nerves (Langer, 1977; Starke, Göthert and Kilbinger, 1989), or as heteroceptors modulating release of other neurotransmitters (especially ACh) from nerve terminals (Paton and Vizi, 1969; Vizi, 1979). An important postsynaptic location of α2-adrenoceptors is that on enterocytes, where they control water and electrolyte absorption. β1- and β2-adrenoceptors are found mainly on smooth muscle cells, but the former may be present on enteric neurons (Bülbring and Tomita, 1987; Ek, Bjellin and Lundgren, 1986) (Table 6.1). Prejunctional adrenoceptors An electrophysiological study on guinea-pig antral myenteric neurons provided evidence for a concentration-dependent inhibition of cholinergic fast EPSPs and non-cholinergic slow EPSPs by application of noradrenaline or clonidine (Tack and Wood, 1992). Yohimbine and phentolamine, but not prazosin, reversed this effect, suggesting an involvement of α2-adrenoceptors. Interestingly, when noradrenaline was applied on cell bodies, no response was observed, in line with the notion that axo-axonal synaptic mechanisms are responsible for the adrenergic inhibitory effect (Tack and Wood, 1992). Another study carried out on myenteric neurons of the guinea-pig gastric corpus (Schemann, 1991) also found inhibition by noradrenaline of fast EPSPs, probably by activation of α2-adrenoceptors on extrinsic vagal fibers. Besides α2-adrenoceptors, it has been suggested that also α1- and β-adrenoceptors may be present on gastric neurons (Allescher et al., 1989; Schemann, 1991; Tack and Wood, 1992). Application of noradrenaline depolarized a small fraction of antral myenteric neurons and enhanced their excitability probably by activation of α1-adrenoceptors on somal membranes (Tack and Wood, 1992). The authors suggested that this α1-adrenoceptor may be located on non-cholinergic inhibitory neurons. In myenteric neurons of the guinea-pig gastric corpus, application of noradrenaline resulted in prolonged excitation in 40% of the neurons tested (Schemann, 1991). This excitatory effect, which is probably mediated by α1-adrenoceptors, seems peculiar to the stomach, since neuronal depolarization is not usually reported in other parts of the gut. Concerning the small bowel, many reports are available on presynaptic α2-adrenoceptors inhibiting neurotransmitter release at this level (Paton and Vizi, 1969; Wikberg, 1978; Drew, 1978; Vizi, 1979; Bauer and Kuriyama, 1982; Reese and Cooper, 1984; Wessler et al., 1987), while the presence of presynaptic β-adrenoceptors having a facilitatory effect


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on neurotransmitter release is less well documented (Alberts, Ögren and Sellstöm, 1985; Kotska et al., 1989). Evidence of a neuronal location of α2-adrenoceptors in the guinea-pig small intestine was provided by binding and functional studies (Drew, 1978; Wikberg and Lefkowitz, 1982): in the denervated longitudinal muscle, [3H]-clonidine binding was virtually abolished, indicating that denervation eliminated α2-adrenoceptors (Wikberg and Lefkowitz, 1982). Electrophysiological studies with intracellular recordings of guinea-pig myenteric neurons showed α2-adrenoceptor-mediated hyperpolarizations (resulting from an increase in potassium conductance) (Morita and North, 1981; Surprenant and North, 1985; Galligan and North, 1991) and inhibition of fast EPSPs (Galligan and North, 1991). Recent studies suggest that α2-autoreceptors and α2-heteroceptors modulating ACh release in the small bowel belong to the α2A/D subtype (Funk et al., 1995; Liu and Coupar, 1997). In the colon, sympathetic nerve stimulation reduces ACh release (Beani, Bianchi and Crema, 1969; Del Tacca et al., 1970) as well as the response to pelvic nerve stimulation (Gillespie and Khoyi, 1977) by activation of presynaptic α2-adrenoceptors (Marcoli et al., 1985), similarly to what has been described above for the small bowel. Indeed, clonidine is one of the most potent inhibitors of the colonic peristaltic reflex (Marcoli et al., 1985), an event requiring the integrity of neural circuits. Presynaptic α2-adrenoceptors can also modulate the release of the non-adrenergic, non-cholinergic transmitter, as indicated by inhibition of inhibitory junction potential amplitude in circular smooth muscle of guineapig caecum (Reilly, Hoyle and Burnstock, 1987) and by the contractile effect observed in colonic longitudinal muscle strips in mice (Fontaine, Grivegnée and Reuse, 1984). Postjunctional adrenoceptors Catecholamines can affect gastric motility through direct activation of smooth muscle adrenoceptors, which may belong to the α1-, α2-, β1-, β2- and β3-class (El-Sharkawy and Szurszewski, 1978; Verplanken, Lefebvre and Bogaert, 1984; Bülbring and Tomita, 1987; Coleman, Denyer and Sheldrick, 1987; McLaughlin and MacDonald, 1991; Cohen et al., 1995). β-mediated responses are usually inhibitory, α2-responses are excitatory, while the responses to α1-adrenoceptor stimulation are variable: both relaxations and contractions have been reported (Sahyoun, Costall and Naylor, 1982; Costall, Naylor and Tan, 1983; Chihara and Tomita, 1987; Bülbring and Tomita, 1987; MacDonald, Kelly and Dettmar, 1990; Mandrek and Kreis, 1992). In circular muscle strips of the porcine pyloric ring, the excitatory responses to catecholamines were unaffected by propranolol or yohimbine but were completely antagonized by prazosin and phentolamine, indicating the involvement of α1-adrenoceptors (Mandrek and Kreis, 1992). Binding studies with [3H]-prazosin and [3H]-yohimbine support the existence of both α1- and α2-adrenoceptors in the guinea-pig stomach, although they do not make it possible to distinguish between neural and smooth muscle receptors (Taniguchi et al., 1988). In some species, such as the rainbow trout, noradrenaline and adrenaline have been reported to induce smooth muscle contraction mainly by a direct action on muscular α2-adrenoceptors (Kitazawa, Kondo and Temma, 1986). Although functional responses can be obtained in the isolated stomach by stimulation of postjunctional α1- and α2-adrenoceptors, their physiological relevance in vivo is still unknown.

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Both α- and β-adrenoceptors have been detected at the postjunctional level in the small bowel. Excitatory α1-adrenoceptors are found especially in the terminal ileum (Bauer, 1981, 1982; Bauer and Kuriyama, 1982), although inhibitory responses probably mediated by these receptors have also been described (Bülbring and Tomita, 1987). Postjunctional α2-adrenoceptors have been detected in a binding study (Ahmad et al., 1991), but their function is still disputed, although Bauer and Kuriyama (1982) suggested an inhibitory effect by observing reduction or suppression of the generation of excitatory junction potentials by clonidine. Postjunctional β-adrenoceptors mediating smooth muscle relaxation were initially characterized as belonging to the β1-subtype (Grassby and Broadley, 1984; Mian, Malta and Raper, 1984; Sim and Lim, 1983). In the rabbit ileum, motor inhibition by perivascular nerve stimulation was reduced by 75% by the β1-adrenoceptor antagonist atenolol, while the remainder of the response was blocked by the β2-adrenoceptor antagonist butoxamine (Greenwood, Davison and Dodds, 1990). Several reports showing low pA2 values of conventional β-blockers have now provided evidence in favour of the presence of β3-adrenoceptors (Salimi, 1975; Bond and Clarke, 1987; Bond and Clarke, 1988; Grassby and Broadley, 1987; Norman and Leathard, 1990; Taneja and Clarke, 1992; Growcott et al., 1993a; Growcott et al., 1993b; MacDonald et al., 1994), which, at least in some preparations, seem to contribute to a significant extent to isoprenaline-induced relaxation (Tesfamariam and Allen, 1994). Postjunctional α-adrenoceptors may belong to the α1- and α2-subtype: the former may be both inhibitory (Fontaine, Grivegnée and Reuse, 1984; Dettmar, Kelly and MacDonald, 1986; Reilly, Hoyle and Burnstock, 1987) and excitatory (Venkova, Milne and Krier, 1994), while activation of α2A-adrenoceptors, at least in the circular smooth muscle of the canine colon, results in a significant increase in contractile force, probably mediated by a decrease in cyclic AMP levels (Zhang et al., 1992). In line with the general rule of a postjunctional location of β-adrenoceptors, most colonic β-sites mediate relaxation by a direct action on smooth muscle cells (Ek, Jodal and Lundgren, 1987; Taniyama, Kuno and Tanaka, 1987; Kwon et al., 1993; Smith et al., 1993), except for a minor population of neuronal β1-adrenoceptors which have been proposed to inhibit the activity of cholinergic neurons (Ek and Lundgren, 1982; Ek, 1985). This hypothesis of a neuronal location was based on the observation that the effect of the partial β1-agonist prenalterol was markedly reduced by the neurotoxin tetrodotoxin and 6-hydroxydopamine pretreatment (Ek, Bjellin and Lundgren, 1986). Binding studies performed in rat colon with [125I]-(−)-pindolol and [3H]-dihydroalprenolol revealed a small population of β1- (20–25%) and a large β2-adrenoceptor population (Ek and Nahorski, 1986; Landi et al., 1992). Although binding studies have failed to detect β3-sites because of the lack of selective ligands (Landi et al., 1992), several functional studies have provided evidence in favour of the presence of β3-sites. Grivegnée, Fontaine and Reuse (1984), studying the relaxatory effect of isoprenaline on the canine colon, concluded that the inhibitory effect of conventional β-blockers on the agonist-induced relaxation was “weak even at high concentrations”. Since this is now an established hallmark for the presence of β3-adrenoceptors, several investigators have assessed their role in the modulation of colonic motility in vitro (Bianchetti and Manara, 1990; McLaughlin and MacDonald, 1990; Landi, Croci and


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Manara, 1993; MacDonald and Lamont, 1993; Koike et al., 1994; De Ponti et al., 1995, 1996) and found concentration-dependent relaxations by using selective β3-agonists such as SR 58611A or CGP 12177 (a partial agonist).

PHARMACOLOGY OF NON-ADRENERGIC, NON-CHOLINERGIC INHIBITORY TRANSMISSION Intrinsic inhibitory transmission in the gut has much attracted the attention of investigators, leading to the recognition that non-adrenergic, non-cholinergic (NANC) inhibitory neural responses can be demonstrated in virtually all sections of the gut. The nature of the transmitter(s) responsible for NANC inhibitory transmission has been a matter of debate: over the years, ATP, VIP-like peptides and NO have been proposed as the main mediators of NANC relaxatory responses. Recently, evidence has begun to accrue that the hypotheses about the identity of the transmitter are not mutually exclusive and that multiple inhibitory mechanisms may coexist (Maggi and Giuliani, 1993; Lefebvre, Smits and Timmermans, 1995; Smits and Lefebvre, 1996; Selemidis, Satchell and Cocks, 1997) and may operate at different gut levels in response to mechanical, chemical or electrical stimuli. NANC inhibitory neurons are involved in the relaxation of sphincter regions, in the accommodation processes in the gastric fundus, small and large intestine, as well as in the descending inhibition in response to localized gut wall distension and peristalsis (Ciccocioppo et al., 1994; Tonini et al., 2000). VIP-LIKE PEPTIDES The VIP family of peptides includes VIP, peptide histidine isoleucine (PHI), pituitary adenylate cyclase-activating peptide (PACAP), glucagon, secretin and gastrin-releasing factor. There is considerable evidence in favour of a transmitter role for VIP in the intestine (Furness and Costa, 1987; McConalogue and Furness, 1994). In the guinea-pig small intestine, VIP is found in short and long descending inhibitory CM motor neurons and cholinergic and non-cholinergic descending interneurons (Furness and Costa, 1979a; Costa et al., 1980a, 1996; Jessen et al., 1980; Schultzberg et al., 1980; Costa and Furness, 1983; Llewellyn-Smith et al., 1988; Brookes, Steele and Costa, 1991b). A small population of VIP-immunoreactive LM motor neurons may exist (Schultzberg et al., 1980; Costa and Furness, 1983). VIP-immunoreactive fibres form synapses on both VIP- and NOSimmunoreactive myenteric neurons (Llewellyn-Smith, Furness and Costa, 1985; Costa et al., 1996). PHI is derived from the same precursor protein as VIP, is colocalized with VIP in vesicles (Agoston et al., 1989), and is thus found in the same populations of neurons as VIP. PACAP was originally isolated from ovine hypothalamus. Two forms occur: a 39-amino acid peptide and a 27-amino acid peptide which show 68% homology with VIP (Miyata et al., 1989; Miyata et al., 1990). PACAP nerve fibres are found in the smooth muscle layers of all regions of the rat gastrointestinal tract (Köves and Arimura, 1990; Nagahama et al., 1998). PACAP-immunoreactive fibres are found in the myenteric plexus and muscularis

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externa of the guinea-pig small and large intestine (Sundler et al., 1992; Portbury et al., 1995). While high affinity PACAP binding sites in the central nervous system do not bind VIP, in the periphery PACAP and VIP appear to share high affinity binding sites (Gottschall et al., 1990; Mao et al., 1998; Teng et al., 1998). Effects of VIP-like peptides on neuromuscular preparations VIP, at concentrations greater than 0.1 nM, enhances the cholinergic twitch and produces contraction of guinea-pig small intestine LM-MP preparations (Kusunoki et al., 1986). The contraction is partly blocked by hyoscine (Kusunoki et al., 1986), suggesting that VIP stimulates ACh and possibly tachykinin release (Katsoulis et al., 1992) from LM motor neurons. More direct evidence has shown that VIP causes increased release of [3H]-ACh from LM-MP preparations, isolated myenteric ganglia and from LM-MP synaptosomes (Yau, Youther and Verdun, 1985; Kusunoki et al., 1986; Yau, Dorsett and Youther, 1986b; Yau, Dorsett and Parr, 1989). Intra-arterial injection of VIP or PACAP1–27 or PACAP1–38 promotes ACh release from canine ileal circular muscle (Fox-Threlkeld et al., 1999). In the guinea-pig ileum CM, VIP produces concentration-dependent relaxations through a direct myogenic effect, which is resistant to apamin (Costa, Furness and Humphreys, 1986). PACAP causes relaxation of rat stomach, duodenum, jejunum, ileum and colon, through a direct action on the LM and CM (Mungan et al., 1992). In the human sigmoid colon, PACAP and VIP concentration-dependently inhibit phasic myogenic contractions of the LM. The effect of PACAP but not VIP is reduced by 80% by apamin. The inhibitory effect of VIP, but not PACAP, was attenuated by 70% by TEA (Schwörer et al., 1992). Similar findings were obtained in the rat colon (Ekblad, 1999), where the potassium channels involved in the VIP action are also sensitive to charybdotoxin (Kishi et al., 2000). These results suggest that PACAP and VIP act at different receptor subtypes which are coupled to different potassium channels, as also observed in the guinea-pig taenia coli (Jin et al., 1994). The receptors involved belong to the PAC1 subtype in the dog ileum (Fox-Threlkeld et al., 1999) and in the rat colon (Ekblad, 1999), whereas only the VIP2 /PACAP3 receptor (now VPAC2 receptor) is apparently expressed in gastric smooth muscle cells of rabbit and guinea-pig (Teng et al., 1998). Electrophysiological effects of VIP-like peptides on neurons and enteric smooth muscle cells VIP depolarizes myenteric neurons and mimics the slow EPSP in AH neurons (Williams and North, 1979; Zafirov et al., 1985). By combining electrophysiological and immunohistochemical methods, Katayama, Lees and Pearson (1986) demonstrated that all VIPimmunoreactive neurons belong to the S type. Non-cholinergic slow EPSPs could be evoked in 77% of VIP-immunoreactive neurons by stimulating orally or anally to the recording site. Thus, VIP neurons receive both ascending and descending inputs. PACAP1–27 or PACAP1–38 were found to depolarize 96% of AH neurons and 36% of S type neurons, respectively (Christofi and Wood, 1993).


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In the rat stomach, the apamin-resistant inhibitory junction potential (IJP) is mediated by VIP (Ito et al., 1990), whereas both VIP and PACAP are involved in the IJP generation in the longitudinal muscle of distal colon (Kishi et al., 1996). VIP produces a slow hyperpolarization of the CM of the guinea-pig ileum, antagonized by VIP10–28 (Crist, He and Goyal, 1992). In the murine gastric fundus circular muscle, the slow component of the IJP is also blocked by VIP10–28. This effect is apparently mediated by blockade of neuronal VIP receptors, which in turn cause release of NO as final transmitter (Mashimo et al., 1996). Role of endogenous VIP-like peptides VIP is a recognized transmitter of inhibitory motor neurons of the gut in a number of mammalian species, which is released upon a variety of physiological stimuli (Grider and Makhlouf, 1988b). In the rat and guinea-pig colon and human jeunum, distension-induced reflex relaxation is associated with increased VIP release and is blocked by VIP10–28 or VIP antibodies (Grider and Makhlouf, 1986, 1988b; Grider and Rivier, 1990; Foxx-Orenstein and Grider, 1996; Grider, Foxx-Orenstein and Jin, 1998). Release of PACAP from rat colon during reflex relaxation has also been documented (Grider et al., 1994). PACAP participates in the inhibitory transmission of guinea-pig teaenia coli, internal anal sphincter of opossum and in the canine ileal circular muscle (Jin et al., 1994; Chadker and Rattan, 1998; Fox-Threlkeld et al., 1999). Antibodies to somatostatin reduce both VIP release and the relaxation; exogenous somatostatin stimulates VIP release (Grider, Arimura and Makhlouf, 1987). Grider and Makhlouf (1986) have therefore proposed that somatostatin interneurons stimulate release of VIP from inhibitory motor neurons in response to distension. NITRIC OXIDE Nitric oxide is now known to act as a transmitter in the central and autonomic nervous system. The synthetic enzyme for NO, nitric oxide synthase (NOS) has been localized immunohistochemically in both central and peripheral neurons (Bredt, Hwang and Snyder, 1990). The previously characterized enzyme, NADPH diaphorase (Hope and Vincent, 1989), appears to be the same as NOS in some tissues and has therefore been used as a marker for NOS-containing neurons (Dawson et al., 1991; Hope et al., 1991). NOS catalyzes the synthesis of NO and L-citrulline from L-arginine via a Ca2+/calmodulindependent mechanism, which also requires the presence of tetrahydrobiopterin and NADPH as cofactors (Bredt and Snyder, 1989; Knowles et al., 1989; Mayer, John and Böhme, 1990). In some studies, concentrations of L-citrulline are used as an index of NOS activity (Currò, Volpe and Preziosi, 1996). NOS immunoreactivity has been demonstrated to be colocalized with VIP in discrete populations of myenteric neurons: descending inhibitory motor neurons and descending interneurons to other myenteric ganglia and submucous ganglia (Costa et al., 1991, 1996). Generally, the colocalization NOS/VIP has been recognized throughout the gastrointestinal tract of various species, from the stomach (Tonini et al., 2000) to the internal anal sphincter. Double immunostaining in the canine bowel revealed NOS and VIP in the same nerve varicosities, but never in the same organelles (Berezin et al., 1994).

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The “receptor” for NO is the soluble form of guanylate cyclase. NO binds to the haeme group of this enzyme, producing a conformational change to the active state. Thus, NO stimulates synthesis of cGMP, which in turn is believed to regulate protein kinases, phosphodiesterases and ion channels (Goy, 1991; Vincent and Hope, 1992). A recent study (Franck et al., 1997) using a novel inhibitor of guanylate cyclase (ODQ), suggested that electrical and mechanical effects of endogenous and exogenous NO in the canine colon are largely due to cGMP synthesis, while no evidence was found in support of a cGMPindependent mechanism of NO action. NO is now believed to act as a NANC inhibitory transmitter in several regions of the gastrointestinal tract in different species. The following sections will concentrate largely on the effects of exogenous and endogenous NO in the guinea-pig small intestine. Effects of NO on neuromuscular preparations Electrically induced NANC relaxations of the CM in the guinea-pig ileum are reduced by approximately 50% by apamin, while the remaining relaxation is blocked by L-N Gnitroarginine methyl ester (L-NAME). Alone, L-NAME reduces the relaxation by 71% and this inhibition can be reversed by L-arginine (Humphreys, Costa and Brookes, 1991). These results, suggest that NO-induced relaxation depends in part on an apamin-resistant mechanism and in part on an apamin-sensitive one. Sodium nitroprusside (SNP), a nitric oxide donor, causes relaxation of the guinea-pig ileum (Osthaus and Galligan, 1992) which is partly apamin-sensitive (which means involvement of small conductance calcium-activated potassium channels). Furthermore, relaxations can be mimicked by 8-bromo-cGMP (Osthaus and Galligan, 1992). The exact mechanism whereby cGMP causes relaxation is not clear, although it is known to vary according to the tissue and species. In smooth muscle cells isolated from canine colon, cGMP increases the probability of opening of potassium channels (Thornbury et al., 1991). The resultant hyperpolarization reduces voltage-dependent calcium influx into the muscle cells, thereby reducing tone. However, not all cGMP-mediated relaxation is associated with hyperpolarization. In vascular smooth muscle, cGMP has been shown to stimulate Ca2+-ATPase activity, thus reducing cytoplasmic calcium concentrations (Lincoln, 1989). Grider and Makhlouf (1992) proposed a complex mechanism for relaxation of intestinal tissue: they reported that NO could be synthesized in isolated myenteric ganglia from the guinea-pig small intestine in response to DMPP and gastrin-releasing peptide (GRP) (Jin and Grider, 1992). Circular smooth muscle cells from rat colon and guinea-pig gastric fundus could also synthesize NO in response to VIP (Grider and Makhlouf, 1992; Grider et al., 1992). L-N G-nitroarginine (L-NNA) inhibited VIP-induced relaxation and suppressed NO production. In a further study, Jin et al. (1992) reported that relaxation induced by SNP could be abolished by a cGMP-dependent protein kinase (protein kinase G) inhibitor, whilst relaxation due to VIP was reduced by 42%. Furthermore, VIP-induced relaxation was reduced by 33% by an inhibitor of cAMP-dependent protein kinase (protein kinase A), whereas the response to SNP was unaffected. Grider et al. (1992) have therefore proposed that VIP is released from inhibitory motor neurons and acts on smooth muscle receptors leading to stimulation of adenylate cyclase and NO production. NO in turn stimulates guanylate cyclase and also feeds back to further stimulate VIP release from the


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inhibitory motor neuron. In the canine colon, both VIP and NO appear to be responsible for the relaxation. Huizinga, Tomlinson and Pintin-Quezada (1992) found that exogenous VIP produced relaxation of the CM which was partly TTX-sensitive. L-NNA also partly blocked the relaxation. The effect of L-NNA was not significantly different from that produced by TTX or TTX and L-NNA, indicating that NO is responsible for the nervemediated component of the relaxation to VIP. Similarly, relaxation produced by 5-HT in the canine terminal ileum and ileocolonic junction is inhibited by L-NNA (Bogers et al., 1991) and VIP- and electrically evoked relaxations of the opossum internal anal sphincter are blocked by L-NNA (Rattan and Chakder, 1992). However, the relaxation produced by VIP in the canine lower oesophageal sphincter (De Man et al., 1991), guinea-pig taenia coli (Grider et al., 1992), and pig, canine and human gastric fundus strips (Lefebvre, Smits and Timmermans, 1995; Bayguinov et al., 1999; Tonini et al., 2000) are not affected by L-NNA. Thus, the degree to which NO mediates relaxation produced by VIP and other transmitters varies according to species and region of intestine.

Electrophysiological effects of NO on neurons and enteric smooth muscle cells In the canine jejunum CM, a single brief pulse of NO produces transient membrane hyperpolarization and relaxation (Stark, Bauer and Szurszewski, 1991). When NO is applied repetitively, the initial hyperpolarization runs down, although spontaneous phasic contractions are still inhibited (Irons, Stark and Szurszewski, 1992). This suggests that NO relaxes the intestine by two mechanisms: one involving hyperpolarization and the other independent of hyperpolarization. Transmural electrical stimulation of guinea-pig ileum in the presence of atropine and SP desensitization produces a fast and slow IJP in the CM (Niel, Bywater and Taylor, 1983b; Bywater and Taylor, 1986). The fast IJP is apamin-sensitive, whereas the slow IJP is apamin-resistant (Niel, Bywater and Taylor, 1983b). L-NNA, a NOS inhibitor, has no effect on the fast IJP (Watson, Bywater and Taylor, 1992), but blocks the slow IJP and this effect is partly reversed by L-arginine (He and Goyal, 1992; Lyster, Bywater and Taylor, 1992), suggesting that NO is involved. The slow IJP is also blocked by VIP10–28 (Crist, He and Goyal, 1992). Furthermore, He and Goyal (1992) demonstrated that the slow hyperpolarizing response to VIP in CM is inhibited by L-NNA, suggesting that NO mediates the response to VIP. Thus, VIP released from inter- or motor neurons may stimulate NO synthesis in inhibitory motor neurons which in turn relaxes and hyperpolarizes the CM. This mechanism has also been proposed for the generation of slow IJPs in circular muscle strips of murine gastric fundus (Mashimo et al., 1996).

Role of endogenous NO The hypothesis that NO may serve as an inhibitory transmitter in the gastrointestinal tract is based on a great deal of evidence accumulated in the last decade. These findings generally indicate that neurogenic relaxations evoked by electrical field stimulation from gastrointestinal specimens of several animal species, including humans, depend on

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a combination of NO and VIP release. Compared to VIP, NO is preferentially released at low frequency of stimulation (Tonini et al., 2000). NO seems to play an important role in maintaining tonic neural inhibition and compliance of the circular muscle. In fact, NO could be viewed as the primary inhibitory transmitter initiating accommodation in the guinea-pig ileum, rabbit colon and human gastric fundus (Waterman, Costa and Tonini, 1994; Ciccocioppo et al., 1994; Tonini et al., 2000). Apart from its action as an inhibitory transmitter at a postjunctional site, NO may also act as a modulator of excitatory transmission. Several reports now indicate that NO may inhibit cholinergic as well as noncholinergic transmitter release in several models (Knudsen and Tøttrup, 1992; Wiklund et al., 1993; Hryorenko, Woskowska and Fox-Threlkeld, 1994; Yunker and Galligan, 1996; Holzer-Petsche et al., 1996; Tonini et al., 2000). ADENOSINE TRIPHOSPHATE The presence of ATP in all cells makes it difficult to identify which neurons use ATP as a transmitter, although a recent study (McConalogue et al., 1996) provided evidence that ATP and its major metabolites are released from a neuronal source in the guinea-pig taenia coli. In other studies (Matsuo et al., 1997) ATP seems to derive mainly from smooth muscle cells. The best evidence that ATP may have a transmitter role in the enteric nervous system comes from studies using synaptosomes from LM-MP preparations. In these preparations, ATP is released in response to stimulation by high potassium, veratridine, ACh, or 5-HT (White, 1982; White and Leslie, 1982; White and Al-Humayyd, 1983; Al-Humayyd and White, 1985). Since the release is calcium-dependent, it is likely to represent a transmitter rather than a metabolic pool (White and Leslie, 1982). ATP is also released from guinea-pig taenia coli in response to electrical field stimulation. Some of this release is TTX-sensitive (Rutherford and Burnstock, 1978; McConalogue et al., 1996) and is therefore neuronal in origin. Relaxations in this preparation, like in many other gastrointestinal tissues, are mediated by P2Y purinoceptors modulating a subset of calcium-activated potassium channels (Kong, Koh and Sanders, 2000). Nevertheless, most of the measurable ATP is not neuronal in origin, since ATP can be released by smooth muscle relaxants such as papaverine and nitroglycerine (Kuchii, Miyahara and Shibata, 1973). The use of synaptosomes overcomes this problem. A further complication, however, is that ATP release from myenteric synaptosomes is reduced by 50–90% when the guinea-pigs are pretreated in vivo with 6-hydroxydopamine, suggesting that the majority of released ATP comes from noradrenergic nerve terminals of extrinsic nerves (White and Al-Humayyd, 1983; Al-Humayyd and White, 1985). ATP depolarizes S neurons, mimicking slow EPSPs, and hyperpolarizes AH neurons, mimicking slow IPSPs (Katayama and Morita, 1989). These effects are likely to be mediated by calcium-dependent potassium channel opening and closing, respectively (Katayama and Morita, 1989). Recent data also indicate that, in addition to ACh, ATP contributes to fast synaptic transmission through P2X receptors in myenteric neurons (Galligan and Bertand, 1994; LePard, Messori and Galligan, 1997). This transmission, which is antagonized by the P2X receptor antagonist PPADS (Lambrecht et al., 1992), predominantly occurs between descending interneurons and inhibitory motor neurons in descending inhibitory reflex pathways of guinea-pig ileum (Bian et al., 2000). Other


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electrophysiological evidence supports convincingly a role for endogenous ATP as an inhibitory transmitter. Single pulse transmural stimulation of the guinea-pig ileum produces a fast IJP which is TTX- and apamin-sensitive (Niel, Bywater and Taylor, 1983b; Crist, He and Goyal, 1992). This IJP, like the fast hyperpolarization induced by exogenous ATP, is antagonized by Reactive blue 2 and desensitization to α,β-methyleneATP, but is unaffected by VIP10–28 (Crist, He and Goyal, 1992). It is likely therefore, that ATP or a related nucleotide mediates the apamin-sensitive component of non-adrenergic, non-cholinergic inhibitory transmission. More recently, ATP was found to mediate fast relaxations or IJPs in response to electrical stimulation in the circular muscle of guinea-pig proximal colon and internal anal sphincter, the fast component of IJPs in the circular muscle of murine gastric fundus, and the IJPs in the circular muscle of porcine ileum via mechanisms sensitive to apamin, desensitization to α,β-methyleneATP, Reactive blue 2, and suramin, a non-selective P2 receptor antagonist (Maggi and Giuliani, 1996; Rae and Muir, 1996; Mashimo et al., 1996; Fernandez et al., 1998). Desensitization to ATP does not significantly alter peristalsis in the guinea-pig ileum (Weston, 1973) and rabbit colon (Tonini et al., 1982). It is therefore not clear whether endogenous ATP plays a role in peristalsis.

PHARMACOLOGY OF SEROTONERGIC TRANSMISSION Serotonin (5-HT) is present in enterochromaffin cells and neurons in the guinea-pig small intestine (Furness and Costa, 1982). Non-neuronally located 5-HT can be released in a calcium-dependent manner in response to increased intraluminal pressure (Bülbring and Lin, 1958; Bülbring and Crema, 1959; Schwörer, Racké and Kilbinger, 1987) or to vagal stimulation (Furness and Costa, 1987). 5-HT is localized in descending interneurons which project to other myenteric ganglia and/or submucous ganglia (Costa et al., 1982; Furness and Costa, 1982; Brookes, Steele and Costa, 1992). These neurons are also immunoreactive for cholineacetyl transferase. Ultrastructural studies indicate that 5-HT neurons form close contacts with other 5-HT neurons and with non 5-HT-immunoreactive Dogiel type I and type II neurons (Young and Furness, 1991). No obvious classification of enteric neurons has been found based on the number of 5-HT inputs they receive (Young and Furness, 1991). Release of 5-HT from these neurons can be demonstrated in response to depolarizing stimuli (Holzer and Skofitsch, 1984). 5-HT has a bewildering range of effects in the intestine, largely due to the presence of multiple receptor subtypes which appear to be present on several classes of myenteric neurons and smooth muscle cells (Table 6.2). The following section summarizes recent studies in which more selective agonists and antagonists have been used. Earlier work has been reviewed on numerous occasions (e.g. Costa and Furness, 1979; Furness and Costa, 1982, 1987). EFFECTS OF 5-HT ON NEUROMUSCULAR PREPARATIONS 5-HT contracts the ileal LM, producing a biphasic concentration-response curve (Buchheit et al., 1985; Eglen et al., 1990; Kilbinger and Wolf, 1992). The first phase has an EC50 for

8-OH-DPAT, buspirone

(±)WAY100635

Gi/o

Selective agonists

Selective antagonists

Effectors

Ketanserin, SB200646 MDL100907 SB204741

Gq/11

GR127935 (5-HT1B/D), SB216641 (5-HT1Bselective), BRL15572 (5-HT1D-selective)

Gi/o

Gq/11

α-Me-5-HT, BW723C86

α-Me-5-HT

sumatriptan

Longitudinal smooth muscle

5-HT2B

Contraction

Smooth muscle

5-HT2A

Contraction

Facilitation of Peristalsis, contraction

Myenteric neurons, Circular smooth muscle2

5-HT1B/D

Ligand-gated ion channel

Ondansetron granisetron

2-Me-5-HT, m-CPBG

Enhanced transmitter release

Myenteric neurons

5-HT3

Gs

Slow EPSP

Myenteric neurons

5-HT1P1

Gs

Go

5-HTP-DP

5-Carboxamido- Hydroxylated tryptamine indalpines

Relaxation

Smooth muscle

5-HT7

SB204070, Methiotepin, GR113808, GR125487 metergoline, SB258719

Renzapride, tegaserod (HTF919), ML10302, BIMU8

Enhanced transmitter release, relaxation

Myenteric neurons, smooth muscle

5-HT4

5-HT1P receptors are not included in the IUPHAR nomenclature of 5-HT receptors; 2 Sumatriptan is the only 5-HT1B/D receptor agonist so far tested and only fragmentary data exist on the possible involvement of these receptor subtypes in the gut.

Reduced transmitter release

Functional response

1

Myenteric neurons

Distribution in the gut

5-HT1A

TABLE 6.2 Synopsis of major 5-HT receptor subtypes in the gut.

234 INNERVATION OF THE GASTROINTESTINAL TRACT


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5-HT of 15 nM, indicating the involvement of a high affinity receptor (Eglen et al., 1990; Buchheit et al., 1985). This phase is selectively blocked by high concentrations of ICS 205–930 and mimicked by 5-MOT, BIMU 1, BIMU 8, cisapride and DAU 6236 (all 5-HT4 receptor agonists; Schuurkes et al., 1985; Eglen et al., 1990; Kilbinger and Wolf, 1992; Rizzi et al., 1992). These characteristics suggest that 5-HT is acting via the 5-HT4 receptor subtype. The first phase is also blocked by atropine, TTX, morphine and [D-Pro4, 7,9 D-Trp ]SP4–11 and augmented by physostigmine (Buchheit et al., 1985; Schuurkes et al., 1985). In the presence of antagonists to block all but the 5-HT4 receptor subtype, 5-HT and 5-MT enhance electrically evoked ACh release and associated twitch, and increase basal ACh release (Kilbinger and Pfeuffer-Friederich, 1985; Craig and Clarke, 1990; Fox and Morton, 1990; Kilbinger and Wolf, 1992). Cisapride also increases electrically evoked ACh release and the associated contraction (Taniyama et al., 1991). Cisapride has no effect on the contraction produced by exogenous ACh (Schuurkes et al., 1985). Consequently, the 5-HT4 receptor is likely to be located on LM motor neurons, where its activation stimulates the release of ACh and SP in a TTX-sensitive manner. The second phase of the concentration-response curve has an EC50 for 5-HT of 1.3 mM, indicating the involvement of a low affinity receptor (Buchheit et al., 1985; Eglen et al., 1990). This phase is blocked by low concentrations of ICS 205–930 (Buchheit et al., 1985; Eglen et al., 1990), suggesting that the 5-HT3 receptor subtype is involved. The contraction is inhibited by TTX, morphine, [D-Pro4, D-Trp7,9]SP4–11 and atropine and enhanced by physostigmine (Buchheit et al., 1985; Fox and Morton, 1989; Eglen et al., 1990). Activation of 5-HT3 receptors enhances basal ACh outflow (Kilbinger and Wolf, 1992). Thus, activation of 5-HT3 receptors may stimulate ACh and SP release from LM motor neurons. 5-HT also stimulates calcium-dependent, TTX-sensitive γ-aminobutyric acid (GABA) release through an action at 5-HT3 receptors (Shirakawa et al., 1989). At high concentrations (>10 µM), 5-HT acts at 5-HT2 receptors (formerly called D receptors) which are present on the smooth muscle and stimulate contraction (Engel et al., 1984; Buchheit et al., 1985; Richardson et al., 1985; Fox and Morton, 1989; Table 6.2). 5-HT produces a TTX-sensitive relaxation of precontracted LM in the presence of antagonists to 5-HT2, 5-HT3 and 5-HT4 receptors (Bill, Dover and Rhodes, 1990; Elswood and Bunce, 1992). This action is mimicked by carboxamidotryptamine and sumatriptan, suggesting that 5-HT1A and 5-HT1D receptors are involved (Bill, Dover and Rhodes, 1990; Elswood and Bunce, 1992). This relaxation may be due to inhibition of excitatory LM motor neurons, since activation of 5-HT1A receptors reduces the amplitude of the tachykinin-mediated twitch by a prejunctional mechanism (Galligan, 1992). It has also been reported that 5-HT1A agonists inhibit electrically evoked ACh outflow (Fozard and Kilbinger, 1985; Kilbinger and Wolf, 1992; Dietrich and Kilbinger, 1996) and reduce cholinergic twitch responses (Fozard and Kilbinger, 1985; Craig and Clarke, 1990). However, these effects may be due in part to an action of the agonists at histamine and muscarinic receptors (Galligan, 1992). 5-HT1A receptors also mediate inhibition of GABA release (Shirakawa et al., 1989). Some authors suggest caution in considering the involvement of 5-HT1A receptors in mediating the effects of 5-HT in the gut on the basis of data obtained with 8-OH-DPAT, a compound previously considered a selective 5-HT1A receptor agonist. After the discovery that 5-HT7 receptors mediate relaxation probably by a direct action on the smooth muscle

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(Carter et al., 1995; Prins et al., 1999) and that 8-OH-DPAT is also a partial agonist at the 5-HT7 receptor, some functional data should probably be reconsidered (Vanhoenacker, Haegeman and Leysen, 2000). Agonists at 5-HT4 receptors (BIMU 8, cisapride and 5-HT) cause a concentrationdependent increase in the amplitude of submaximal cholinergic twitch responses. This effect is inhibited by micromolar concentration of ICS 205–930 and DAU 6285 (Tonini et al., 1992a), confirming that 5-HT4 receptors are involved. In addition to enhancing electrically evoked twitch responses, cisapride enhances the ascending reflex contraction produced by balloon distension of the ileum (Tonini, 1992). Since the drug was added to the anal compartment of a partitioned organ bath, which contains the sensory neurons and some interneurons involved in the reflex, the effect of cisapride may be due to enhancement of transmission between these neuronal classes. Electrophysiological studies (see below) indicate that cisapride enhances nicotinic transmission. The ability of 5-HT to enhance peristalsis has been known for many years (Bülbring and Crema, 1958; Bülbring and Lin, 1958). The receptor involved in this process has been the subject of several investigations. Renzapride (a 5-HT4 receptor agonist) and 5-HT initiate peristalsis in the guinea-pig ileum and this effect is blocked by high concentrations of ICS 205–930 (Craig and Clarke, 1991). Using a slightly different preparation, Tonini, Galligan and North (1989) and Rizzi et al. (1992) showed that the frequency of emptying was increased by cisapride, BIMU 1, BIMU 8 and DAU 6236; on close inspection of the recordings, the compliance of the intestinal wall also appears to have been decreased. These effects were inhibited by high concentrations of ICS 205–930, but not by ondansetron, indicating the involvement of 5-HT4 receptors. There was no effect of 5-HT4 receptor agonists on LM muscle contraction during the preparatory phase or on the maximal ejection pressure (Tonini, Galligan and North, 1989a; Rizzi et al., 1992). 5-HT4 receptor agonists could conceivably reduce the threshold volume required to trigger emptying of the intestine and the compliance of the intestinal wall, by stimulating excitatory motor neuron pathways or inhibiting inhibitory motor neuron pathways (Waterman, Costa and Tonini, 1992). The former hypothesis seems more likely, since 5-HT4 receptor agonists can enhance neuronal release of ACh and SP (Kojima and Shimo, 1996), while the inhibitory effects of 5-HT4 receptor agonists on an inhibitory pathway is less well documented. However, other mechanisms may be involved: first, enhancement of ACh and SP release from LM-MP preparations does not necessarily mean that these agonists enhance transmitter release from CM motor neurons. Second, enhancement of the cholinergic twitch response in CM by 5-HT4 agonists could equally well be due to removal of an inhibitory input to the muscle as to stimulation of excitatory motor neurons. Therefore, even though the net effect is prokinetic, it is unclear whether the underlying mechanism is one of excitation or inhibition. Interestingly, there are animal (Grider, Kuemmerle and Jin, 1996) and human (FoxxOrenstein, Kuemmerle and Grider, 1996) data suggesting that 5-HT released by mucosal stimulation initiates a peristaltic reflex by activating 5-HT4 receptors on sensory neurons containing calcitonin gene-related peptide (CGRP). These effects are mimicked by mucosal application of selective 5-HT4 receptor agonists (prucalopride and tegaserod: Grider, Foxx-Orenstein and Jin, 1998). Experimental evidence for this mechanism in humans is so far limited to the small bowel.


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Important differences may exist among different species/different gut levels. 5-HT4 receptor-mediated prokinesia may result from increased release of ACh (and tachykinins) from excitatory neurons and may operate in human small bowel and stomach (SakuraiYamashita et al., 1999; Schuurkes et al., 1991), whereas this pathway does not seem to operate in the human colonic circular muscle (Burke and Sanger, 1988; Burleigh and Trout, 1985). In addition, it should be noted that, in contrast with what observed in the one of the most widely used models [the guinea-pig colon, where neuronal 5-HT4 receptors mediate contractile responses that are mainly cholinergic in nature (Briejer et al., 1993b; Wardle and Sanger, 1993)], human colonic circular muscle strips are endowed with 5-HT4 receptors located on smooth muscle cells, where they mediate relaxation (McLean and Coupar, 1996; Prins et al., 2000b; Sakurai-Yamashita et al., 2000; Tam et al., 1995). A recent report (Prins et al., 2000a) suggests the presence of 5-HT4 receptors on cholinergic neurons supplying the longitudinal muscle in the human colon. Figure 6.2 illustrates the distribution of 5-HT4 receptors on effector cells and intrinsic neuronal pathways (i.e. myenteric plexus), which mediate propulsive activity in the gastrointestinal tract. 5-HT1B/D receptors are now emerging as possible targets of 5-HT action in the gut (Borman and Burleigh, 1997; Coulie et al., 1997; Coulie et al., 1999; Tack et al., 2000). However, sumatriptan is the only 5-HT1B/D receptor agonist so far tested and, to the best of our knowledge, no formal assessment of the involvement of this receptor subtype by the use of selective antagonists has been performed (Cipolla et al., 2001). A study carried out

Figure 6.2 Modulation of intestinal motility by serotonergic 5-HT4 receptors. These receptors, which are distributed at multiple neuronal sites, have an excitatory effect (+) on enteric neurons leading to transmitter release and facilitation of propulsion. 5-HT4 receptors are also located on smooth muscle cells where they produce relaxation (–).

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in the human ileum suggested that, in the circular muscle, 5-HT-induced contraction is mediated via a receptor of the 5-HT1D subtype, whereas a receptor of the 5-HT2B subtype mediates the contractile response to 5-hydroxytryptamine of longitudinal muscle layer (Borman and Burleigh, 1997). Selective 5-HT1D receptor antagonists were not available at the time of this study to confirm the hypothesis. Finally, preliminary data suggest that 5-HT1B/D receptors are involved in mediating the facilitatory effect of sumatriptan on gastric accommodation in an in vivo canine model (De Ponti, 2000). ELECTROPHYSIOLOGICAL EFFECTS OF 5-HT ON NEURONS The electrophysiological effects of 5-HT on enteric neurons has been reviewed by Galligan (1995). 5-HT1A receptors are located on the cell bodies of AH neurons and on nerve terminals of cholinergic neurons (Galligan et al., 1988). Agonists at these receptors produce membrane hyperpolarization in AH neurons, but not S neurons, due to an increase in potassium conductance (Galligan et al., 1988; Galligan and North, 1991). These receptors also mediate presynaptic inhibition of ACh release which is recorded electrophysiologically as an inhibition of fast EPSPs (North et al., 1980; Galligan et al., 1988; Galligan and North, 1991). The amplitude of slow EPSPs is also reduced (Galligan et al., 1988; Galligan and North, 1991). 5-HT1P receptors are present on AH neurons where they mediate a slow depolarization associated with an increase in input resistance (Mawe, Branchek and Gershon, 1986). These receptors have been reported to be specifically antagonized by dipeptides of 5-hydroxytryptophan (5-HTP-DP) (Takaki et al., 1985a; Mawe, Branchek and Gershon, 1986). Activation of 5-HT3 receptors by 2-methyl-5-HT produces a fast depolarization in AH neurons which is associated with a decrease in input resistance, due to the opening of a cation channel (Mawe, Branchek and Gershon, 1986; Surprenant and Crist, 1988; Derkach, Surprenant and North, 1989; Mawe, Branchek and Gershon, 1989). This effect is blocked by low concentrations of ICS 205–930 (Mawe, Branchek and Gershon, 1986). Cisapride increases the amplitude of fast EPSPs through a presynaptic action, since it has no effect on the fast depolarization produced by exogenous ACh. This is likely to be due to stimulation of ACh release (Tonini, Galligan and North, 1989a). Cisapride had no effect on the resting membrane potential in several studies (Mawe, Branchek and Gershon, 1989; Tonini, Galligan and North, 1989a), but was reported to produce depolarization or hyperpolarization in a minority of cells (Nemeth et al., 1985; Nemeth and Gullikson, 1989). Cisapride does not alter non-cholinergic slow EPSPs in AH neurons (Tonini, Galligan and North, 1989a). ROLE OF ENDOGENOUS 5-HT Although 5-HT is present in and can be released from enterochromaffin cells and neurons in the guinea-pig ileum, it is difficult to prove its physiological role. 5-HT3 receptors are ligand-gated cation channels and mediate rapid depolarization of myenteric neurons (Surprenant and Crist, 1988; Derkach, Surprenant and North, 1989; Mawe, Branchek and Gershon, 1989). It is conceivable therefore, that at least some of the


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fast EPSPs recorded in the myenteric plexus are due to the action of an endogenous 5-HT-like substance on 5-HT3 receptors rather than the action of ACh at nicotinic receptors. This possibility has received little attention. However, Tonini, Galligan and North (1989a) found that the 5-HT3 receptor antagonists, ICS 205–930 and ondansetron, had no effect on the amplitude of fast EPSPs. It has been proposed that 5-HT mediates some or all slow EPSPs in the myenteric plexus. The latter hypothesis is unlikely, since lesioning 5-HT inputs to myenteric ganglia does not significantly alter the probability of recording slow EPSPs (Bornstein et al., 1984). However, 5-HT may be responsible for a proportion of slow EPSPs. Takaki et al. (1985b) and Mawe, Branchek and Gershon, (1986, 1989) reported that electrically evoked slow EPSPs could be blocked by 5-HTP-DP and renzapride but not by low concentrations of ICS 205–930. Some slow EPSPs may therefore be mediated by the action of 5-HT-like substances at 5-HT1P receptors. Since exogenous 5-HT can elicit fast and slow EPSPs, it is reasonable to propose that endogenous 5-HT might be responsible for non-cholinergic transmission. Such transmission has been shown to occur in the ascending excitatory reflex response to distension (Holzer, 1989; Tonini and Costa, 1990). However, endogenous 5-HT-like substances acting at 5-HT1, 5-HT1P, 5-HT3 and 5-HT4 receptors do not mediate the non-cholinergic component of ganglionic transmission in this reflex (Tonini et al., 1992b). Nevertheless, it is not known whether 5-HT may play a role in modulating cholinergic transmission in this reflex pathway through an action at presynaptic receptors. Endogenous 5-HT may play a role in the ascending excitatory reflex response to mucosal distortion, rather than distension. Bülbring and Crema (1958) and Bülbring and Lin (1958) originally proposed that 5-HT is involved in sensitizing mucosal processes of sensory neurons in the intestine. This was based on these pieces of evidence: first, 5-HT is present in enterochromaffin cells which are near the mucosal processes of presumed sensory neurons. Second, 5-HT could be released into the lumen of the intestine and this release is increased by increasing intraluminal pressure. Third, mucosal application of 5-HT stimulates peristalsis. More recently, Kirchgessner, Tamir and Gershon (1992) reported that stimulation of the mucosa with puffs of nitrogen gas results in c-fos expression in some myenteric and submucous neurons. Expression of c-fos is inhibited by dipeptides which are believed to inhibit 5-HT1P receptors. These investigators also demonstrated that antibodies to 5-HT1P receptors label submucosal calbindin-immunoreactive neurons, the proposed sensory neurons. The investigators therefore suggested that the response to nitrogen puffs may be due to release of 5-HT from enterochromaffin cells, which stimulates sensory neurons through an action at 5-HT1P receptors. However, these results await to be confirmed by other research groups. Furthermore, even if the conclusion drawn by Kirchgessner, Tamir and Gershon (1992) is correct, it does not necessarily mean that this mechanism operates during peristalsis. In fact, studies by other investigators, provide evidence that 5-HT is not necessarily involved. Schwörer, Racké and Kilbinger (1989) measured 5-HT release from enterochromaffin cells in the guineapig ileal mucosa in response to increased pressure in the lumen. If the intestine was infused with fluid at 22° C instead of 37° C, release of 5-HT was inhibited. However, there were no concomitant changes in peristalsis, thereby suggesting that 5-HT released from mucosal enterochromaffin does not play a significant role in peristalsis. Furthermore,

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antagonists at 5-HT3 and 5-HT4 receptors do not appear to have any effect on peristalsis (Craig and Clarke, 1991), suggesting that endogenous 5-HT has little, if any, role in peristalsis through an action at these receptors. However, the facilitatory effect of mucosally applied 5-HT to reduce the peristaltic threshold in the guinea-pig ileum is probably mediated by 5-HT3 receptors located on the mucosal side (Tuladhar, Kaisar and Naylor, 1997), and in particular on the terminals of intrinsic primary afferent neurons (Bertrand et al., 2000).

PHARMACOLOGY OF AMINO ACIDS AS ENTERIC NEUROTRANSMITTERS γ-AMINOBUTYRIC ACID (GABA) The presence of GABA in the guinea-pig small intestine has been demonstrated by immunohistochemistry (Jessen, Hills and Saffrey, 1986; Saito and Tanaka, 1986; Pompolo and Furness, 1990) and autoradiography (Krantis and Kerr, 1981). GABA is contained in short inhibitory CM motor neurons and LM motor neurons (Krantis, Kerr and Dennis, 1986; Saito and Tanaka, 1986; Hills, Jessen and Mirsky, 1987; Furness et al., 1989b). Myenteric neurons also express the synthetic enzyme for GABA, glutamic acid decarboxylase (GAD) (Miki et al., 1983; Williamson et al., 1995) and a high affinity uptake mechanism for the neurotransmitter (Krantis and Kerr, 1981; Saffrey et al., 1983; Krantis, Kerr and Dennis, 1986). GABA is released from myenteric synaptosomes of the guinea-pig small intestine in response to depolarization by high potassium and veratridine in a calcium-dependent manner (Yau and Verdun, 1983). The release of GABA from LM-MP preparations of the guinea-pig small intestine has also been demonstrated during either high potassium or electrically-evoked stimulation (Taniyama et al., 1983a; Wiley, Lu and Owyang, 1991). Somatostatin, SP, 5-HT, neurotensin and cholecystokinin (CCK) can stimulate GABA release from LM-MP preparations (Kerr and Krantis, 1983; Taniyama et al., 1983a; Tanaka and Taniyama, 1985; Nakamoto, Tanaka and Taniyama, 1987; Sano, Taniyama and Tanaka, 1989; Shirakawa et al., 1989; Takeda et al., 1989). This release is blocked by TTX and low Ca2+ concentrations. GABA release can be inhibited presynaptically by agonists at M1 receptors and α2-adrenoceptors (Hashimoto, Tanaka and Taniyama, 1986). Immunohistochemical evidences have been provided for the presence of both GABAA and GABAB receptors in the enteric nervous system (Krantis et al., 1995; Nakajima et al., 1996). In particular, GABAA receptor immunoreactivity has been detected on the soma of myenteric ganglion cells in the rat distal colon (Krantis et al., 1995). In addition, molecular biological investigations have demonstrated an abundant and widespread distribution of different GABAA receptor subunit mRNAs in the rat’s small and large intestines (Zeiter, Li and Broussard, 1996; Poulter et al., 1999). GABAB immunoreactivity was found on the soma of neurons in both submucosal and myenteric ganglia at different levels in the rat gut (Nakajima et al., 1996).


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Effects of GABA on neuromuscular preparations GABA is not effective on nerve-free preparations. In the LM of the guinea-pig small intestine, GABA produces a transient contraction followed by a relaxation (Inouye et al., 1960; Krantis et al., 1980; Giotti et al., 1983; Taniyama et al., 1983b). Both effects are blocked by TTX (Krantis et al., 1980), indicating that GABA is not directly acting on the LM. GABA-induced contractions are mimicked by muscimol and associated with an increase in ACh release (Kleinrok and Kilbinger, 1983; Tonini et al., 1987). The enhancements both in tension and in ACh release are blocked by bicuculline, picrotoxin, hyoscine and atropine, suggesting the involvement of GABAA receptors. Hexamethonium reduced the phasic GABAA-mediated contraction by 20% in one study (Tonini et al., 1987), suggesting that GABA is capable of stimulating cholinergic interneurons. GABA has also been shown to enhance ACh release from synaptosomes isolated from LM-MP preparations of the guinea-pig small intestine, suggesting that the stimulatory effect of GABA is due, at least in part, to an action on nerve terminals (Yau and Verdun, 1983). Tonini et al. (1987) found that GABA-induced contractions were also reduced by [D-Pro4, D-Trp7,9]SP4–11, indicating that GABAA receptors are also involved in the stimulation of tachykinin release from motor neurons and interneurons in the LM pathways of the guinea-pig ileum. The presence of GABAA receptors is also reported on inhibitory neurons and their stimulation induces relaxant responses (Minocha and Galligan, 1993; Boeckxstaens et al., 1991). Several reports are available to suggest that GABA may also inhibit both electricallyand chemically-evoked contractions by acting prejunctionally to reduce ACh release, in a bicuculline- and picrotoxin-insensitive manner (Hobbiger, 1958a; Hobbiger, 1958b; Florey and McLennan, 1959; Inouye et al., 1960; Kleinrok and Kilbinger, 1983; Ong and Kerr, 1983). This effect is mimicked by homotaurine (3-APS) and baclofen, but not by muscimol (Kleinrok and Kilbinger, 1983; Hills et al., 1989). δ-Aminovaleric acid (DAVA), but not bicuculline, reversed the action of both GABA and baclofen (Ong and Kerr, 1983). On the whole, these observations suggest that inhibitory effects of GABA on contractile responses are due to inhibition of ACh release from motor neurons mediated via GABAB receptors. GABA does not have any significant effect on electrically stimulated contraction of the CM, however it reduces the frequency of spontaneous CM contractions (Ohkawa, 1987).

Electrophysiological effects of GABA on neurons Ionophoretic application of GABA to myenteric neurons of the guinea-pig ileum determines a depolarizing response of AH neurons in a bicuculline-sensitive manner. This effect is associated with an increase in chloride conductance and is prone to desensitization suggesting the involvement of GABAA receptors (Grafe, Galvan and Mayer, 1979; Cherubini and North, 1984a). Data from studies in acutely isolated myenteric plexus preparations and in myenteric neurons maintained in primary culture suggest that GABAA-mediated responses are potentiated by a number of drugs acting at benzodiazepine and barbiturate binding sites (Cherubini and North, 1985a; Bertrand and Galligan, 1992; Zhou and Galligan, 2000). Electrophysiological and immunohistochemical investigations

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have recently demonstrated that myenteric neurons expressing calbindin immunoreactivity, a marker for AH neurons, have a high density of GABAA receptors (Zhou and Galligan, 2000). In addition to rapid depolarizing responses induced by ionophoretic application of GABA, superfusion of the agonist produces depolarizations in AH myenteric neurons which are mimicked by baclofen and are insensitive to bicuculline. This effect is associated with a smaller increase in conductance than that evoked by ionophoresis of GABA and does not desensitize. On the whole these observations suggest that this latter effect is mediated via activation of GABAB receptors probably located on nerve processes rather than on neuronal somata (Cherubini and North, 1984a). In isolated myenteric ganglia of the guinea pig ileum, GABA does not alter the resting membrane potential or conductance of S neurons (Cherubini and North, 1984b). However, GABA reduces the amplitude of evoked fast EPSPs without altering the response to ionophoretic application of ACh (Grafe, Galvan and Mayer, 1979; Cherubini and North, 1984b). This effect is mimicked by baclofen and resistant to bicuculline, suggesting the involvement of a GABAB receptor in the response (Cherubini and North, 1984b). In the same experimental model, GABAB receptors have also been shown to mediate the inhibition of slow EPSPs (Cherubini and North, 1984b). Thus, inhibition of both fast and slow EPSPs in S myenteric neurons is accomplished through GABAB receptors, which have been suggested to be located presynaptically to inhibit the release of both ACh and of a non-cholinergic excitatory transmitter.

Role of endogenous GABA Pharmacological studies have been described on the possible involvement of GABA receptors in the modulation of peristalsis with contrasting results. Indeed, early studies reported a GABA-mediated inhibition of peristalsis in a picrotoxin-sensitive manner (Hobbiger, 1958b; Inouye et al., 1960), whereas in a later study Schwörer and Kilbinger, (1988) could not demonstrate any significant effect of exogenous GABA on this functional parameter. In guinea-pig ileum, the amplitude of cholinergic twitches in the LM and the efficiency of peristalsis were enhanced by bicuculline (Schwörer and Kilbinger, 1988; Tonini et al., 1989b). However, these effects were not mimicked by picrotoxin or SR 95531, which also act on the GABAA receptor complex. These evidences might be explained on the basis of a non-specific effect of bicuculline (Tonini et al., 1989b), which might explain the discrepancy between an apparent effect of endogenous GABA on peristalsis (as deduced from the effect of bicuculline) and the lack of effect of exogenous GABA. However, in the guinea-pig colon, bicuculline has been shown to enhance the efficiency of peristalsis in normal but not in GABA-desensitized preparations, suggesting an action of bicuculline entirely dependent on the availability of functional GABA receptors (Frigo et al., 1987). In the same study bicuculline enhanced both cholinergic excitatory and non-adrenergic, non-cholinergic inhibitory responses to transmural stimulation and the release of ACh. These effects were completely abolished after desensitization with GABA (Frigo et al., 1987).


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SP- and neurotensin-induced stimulation of ACh release is partly reduced by bicuculline (Tanaka and Taniyama, 1985; Nakamoto, Tanaka and Taniyama, 1987). The authors concluded that endogenous GABA, which is released in response to SP and neurotensin, acts on GABAA receptors to stimulate ACh release. However, it seems likely that these effects are also due to non-specific actions of bicuculline, since the concentrations used in these studies (1–30 µM) were the same as those employed by Tonini et al. (1989b). Other evidence for non-specific effects of bicuculline has previously been reported in isolated rat atria, guinea-pig ileum and mouse vas deferens (Bartolini et al., 1985). Somatostatin-induced relaxation of the LM and the associated inhibition of ACh release, are blocked by phaclofen (Takeda et al., 1989). Somatostatin can stimulate GABA release (Takeda et al., 1989); therefore these results are likely to be due to a somatostatininduced GABA release which acts at GABAB receptors to inhibit ACh release and LM contraction. Since GABA is present only in motor neurons, and not in sensory neurons or interneurons, these results suggest that GABA may be released at the neuroeffector junction and act on local nerve terminals to inhibit (via GABAB receptors) ACh release. Exogenous GABA has a similar action on cholinergic nerve terminals in the myenteric plexus. Spontaneous neurogenic contractions of the guinea-pig ileum LM can be inhibited by atropine, bicuculline, picrotoxin, RU 5135, desensitization to GABA, 3-mercaptopropionic acid and cortisol (Ong and Kerr, 1984, 1989). Together with other results, these findings suggest that endogenous GABA is released from LM motor neurons and acts on local nerve terminals via GABAA receptors to stimulate ACh release. Immunohistochemical evidences have been provided for the presence also of inhibitory neurons co-expressing GABA and NOS immunoreactivity in the guinea-pig small intestine (Williamson, Pompolo and Furness, 1996). EXCITATORY AMINO ACIDS The concept that glutamate, the major excitatory neurotransmitter in the central nervous system (Ozawa, Kamiya and Tsuzuki, 1998), plays a role as a neurotransmitter also in the enteric nervous system is gaining increased acceptance (Liu et al., 1997; Sinsky and Donnerer, 1998). Immunoreactivity for glutamate has been detected in subsets of neurons in both the myenteric and submucosal plexus of the rat and guinea-pig ileum (Liu et al., 1997). In particular, glutamate immunoreactivity was found in enteric cholinergic neurons colocalized with substance P and/or calbindin (Liu et al., 1997). Immunohistochemical evidence has also been provided for glutamate storage into terminal axonal varicosities of myenteric neurons in the guinea-pig ileum (Liu et al., 1997). At this level, L-glutamate can be synthesized from the precursor, L-glutamine (Wiley, Lu and Owyang, 1991) and released during high potassium (Wiley, Lu and Owyang, 1991) or electrical-evoked (Sinsky and Donnerer, 1998) depolarization in a Ca2+-dependent manner. A recent study has demonstrated that both N-type and P-type calcium channels control most of stimulated glutamate release from LM-MP preparations of the guinea-pig ileum (Reis et al., 1999). Both ionotropic and metabotropic glutamate receptors have been found to be abundantly expressed in the enteric nervous system. Immunoreactivity for ionotropic receptors of the

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N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and of the kainate type, and for group I metabotropic receptors of the mGluR5 type has been detected in both the myenteric and submucosal plexus of the guinea-pig ileum (Kirchgessner, Liu and Alcantara, 1997; Liu et al., 1997; Liu and Kirchgessner, 2000). In addition, expression of mRNAs encoding for NMDA and mGluR2/3 metabotropic receptors has been demonstrated within enteric ganglia at different levels in the guinea-pig and rat gut (Broussard et al., 1994, Burns, Stephens and Benson, 1994; Larzabal et al., 1999). Enteric neurons are also endowed with a glutamate-inactivating mechanism, represented by the high affinity neuronal transporter, EAAC1 whose expression along the gut is particularly extensive (Liu et al., 1997). Effects of excitatory amino acids on neuromuscular preparations Evidence from functional studies suggests that L-glutamate mediates contraction of the longitudinal muscle in the guinea-pig ileum through activation of NMDA receptors, probably located on excitatory cholinergic motor neurons (Luzzi et al., 1988; Shannon and Sawyer, 1989; Wiley, Lu and Owyang, 1991; Sinsky and Donnerer, 1998). Indeed, in LM-MP preparations of the guinea-pig ileum, L-glutamate, L-aspartate and NMDA determine contractile responses which are competitively antagonized by NMDA receptor antagonists and noncompetitively blocked by Mg2+ (Moroni et al., 1986; Luzzi et al., 1988; Shannon and Sawyer, 1989; Wiley, Lu and Owyang, 1991). An NMDA receptor has been suggested to be involved in the contraction of the guinea-pig ileum also since the action of glutamate is potentiated by glycine in a strychnine-insensitive manner (Luzzi et al., 1988). In addition, glutamate-induced contractions are inhibited by TTX and hyoscine, but not by hexamethonium (Moroni et al., 1986; Luzzi et al., 1988; Wiley, Lu and Owyang, 1991). On the whole, this evidence suggests that L-glutamate stimulates ACh release from LM motor neurons which in turn causes the contraction via activation of postjunctional muscarinic receptors. Indeed, pharmacological evidence has been provided for an NMDA-mediated enhancement of ACh release from myenteric neurons in the guinea-pig ileum and colon (Wiley, Lu and Owyang, 1991; Cosentino et al., 1995). At this latter level, application of NMDA enhances also the release of an inhibitory neurotransmitter such as noradrenaline. This effect has been correlated to the NMDA-mediated inhibition of peristalsis in the colon (Cosentino et al., 1995). The possible involvement of AMPA and kainate receptors in the modulation of motor function in the gastrointestinal tract has not yet been completely clarified. AMPA and kainate receptors do not seem to mediate a contractile response in the guinea-pig ileum, since neither quisqualate nor kainate are effective in producing contractions of LM-MP preparations at concentrations up to 1 mM (Moroni et al., 1986; Luzzi et al., 1988; Shannon and Sawyer, 1989). Recent pharmacological investigations have demonstrated that activation of AMPA receptors inhibits both electrically-induced contractions of the circular muscle and ACh release in the guinea-pig distal colon (Giaroni et al., 2000). In the same study, AMPA has been described to enhance the efficiency of peristalsis. On the basis of these apparently contrasting results, the authors suggested that AMPA receptors might have a more prominent role in the modulation of inhibitory inputs to the colon (Giaroni et al., 2000). Furthermore, in accordance with the inability of kainate to induce LM-MP


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contractions in the guinea-pig ileum (Luzzi et al., 1988), this compound was ineffective in modulating both ACh release and peristalsis in the colon of the same species (Giaroni et al., 2000). Electrophysiological effects of excitatory amino acids on neurons Electrophysiological studies of glutamate effects on enteric neurons have been described with contrasting results for the occurence of depolarizing responses and identification of glutamate receptors involved in the response. Grafe, Galvan and Mayer (1979) found no significant electrophysiological effect of L-glutamate in seven myenteric neurons studied, whereas Liu et al. (1997) found that application of glutamate evokes depolarizing responses in myenteric neurons that had fast and slow components. The fast component was mimicked by AMPA, whereas the slow component was mimicked by NMDA. In the same study, glutamate and ACh have been suggested to represent excitatory co-transmitters, both contributing to mediate fast ganglionic neurotransmission in AH/type 2 neurons. However, contrasting data have been recently reported suggesting that glutamate evokes only slowly activating depolarizing responses in submucous and myenteric neurons of the guinea-pig small intestine by activation of group I metabotropic receptors (Ren et al., 2000).

PHARMACOLOGY OF HISTAMINERGIC TRANSMISSION Histidine decarboxylase (HDC, a marker for histamine containing neurons) and its mRNA have been demonstrated in neurons in the rat central and peripheral nervous systems (Watanabe et al., 1984; Karhula et al., 1990). HDC immunoreactivity has been demonstrated in myenteric neurons in the rat small intestine (Ekblad et al., 1985). However, since the antibody used in these studies cross-reacts with guinea-pig L-dopa decarboxylase, the distribution of HDC in the myenteric neurons in the guinea-pig small intestine could not be determined (Ando-Yamamoto et al., 1986). In the intestine, histamine interacts with three types of receptor, namely H1, H2 and H3 receptors, for which there are currently available selective agonists and antagonists (Hill et al., 1997). H1 receptors are located postjunctionally where they mediate contraction (Zavecz and Yellin, 1982; Trzeciakowski, 1987; Barocelli et al., 1993). H2 receptors are present on LM motor neurons of guinea-pig small intestine where they facilitate the release of ACh and tachykinins (Zavecz and Yellin, 1982; Barker and Ebersole, 1982; Poli, Loruzzi and Bertaccini, 1990). These receptors excite myenteric AH neurons partly by enhancing chloride conductance (Starodub and Wood, 2000). H3 receptors are mainly prejunctional and inhibit the release of ACh and tachykinins from LM motor neurons (Trzeciakowski, 1987; Hew, Hodgkinson and Hill, 1990; Menkveld and Timmerman, 1990; Taylor and Kilpatrick, 1992). In addition, H3 receptors have been demonstrated to mediate inhibition of noradrenaline release from intestinal sympathetic nerves (Blandizzi et al., 2000; Liu et al., 2000). The effects mediated by histamine receptors have been recently assessed in distensionevoked ascending excitatory transmission in the guinea-pig ileum. Histamine acting at H1

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receptors depressed synaptic transmission at low concentrations, whereas increased it at high concentrations. Activation of H2 receptors by the agonist dimaprit was associated with facilitation of transmission, whereas activation of H3 receptors by α-methylhistamine inhibited synaptic transmission (Izzo et al., 1998). Histamine is known to exist in mast cells and basophils in the intestinal mucosa, but the role of endogenous histamine in intestinal motility is unknown.

PHARMACOLOGY OF PEPTIDES AS ENTERIC NEUROTRANSMITTERS OPIOID PEPTIDES The guinea-pig small intestine contains peptides derived from proenkephalin and prodynorphin but not pro-opiomelanocortin (Steele et al., 1992). The presence of enkephalinlike and dynorphin-like immunoreactivity in the myenteric plexus has been reported by a number of researchers (Furness and Costa, 1987). Opioids are found in orally and anally directed interneurons, excitatory and inhibitory CM motor neurons and rarely in LM motor neurons (Brookes, Steele and Costa, 1991b, 1992; Steele et al., 1992; Costa et al., 1996). Recently, the endogenous opioid endomorphin-1 and endomorphin-2 have been detected in brain (Zadina et al., 1997) and spinal cord of various species, including man. However, endomorphins have not been found yet in the gut. Ligand binding and functional studies indicate that µ and κ receptors, and to a lesser extent δ receptors, are present in the myenteric plexus of mammals. The presence of δ receptors has been detected in the ileal submucosal plexus, at least in the guinea-pig. cDNA encoding an “orphan” receptor has been identified which has a high degree of homology to the classical opioid receptors. This receptor has been named ORL1 (opioid receptor like) (Mollereau et al., 1994; Nicholson et al., 1998). The endogenous peptide agonist for ORL1, nociceptin referred also as orphanin FQ, has been detected in porcine, rat and guinea-pig gastrointestinal tract (Osinski et al., 1999; Yazdani et al., 1999; O’Donnell et al., 2001). In the guinea-pig myenteric plexus, nociceptin immunoreactivity is expressed preferentially in excitatory motor neurons projecting to the longitudinal and circular muscle layers, as well as a small subgroup of descending interneurons (O’Donnell et al., 2001). Effects of opioids on neuromuscular preparations In the guinea-pig ileum, agonists at µ (morphine, normorphine, enkephalins, DAMGO, and endomorphin-1,2), κ receptors (dynorphin and U-50488H), and ORL1 receptors (nociceptin) inhibit electrically-induced longitudinal muscle cholinergic contractions by presynaptic inhibition of ACh release (Greenberg, Kosterlitz and Waterfield, 1970; Chavkin and Goldstein, 1981; Vizi et al., 1984; Kojima et al., 1994; Tonini et al., 1998; Sternini et al., 2000; O’Donnell et al., 2001). Opioids acting at µ and κ receptors have been shown directly to decrease ACh release evoked by substance P, neurotensin and caerulein (Yau, Verdun and Youther, 1983; Yau, Verdun and Youther, 1983; Yau, Dorsett and Youthe, 1986b; Huidobro-Toro et al., 1984). They can inhibit hyoscine-resistant,


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substance P-mediated contractions (Gintzler and Scalisi, 1982; Barthó et al., 1982b) and have been shown to reduce substance P release (Holzer, 1984). The action of agonists at ORL1 receptors is region specific as observed in the rat gastrointestinal tract. In this species, nociceptin inhibits cholinergic transmission in the stomach and small intestine, whereas it stimulates colonic contractions by inhibiting a neurogenic inhibitory pathway within the myenteric plexus (Yazdani et al., 1999). No specific effect of δ receptor agonists on excitatory LM motor neurons has been demonstrated. The agonists DPLPE and DPDPE were found to reduce LM contraction by 50% (Mosberg et al., 1983; Galligan et al., 1984; Ersparmer et al., 1989) or to be ineffective (Kojima et al., 1994). Whilst these effects were reversed by naloxone (Mosberg et al., 1983; Galligan et al., 1984) and the µ receptor antagonist CTP, (Shook et al., 1987) they were not antagonized by the specific δ receptor antagonist, ICI 174864 (Shook et al., 1987). Agonists at µ and κ receptors inhibit CM contraction in the whole wall, CM-MP and CM-axons preparations evoked by 5-HT (Harry, 1963; Johnson et al., 1987; Johnson, Costa and Humphreys, 1988). The δ agonist, DPLPE, partly inhibited circular muscle contraction at very high concentrations (3 mM); however this effect was not antagonized by the δ antagonist, ICI 174864. Morphine inhibits ascending excitatory reflex contractions when added to the oral, intermediate or anal compartments of a partitioned organ bath (Tonini et al., 1992c). This suggests that µ receptors are present on CM motor neurons and interneurons, where they inhibit transmitter release. Selective agonists at µ and κ receptors (PLO17 and U-10488, respectively) concentration-dependently inhibit the ascending excitatory reflex response in the rat ileum, whereas the δ agonist DPDPE is ineffective (Allescher et al., 2000). However, δ receptors seem to be involved together with µ receptors in the timing of the reflex response. The effects of opioid receptor stimulation on NANC inhibitory transmission have been investigated in rat, guinea-pig, canine and human gastrointestinal tract. Morphine was found to inhibit electrically evoked NANC inhibitory responses in rat gastric fundus strips (Dehpour et al., 1994), whereas loperamide (a non-selective µ receptor agonist) inhibited the 5-HT-induced NANC relaxation in isolated intact stomach from guinea-pig (Meulemans, Helsen and Schuurkes, 1993). Agonists acting at µ and κ receptors decreased the amplitude of NANC IJPs evoked by transmural nerve stimulation in the circular muscle of canine duodenum (Bauer and Szurszewski, 1991). In the circular muscle of guinea-pig and human colon, NANC inhibitory responses are reduced by activation of δ receptors (Hoyle et al., 1990; Zagorodnyuk and Maggi, 1994). Electrophysiological effects of opioids on neurons Early studies involving intracellular recordings of myenteric neurons showed that opioid agonists acting at µ receptors, such as met- and leu-enkephalin, morphine and normorphine hyperpolarize cell membranes (North and Tonini, 1977; Sakai, Hymson and Shapiro, 1978; North, Katayama and Williams, 1979; Cherubini and North, 1985b) via stereospecific and naloxone-reversible mechanisms, whereas opioids that are selective for κ receptors (dynorphin, U-50488H) do not (Cherubini and North, 1985b). Opioids acting at both receptor subtypes caused presynaptic inhibition of ACh release in the myenteric

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plexus, depressing the amplitude of fast EPSPs (Cherubini and North, 1985b). However, µ and κ opioids inhibit transmitter release by different presynaptic inhibitory mechanisms; µ receptor activation results in hyperpolarization due to increased potassium conductance, whereas κ receptor activation may depress the release of ACh by directly reducing calcium entry into the nerve terminals (Cherubini and North, 1985b). A comprehensive study of the electrophysiological effects of DAMGO was undertaken by Pillai and Johnson (1991). This study demonstrated that 42% of S neurons are hyperpolarized by DAMGO. The magnitude of the hyperpolarization increased with increasing concentrations of DAMGO and was prevented by pre-application of naloxone. Fast EPSPs were significantly diminished in amplitude in 48% of S cells; this effect occurred in some cells which had not been hyperpolarized by DAMGO. This effect is probably due to a presynaptic action of opioids, since fast EPSPs evoked by exogenous nicotine are unaffected (Cherubini and North, 1985b; Cherubini, Morita and North, 1985). In AH cells, there was no effect of DAMGO on the resting membrane potential, input resistance, action potential duration or amplitude, nor on the amplitude or duration of the fast and slow after-hyperpolarizations. Propagation of action potentials to the soma of AH neurons was also unaffected by DAMGO. Ito and Tajima (1980) provided electrophysiological evidence that µ agonists do not act directly on the muscle. Morphine reduced the amplitude of the excitatory junction potential, but did not change the membrane potential, resistance or electrical threshold for activation of spikes in circular or longitudinal muscle. Thus the site of action of morphine was prejunctional. There is no evidence for inhibition of inhibitory junction potentials by opioids (Ito and Tajima, 1980). Role of endogenous opioids Trendelenburg (1917) was the first to demonstrate inhibition of peristalsis by morphine in the guinea-pig small intestine in vitro. Morphine, fentanyl and met-enkephalin inhibit both longitudinal and circular muscle contraction during the preparatory and emptying phases respectively (Schaumann, 1955; Kosterlitz and Robinson, 1957; Fontaine, Reuse and Van Nueten, 1973; Van Nueten, Van Ree and Vanhoutte, 1977). Fontaine, Reuse and Van Nueten (1973) reported a preferential effect of morphine and fentanyl on longitudinal muscle contraction. At submaximal doses, morphine, fentanyl and met-enkephalin increased the pressure at threshold, reduced longitudinal and circular muscle activity, increased filling of the intestine, reduced the volume expelled per wave and reduced the frequency of peristaltic waves (Fontaine, Reuse and Van Nueten, 1973; Van Nueten, Van Ree and Vanhoutte, 1977). Similarly, Kromer and co-workers (Kromer, Pretzlaff and Woinoff, 1981; Kromer, Steigemann and Shearman, 1982) have shown a dose-dependent inhibitory effect of normorphine in terms of the frequency of peristaltic waves. Inhibition was also produced by D-Ala2-Leu-enkephalinamide, β-casomorphin, DAMGO, ketocyclazocine and dynorphin (Kromer, Pretzlaff and Woinoff, 1980; Kromer, 1990). All of these results were obtained using the Trendelenburg preparation. However, peristalsis was also abolished by dermorphin, morphine, D-Ala2-D-Met5-enkephalin, FK 33–824 and dynorphin in the vascularly-perfused preparation (Holzer and Lembeck, 1979; Barthó et al., 1982a; Donnerer and Lembeck, 1985). At low concentrations, these opioids reduced


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both the frequency of peristaltic waves and the maximal ejection pressure. Barthó et al. (1982a) also demonstrated that FK 33–824 could abolish atropine-resistant peristalsis. A more recent study (Waterman, Costa and Tonini, 1992) has demonstrated that µ opioid receptor agonists reduce the compliance of the intestinal wall, the LM contraction during the preparatory phase and increase the threshold volume required to trigger emptying. Kappa receptor agonists reduce the maximal ejection pressure during the emptying phase and LM contraction and increase the threshold volume. Delta receptors also modulate peristalsis in the guinea-pig ileum. In agreement with studies by Kromer (1990), Waterman, Costa and Tonini (1992) demonstrated that δ agonists reduce the maximal ejection pressure, LM contraction during the preparatory phase and increase the threshold volume. In 1977, Schulz et al. reported that material with enkephalin-like activity was released from LM-MP strips. Furthermore, the enkephalin content of LM-MP strips was reduced by high frequency stimulation (1 and 10 Hz) – but not low frequency (0.1 Hz) – stimulation (Hughes, Kosterlitz and Sosa, 1978). Oka and Sawa (1979) showed that this release was calcium-dependent. The first direct measurement of met-enkephalin release from LM-MP strips was not achieved until 1986 (Glass, Chan and Gintzler, 1986). Enkephalin release was detectable following electrical stimulation at frequencies between 5 and 80 Hz. The release was tetrodotoxin-sensitive and calcium-dependent. Enkephalin release was enhanced in the presence of naloxone, but reduced by morphine pretreatment. This suggests that endogenous opioids can modulate their own release (Sublette and Gintzler, 1992). Preparations of synaptosomes from guinea-pig LM-MP strips have been shown to release met- and leu-enkephalin. Release increased following depolarization by potassium. Met-enkephalin release was detectable only in the presence of naloxone or atropine. Leu-enkephalin release was detectable only in the presence of naloxone, atropine or a purine receptor antagonist (1,3-dipropyl-8-p-sulphophenylxanthine) (Christofi, McDonald and Cook, 1990). Waterfield and Kosterlitz (1975) reported that naloxone stereospecifically increased the release of ACh in response to low frequency (0.017 Hz) electrical stimulation of the LM-MP preparation. The effect was less marked at a stimulation frequency of 10 Hz. Puig et al. (1977) combined different stimulation frequencies and showed that longitudinal muscle contraction evoked by stimulation at 0.1 Hz was reduced following stimulation at 10 Hz for 5 min. This inhibitory effect was antagonized in a dose-dependent manner by naloxone and naltrexone. Similar results have since been reported by Horàcek and Kadlec (1984). Gintzler and Scalisi (1982) reported that naloxone increased atropine-resistant contractions of the longitudinal muscle evoked by electrical stimulation at 20 Hz. This effect was unaltered by the addition of hexamethonium, but blocked by prior desensitization of the preparation to SP. Barthó et al. (1982b) similarly showed that naloxone enhanced atropineresistant contractions of the longitudinal muscle. These studies indicate that endogenous opioid peptides released by high frequency electrical stimulation can inhibit the release of SP from excitatory longitudinal muscle motor neurons. More direct evidence for this was provided by Holzer (1982). He directly measured SP release from LM-MP strips and showed that this was increased in the presence of naloxone. Research by Garzon et al. (1985, 1987) also indicated that endogenous opioid peptides inhibit the release of ACh and SP. They showed that contraction of LM-MP strips in response to a variety of peptides

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(corticotropin-releasing factor, CCK, neurotensin, bombesin, angiotensin II, bradykinin and SP) was enhanced by naloxone in the presence and absence of atropine. In the guinea-pig colon, naloxone was found to increase resting and electrically evoked ACh release and electrically evoked noradrenaline release, and to enhance NANC relaxations in the circular muscle. The functional relevance of endogenous opioids appears to be enhanced after chronic sympathetic denervation (Marino et al., 1993). In the guinea- pig small intestine, naloxone enhances the amplitude of ascending excitatory reflex contractions when added to the oral compartment of a partitioned organ bath (Tonini et al., 1992c). Naloxone increases the rate of peristaltic contractions in the rat small intestine (Coupar, 1995). In the same preparation, CTOP and nor-binaltorphimine (nor-BNI), selective antagonists at µ and κ receptors respectively, increase the ascending excitatory reflex, whereas the selective δ antagonist ICI 174864 is ineffective (Allescher et al., 2000). In the rat colon, naloxone enhances distension evoked descending relaxation and VIP release (Grider and Makhlouf, 1987a). Together these findings suggest that endogenous opioids acting preferentially at µ and κ receptors modulate the release of excitatory and inhibitory transmitters in the gastrointestinal tract. Peristaltic activity rapidly “fatigues” when the Trendelenburg preparation is used. In this preparation, the oral end of the intestine is occluded and fluid enters the anal end of the intestine. The intestine empties fluid against the inflow head of pressure. Van Nueten, Janssen and Fontaine (1976) showed that the opioid receptor antagonist, naloxone, restored peristaltic activity in fatigued preparations. Furthermore, the organ bath solution surrounding a fatigued preparation inhibited peristalsis in non-fatigued segments. The latter effect was antagonized by naloxone. Van Nueten and colleagues therefore concluded that fatigue was due to the release of endogenous opioid peptides. In a similar study, Kadlec and Horàcek (1980) demonstrated that inhibition of peristalsis by a “stress stimulus” (12 cm H2O intraluminal pressure and 3 g longitudinal tension) was reversed by naloxone. Davison and Najafi-Farashah (1982) demonstrated that naloxone restored peristaltic activity in fatigued segments of intestine. At higher distending pressures, when intermittent myogenic activity was recorded, naloxone also restored continuous peristaltic activity. In a series of studies, Kromer and colleagues have shown that naloxone stereospecifically increases the frequency of peristaltic waves, reduces the number and duration of peristalsis-free intervals and transiently reduces the volume expelled (Kromer and Pretzlaff, 1979; Kromer and Woinoff, 1980; Kromer, Pretzlaff and Scheiblhuber, 1980; Kromer, Pretzlaff and Woinoff, 1980). Increasing the extracellular calcium concentration attenuated the effects of naloxone (Kromer, Scheiblhuber and Illes, 1980). These effects were observed in segments of ileum and to a lesser extent in the duodenum and jejunum from adult, fetal and pregnant guinea-pigs (Kromer and Pretzlaff, 1979; Kromer, Pretzlaff and Woinoff, 1980). A similar study (Kromer, Steigemann and Shearman, 1982) showed that peristaltic activity was also enhanced by the µ antagonist and proposed σ receptor agonist, SKF 10047. However more recently, Kromer (1990) reported that the µ antagonist, CTOP, did not significantly alter peristaltic activity. Instead, peristalsis was enhanced by the κ antagonist, nor-BNI. In more physiological preparations, in which both ends of the intestine are open and fluid is infused through the oral end, intra-arterial administration of naloxone has no effect


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on peristalsis (Holzer and Lembeck, 1979; Donnerer and Lembeck, 1985). Nevertheless, dynorphin is released into the venous effluent of the vascularly-perfused guinea-pig small intestine during peristalsis (Donnerer, Holzer and Lembeck, 1984). Dynorphin release increases during peristalsis. Donnerer, Holzer and Lembeck (1984) also showed that naloxone enhances the release of substance P and somatostatin during peristalsis, but not under resting conditions. Thus endogenous opioids may inhibit the release of SP and somatostatin during peristalsis. Another study, using more physiological conditions than the Trendelenburg technique, found that the power generated by the intestine when emptying was increased in the presence of nor-BNI, under certain conditions (Waterman, Costa and Tonini, 1992). Thus, endogenous opioids acting at κ receptors, inhibited peristalsis when the intestine was forced to empty against a high resistance. SOMATOSTATIN Somatostatin is a tetradecapeptide which is present in neuronal cell bodies in the myenteric and submucous plexuses of the guinea-pig small intestine (Costa et al., 1977, 1980b; Furness et al., 1980; Schultzberg et al., 1980; Endo, Uchida and Kobayashi, 1986; Portbury et al., 1995; Song et al., 1997). In the myenteric plexus, somatostatin is contained in 4% of neurons, which project up to 70 mm in an anal direction and form very long interneuronal chains (descending interneurons) that connect with both myenteric and submucous neurons (Song et al., 1997; Mann et al., 1997b). At the electron microscope level, the neurons can be seen to form axosomatic synapses with somatostatin-positive and -negative neurons (Endo, Uchida and Kobayashi, 1986). Somatostatin-containing neurons do not project to either the circular or longitudinal muscle layers (Costa et al., 1977, 1980b; Endo, Uchida and Kobayashi, 1986). Somatostatin is colocalized with ChAT, SP and CCK in descending interneurons (Brookes, Steele and Costa, 1992; Portbury et al., 1995; Costa et al., 1996; Mann et al., 1997b; Hens et al., 2000). Effects of somatostatin on neuromuscular preparations Somatostatin reduces the amplitude of the cholinergic twitch of the LM evoked by electrical field stimulation in LM-MP and in whole wall preparations of guinea-pig ileum (Guillemin, 1976; Cohen et al., 1978, 1979; Furness and Costa, 1979b; Jhamandas and Elliot, 1980). Contractions induced by neurotensin and caerulein, but not by SP, are also inhibited (Monier and Kitagbi, 1981; Teitelbaum et al., 1984; Yau, Lingle and Youther, 1983; Yau, Dorsett and Youther, 1986b). Somatostatin has no effect on LM contractions elicited by exogenous ACh (Guillemin, 1976; Cohen et al., 1978), carbachol (Furness and Costa, 1979b) or histamine (Cohen et al., 1979), indicating that somatostatin acts by inhibiting the release of ACh rather than by interfering with its action on the smooth muscle. Inhibition of resting and evoked ACh release by somatostatin has been measured directly (Yau, Lingle and Youther, 1983; Yau, Dorsett and Youther, 1986b; Teitelbaum et al., 1984; Takeda et al., 1989; Milenov and Atanassova, 1993). More recently, somatostatin was found to possess both excitatory and inhibitory effects on myenteric cholinergic transmission, which partly involve the release of endogenous GABA. This transmitter, in turn, may enhance or inhibit ACh release via activation of GABAA or GABAB receptors,

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respectively (Roberts, Hasler and Owyang, 1993). The effect of somatostatin on the CM of the guinea-pig ileum has not been studied in detail. Kromer and Woinoff (1981) reported that somatostatin did not have any effects on the circular muscle in the presence of TTX, atropine, hexamethonium or 5-HT desensitization. Thus, somatostatin does not act directly on the CM. In the guinea-pig colon, somatostatin stimulates inhibitory motor neurons, since it produces a TTX-sensitive relaxation of the LM (Furness and Costa, 1979b). This relaxation is unaffected by guanethidine, and is therefore mediated by non-adrenergic neurons (Furness and Costa, 1979b). It is not clear whether somatostatin stimulates both the apamin-sensitive and -insensitive components of inhibitory transmission. In isolated smooth muscle cells from the stomach and colon of guinea-pig, somatostatin was found to possess a direct contractile activity (Corleto et al., 1997). In these cells, five somatostatin (SST) receptors have recognized (Corleto, Weber and Jensen, 1999), but the contractile effect was predominantly mediated by SST3 receptors in gastric cells and by SST5 receptors in colonic cells (Corleto et al., 1997). Electrophysiological effects of somatostatin on neurons Extracellular recordings of unit activity in the myenteric plexus indicate that somatostatin reduces the firing frequency of neurons (Williams and North, 1978). This effect is independent of extracellular calcium levels. Thus the action of somatostatin is likely to be a direct one on the neurons whose activity was being recorded. Intracellular recording demonstrated that somatostatin can depolarize and hyperpolarize both AH and S type neurons. The proportion of S neurons depolarized varied from 17–34% and those hyperpolarized varied from 20–31%, depending on whether the somatostatin was applied by perfusion or iontophoretically. Similarly, the proportion of AH neurons depolarized and hyperpolarized respectively was 9–29% and 8–20%. Depolarization was associated with an increase in input resistance and had a reversal potential near the equilibrium potential of potassium, suggesting that somatostatin acts by closing a potassium channel. The somatostatin-induced hyperpolarization was associated with an decrease in input resistance (Katayama and North, 1980; Liu et al., 2000). Role of endogenous somatostatin Endogenous somatostatin is released into the venous effluent during peristalsis in various species (Donnerer, Holzer and Lembeck, 1984; Schmidt, Rasmussen and Holst, 1993). This release was partly TTX-sensitive and therefore nerve mediated. However, a significant proportion is likely to represent release from mucosal endocrine cells. Somatostatin release could be increased by DMPP in a TTX-sensitive manner and by CCK-8 in TTXinsensitive manner. Somatostatin is released from isolated myenteric ganglia in response to nicotinic receptor stimulation by DMPP (Grider, 1989). The amount of somatostatin release is decreased by VIP and can be augmented by a VIP receptor antagonist (VIP10–28; Grider, 1989). This suggests that the release of somatostatin may be inhibited by endogenous VIP. Somatostatin has been reported to have a variety of effects on peristalsis. Holzer and Lembeck (1979) found that somatostatin had no immediate effect when infused intra-


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arterially. However, 12 minutes after the end of infusion, peristalsis became increasingly irregular and the volume of fluid expelled was decreased. This effect appeared to be irreversible (Holzer and Lembeck, 1979). Kromer and Woinoff (1981) studied peristalsis using the Trendelenburg method, in which the intestine “fatigues” and ceases to undergo normal peristalsis. They found that somatostatin stimulated peristalsis when it had been highly intermittent and inhibited it when it had been less intermittent. In the rat colon, somatostatin release increases along with VIP in response to the descending inhibitory reflex (Grider, Arimura and Makhlouf, 1987). Anti-somatostatin antibodies reduce VIP release and the reflex relaxation, whereas exogenous and endogenous somatostatin enhances both VIP/NO release and the reflex relaxation (Grider, 1994a). The latter effects are partly achieved through a decrease of met-enkephalin release, which exerts a tonic inhibitory influence on descending pathways. GASTRIN, CHOLECYSTOKININ AND CAERULEIN Cholecystokinins consist of a group of peptides which are C-terminal fragments of CCK-33 (Williams, 1982). Cholecystokinins, gastrin, caerulein (also known as ceruletide) and phyllocaerulein share a common pentapeptide sequence at their C-terminus and act with varying affinities at the same receptors, i.e. CCKA and CCKB receptors (Alexander and Peters, 2000). Immunohistochemical studies have demonstrated that CCK is localized in specific subpopulations of myenteric neurons in the guinea-pig small intestine: anally directed interneurons in the myenteric plexus, neurons projecting to the mucosa, intestinofugal neurons and neurons projecting from the myenteric to the submucous plexus (Furness and Costa, 1987; Brookes, Steele and Costa, 1992). In descending interneurons, CCK is colocalized with somatostatin, ChAT and SP. CCK is not present in motor neurons in either the CM or LM. Electrophysiological and immunohistochemical studies have demonstrated the presence of both CCKA and CCKB receptors in rat and guinea-pig myenteric neurons (Schutte, Akkermans and Kroese, 1997; Sternini et al., 1999a; Sayegh and Ritter, 2000). CCKA receptors are predominantly expressed in neurons containing VIP, but they are also expressed in neurons containing SP (Sternini et al., 1999a). Effects of CCK-like peptides on neuromuscular preparations Gastrin produces a contraction of the guinea-pig LM which is unaffected by hexamethonium but abolished or reduced by atropine, suggesting that the agonist acts on cholinergic LM motor neurons (Bennett, 1965; Yau et al., 1974; Yau, 1978). Caerulein and CCK-8 induce a pronounced and sustained TTX-sensitive contraction of the guinea-pig ileum LM. This contraction is partly inhibited by atropine and potentiated by eserine, suggesting that these peptides also stimulate release of ACh from LM motor neurons (Del Tacca et al., 1970; Hedner, 1970; Yau et al., 1974; Zetler, 1979; Hutchison and Dockray, 1981; Garzón et al., 1985). Increased ACh release in response to CCK-8, pentagastrin, gastrin and caerulein has been confirmed by direct measurement (Del Tacca, Soldani and Crema, 1970; Vizi et al., 1972, 1973; Yau, Lingle and Youther, 1983; Teitelbaum et al., 1984; Sano, Taniyama and Tanaka, 1989; Milenov and Atanassova, 1993). The

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atropine-resistant contraction is abolished by SP desensitization (Hutchison and Dockray, 1981), suggesting that SP or a related tachykinin is involved. Circumstantial evidence in favour of this hypothesis was provided by Holzer (1984), who showed that CCK-8 stimulates the release of SP from LM-MP preparations in a TTX-sensitive manner. In some experiments, the hyoscine-resistant contraction is reduced by about 20–35% by hexamethonium (Del Tacca, Soldani and Crema, 1970; Hedner, 1970), indicating that caerulein and CCK-8 also stimulate cholinergic interneurons. Sano, Taniyama and Tanaka (1989) found that CCK-induced release of ACh from guinea-pig LM-MP preparations was inhibited by 42% by bicuculline, suggesting that part of the effect of CCK is indirect, via release of GABA which acts on GABAA receptors. Nevertheless, neurally-mediated effects may be contractile (via release of ACh/tachykinins) or relaxant (via release of VIP/NO) as demonstrated in the CCK-mediated relaxation of gastric fundus and emptying (Grider, 1994b). CCK also appears to act directly on the LM, since higher concentrations (0.1 mM) can produce contractions in the presence of TTX. A direct effect of CCK has been demonstrated in gastric fundus and in caecal smooth muscle cells where are present both receptor subtypes, which mediate a contractile response (Grider and Makhlouf, 1987b; de Weerth et al., 1997; Motomura et al., 1997). Caerulein induces TTX-sensitive, rhythmic contractions superimposed on increased tone of the guinea-pig ileum CM (Holzer, Lembeck and Donnerer, 1980; Barthó et al., 1987a). These contractions are blocked by atropine but not hexamethonium (Holzer, Lembeck and Donnerer, 1980), suggesting that caerulein acts by stimulating ACh release from CM motor neurons. The contractions are partly or completely inhibited by desensitization to SP, indicating that tachykinin release mediates part of the contractile response. Caerulein and pentagastrin lower the threshold distension required to trigger emptying of the gut and consequently increase the frequency of emptying when the intestine is infused with fluid at a constant rate (Frigo et al., 1971; Chijikwa and Davison, 1978; Tonini et al., 1989b). Similarly, CCK-8 produces a concentration-dependent increase in the frequency of peristaltic waves and in the volume of fluid expelled (Barthó et al., 1982a). Caerulein can initiate peristalsis in the undistended intestine (Holzer and Lembeck, 1979) and restore peristalsis which has been impaired by atropine or hexamethonium (Frigo et al., 1971; Barthó et al., 1989). This effect is likely to be due to the ability of CCK to stimulate release of ACh from CM motor neurons. Electrophysiological effects of CCK-like peptides on neurons The electrophysiological effects of CCK-like peptides in the guinea pig ileum have received little attention. CCK-8 mimics slow EPSPs in 68% of S neurons and 52% of AH neurons (Nemeth, Zafirov and Wood, 1985). CCK-8 also hyperpolarizes 17% of AH neurons and 5% of S neurons. These responses were unaffected by proglumide. Caerulein had similar effects to CCK-8, although hyperpolarizing responses were not recorded (Nemeth, Zafirov and Wood, 1985). Pentagastrin had no significant effect on AH neurons, and was not tested on S neurons (Nemeth, Zafirov and Wood, 1985). In another study, CCK-8 was found to depolarize 83% of S neurons. Depolarization was also obtained with the CCKB receptor agonist CCK-8NS. Use of CCKA and CCKB selective antagonists indicated that some neurons possess exclusively the CCKA and CCKB receptor subtype,


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but others possess both subtypes (Schutte, Akkermans and Kroese, 1997). Since selective antagonists for CCKA and CCKB (L-364,78 and L-365,260, respectively) were found to inhibit slow EPSPs in myenteric neurons, this provides evidence that neurally released CCK is involved in the mediation of slow EPSPs via CCKA and CCKB receptors (Schutte et al., 1997). Role of endogenous CCK-like peptides CCK-8 is released into the venous effluent in response to distension of the intestine during peristalsis (Donnerer et al., 1985). The nicotinic receptor agonist, DMPP, did not produce a significant increase in release above basal levels (Donnerer et al., 1985). It is therefore unlikely that CCK is released from neurons during peristalsis in response to excitation by cholinergic nicotinic transmission. The role of endogenous CCK in peristalsis is not known. Peristalsis was not significantly altered by the CCK receptor antagonist, CR 1409 (Barthó et al., 1987a). Similarly, lorglumide did not produce any consistent, significant effects on hexamethonium-resistant peristalsis other than an increase in the pressure at threshold (Barthó et al., 1989). The latter may reflect a decrease in the compliance of the intestinal wall, due to either a decrease in inhibitory neuronal tone or an increase in excitatory neuronal activity (Waterman, Costa and Tonini, 1992). Thus, endogenous CCK may stimulate inhibitory CM motor neurons or inhibit excitatory CM motor neurons. Both effects are possible, given the ability of CCK to depolarize and hyperpolarize myenteric neurons. In the duodenum of rat, use of selective CCKA and CCKB receptor antagonists (L-364,718 and L-365,260, respectively) had no effect on the ascending excitation to electrical field stimulation of the mucosa, but abolished the inhibitory effect of CCK-8 on electrical stimulation (Giralt and Vergara, 2000). The role of endogenous CCK in the descending inhibitory reflex has not been investigated. Since CCK is present in descending interneurons and not in motor neurons (Furness and Costa, 1987; Brookes, Steele and Costa, 1992), the most likely role for CCK would be in the descending inhibitory reflex, rather than the reflex pathways studied previously. NEUROPEPTIDE Y (NPY) AND PEPTIDE YY (PYY) NPY is a 36 amino acid peptide which has some sequence homology to PYY and pancreatic polypeptide (PP) (Tatemoto, Carlquist and Mutt, 1982). NPY and PYY are found in 5% of myenteric neurons, including anally projecting interneurons and short descending inhibitory motor neurons to the CM (Furness and Costa, 1987; Brookes, Steele and Costa, 1991b, 1992). NPY-immunoreactive nerve terminals synapse on non NPY-immunoreactive somata and fibres (Fehér and Burnstock, 1986). PYY-like immunoreactivity was not detectable by radioimmunoassay of LM-MP synaptosome samples (McDonald et al., 1988). In the guinea-pig gastric fundus and corpus, NPY has been localized on both ascending and descending pathways projecting to the mucosa (Pfannkuche et al., 1998; Reiche and Schemann, 1999). NPY is also present in secremotor neurons in the guineapig small intestine (Lomax, Bertrand and Furness, 1998). Five functional NPY receptors (Y receptors) have been recognized so far.

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Effects of NPY on neuromuscular preparations NPY inhibits contractions of the LM induced by CCK-8 or electrical field stimulation (Garzón, Höllt and Sánchez-Blázquez, 1986; Holzer et al., 1987). This is not due to a direct inhibitory effect on the LM, since NPY does not alter concentration-response curves to ACh or carbachol in the presence of TTX (Garzón, Höllt and Sánchez-Blázquez, 1986; Holzer et al., 1987). Instead, NPY inhibits the release of ACh and a noncholinergic transmitter, probably a tachykinin, from LM motor neurons (Holzer et al., 1987; Takahashi, Yamamura and Utsunomiya, 1992). Similar results have been found in rabbit ileum, in which inhibition of ACh release is mediated by the Y5 receptor (Pheng et al., 1997). In the guinea-pig colon, PYY and NPY inhibit the twitch contractions mediated by the stimulation of cholinergic neurons and the resulting release of ACh. The inhibitory effect of PYY is mediated by a receptor located directly on cholinergic neurons, whereas the effect of NPY is mediated by noradrenaline release due to stimulation of a receptor located on adrenergic neurons (Sawa et al., 1995). NPY abolishes phasic TTX-sensitive contractions of the CM induced by DMPP or caerulein (Holzer et al., 1987). However, TTX-insensitive contractions evoked by carbachol are unaffected. Thus, NPY inhibits CM contractions through an action on excitatory CM motor neurons rather than by a direct effect on the CM. Such an action may also underlie the reduction in basal CM tone produced by NPY in some preparations (Holzer et al., 1987). NPY inhibits the ascending excitatory reflex contraction of the CM in response to balloon distension (Holzer et al., 1987). Both the atropine-sensitive and atropine-resistant components of the response are inhibited. Although these effects are likely to be due to inhibition of excitatory CM motor neurons, NPY needs to be tested in a partitioned organ bath to rule out the possibility that it also inhibits interneurons or sensory neurons. The inhibitory effect of NPY on the ascending excitatory reflex is significantly reduced by apamin (Holzer et al., 1987). NPY may therefore inhibit neurons by opening a calcium-dependent potassium channel in the neuronal membrane. Alternatively, NPY may stimulate inhibitory CM motor neurons which relax the muscle through an apamin-sensitive mechanism. At variance with the effects observed in the guinea-pig intestine, NPY enhances ACh release in the rat colon by acting on Y2 and Y4 receptors (Pheng et al., 1999). Y2 receptors have been also characterized in colonic smooth muscle cells (Feletou et al., 1998). Electrophysiological effects of NPY and related peptides on neurons NPY and PPY and bovine PP suppress nicotinic fast EPSPs (and ACh release) in guineapig gastric myenteric neurons via a mechanism insensitive to α-adrenoceptor blockade (Schemann and Tamura, 1992). These peptides have been found to inhibit fast EPSPs in myenteric neurons of descending colon through a mechanism involving activation of presynaptic Y2 receptors (Browning and Lees, 2000).

CALCITONIN GENE-RELATED PEPTIDE CGRP is a 37-aminoacid peptide localized in blood vessels and nerves of the gastrointestinal tract. Based upon the differential biological activity of various CGRP analogs, CGRP


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receptors have been classified into two major classes, namely the CGRP1 and CGRP2 subtypes (van Rossum, Hanisch and Quirion, 1997). In the guinea-pig ileum, CGRP is present in intrinsic and extrinsic fibres. The intrinsic fibres supply only the mucosa (Costa, Furness and Gibbons, 1986). Fibres innervating the myenteric plexus which are immunoreactive for CGRP are of extrinsic origin (Costa, Furness and Gibbons, 1986a). During peristalsis, CGRP may potentially be released from both sources. In fact, at least in the rat colon, extrinsic sensory pathways, which mediate the peristaltic response to muscle stretch, and intrinsic sensory pathways, which are involved in the peristaltic response to muscular stimulation, utilize CGRP as a sensory transmitter (Grider, 1994c). In the guinea-pig small intestine, however, extrinsic sensory neurons do not participate in the neural pathway mediating peristalsis, but stimulate peristaltic activity following capsaicin stimulation (Barthó and Holzer, 1995). CGRP produces a transient contraction followed by a longer relaxation of the guineapig ileum LM-MP preparation (Barthó et al., 1987b; Sun and Benishin, 1991). The contraction is blocked by TTX, atropine, 2-chloroadenosine, mepyramine and clonidine (Tippins et al., 1984; Sun and Benishin, 1991), suggesting that the contraction is mediated by ACh released from LM motor neurons and acting at muscarinic receptors. Histamine may also be involved in this response. The CGRP-induced relaxation is partly nerve-mediated, since it is partly blocked by TTX. However, CGRP also has a direct relaxing effect (Barthó et al., 1987b; Sun and Benishin, 1991) which is not antagonized by apamin (Holzer et al., 1989). CGRP has also been shown to inhibit electrically induced cholinergic contractions of the guinea-pig ileum LM-MP preparation (Barthó et al., 1987b) via CGRP1-like receptor activation (Tomlinson and Poyner, 1996). In smooth muscle cells isolated from the circular and longitudinal layers of guinea-pig ileum, CGRP triggers different intracellular pathways to induce relaxation: cAMP is involved in cells from both layers while NO is involved only in relaxation of circular smooth muscle cells (Rekik et al., 1997). CGRP induces phasic contractions of the guinea-pig ileum CM by stimulating ACh release from cholinergic motor neurons (Holzer et al., 1989). Part of the action of CGRP is likely to be on cholinergic interneurons, since the phasic contractions are reduced by hexamethonium (Holzer et al., 1989). CGRP modulates peristalsis by increasing the pressure which occurs at threshold (Holzer et al., 1989). This probably reflects a decrease in the compliance of the intestinal wall and increased CM tone due to either an increase in excitatory neuronal activity or a decrease in inhibitory neuronal activity. The former is more likely given the excitatory effect of CGRP on cholinergic motor neurons mentioned previously and that CGRP has not been reported to inhibit neurons. In agreement with this, CGRP was shown to enhance the amplitude of the ascending excitatory reflex (Holzer et al., 1989). In guinea-pig distal colon CGRP inhibits electrically-evoked NANC contractions through a postjunctional mechanism (Kojima, 1997). In electrophysiological studies, CGRP mimicked the slow EPSP in all myenteric AH neurons when applied by superfusion and pressure ejection. Depolarization was associated with an increase in input resistance, suppression of post-spike hyperpolarization and enhanced excitability (Palmer et al., 1986a). This effect may be mediated by a decrease in

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the calcium-activated potassium conductance which is active in resting AH neurons (Palmer et al., 1986a). NEUROTENSIN Neurotensin immunoreactivity in the guinea-pig small intestine occurs mainly in the mucosa (Helmstaedter et al., 1977; Sundler et al., 1977; Schultzberg et al., 1980; Leander et al., 1984). In these studies, neurotensin-like immunoreactivity was not found in myenteric neurons of the guinea-pig ileum. However, other researchers have reported neurotensin-like immunoreactivity in these neurons (Reinecke et al., 1983; Tange, 1983). Neurotensin has been measured by radioimmunoassay in the external muscle layers of the guinea-pig ileum by one research group (Holzer et al., 1982) but was not found by another (Furness et al., 1982). It is possible that the discrepancies relate to the specificity of the antibodies used. Consequently, a peptide similar to neurotensin rather than authentic neurotensin may be present in the guinea-pig small intestine. Neurotensin binding sites have been demonstrated in the CM (Goedert, Hunter and Ninkovic, 1984). Recently, two neurotensin receptors have been identified in various tissues and selective antagonists for neurotensin-1 (NTS1) receptors have been developed (Vincent, Mazella and Kitabgi, 1999). Pharmacological experiments have demonstrated that neurotensin induces relaxation and then contraction of the guinea-pig LM-MP preparation (Kitagbi and Freychet, 1978, 1979a; Monier and Kitagbi, 1980; Goedert, Hunter and Ninkovic, 1984; Wagner and Wahl, 1986). The contraction is nerve-mediated, since it is TTX-sensitive (Kitagbi and Freychet, 1978, 1979a; Zetler, 1980; Garzón et al., 1985) and may be mediated by the release of ACh (Kitagbi and Freychet, 1978, 1979b; Zetler, 1980; Monier and Kitagbi, 1981; Huidobro-Toro and Way, 1982; Yau, Verdun and Youther, 1983; Teitelbaum et al., 1984; Garzón et al., 1985; Yau, Dorsett and Youther, 1986b; Nakamoto, Tanaka and Taniyama, 1987; Rakovska, 1993) and SP (Monier and Kitagbi, 1980). Neurotensin stimulates the release of ACh and SP from LM-MP preparations in a TTX-sensitive and calciumdependent manner (Yau, Verdun and Youther, 1983; Holzer, 1984; Yau, Dorsett and Youther, 1986b; Nakamoto, Tanaka and Taniyama, 1987). The neurotensin enhancement of ACh release is inhibited by stimulation of α2-adrenoceptor agonists (Rakowska, 1993). Stimulation of ACh release is unaffected by hexamethonium, hyoscine or SP receptor antagonists (Kitagbi and Freychet, 1979b; Nakamoto, Tanaka and Taniyama, 1987), suggesting that neurotensin may directly stimulate excitatory LM motor neurons. The action of neurotensin is likely to be at least partly at the level of the nerve terminals, because neurotensin is capable of stimulating ACh release from myenteric synaptosomes (Yau, Lingle and Youther, 1983). Neurotensin-induced contraction is partly inhibited by bicuculline. Since neurotensin also stimulates GABA release, it has been proposed that neurotensin may stimulate ACh release directly and also via release of GABA (Nakamoto, Tanaka and Taniyama, 1987). Neurotensin produces a direct relaxation of the guinea-pig ileum LM (Kitagbi and Freychet, 1978, 1979a; Goedert, Hunter and Ninkovic, 1984). This effect does not appear to involve cAMP or cGMP, since levels of these nucleotides are unaffected by neurotensin (Kitagbi and Freychet, 1979a). The relaxation is however, inhibited by apamin (Holzer


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et al., 1989), suggesting that neurotensin may open small conductance calcium-activated potassium channels. Neurotensin induces a direct relaxation of the guinea-pig ileum CM, which is apaminsensitive and blocked by antibodies to neurotensin. Furthermore, relaxation induced by nerve stimulation is partly blocked by neurotensin antibodies (Goedert, Hunter and Ninkovic, 1984). This led to the proposal that neurotensin is a NANC inhibitory transmitter in the guinea-pig ileum. In the rat stomach strips, neurotensin causes a contractile response, which results from direct activation of smooth muscle receptors (Nguyen et al., 1997). In canine ileal circular muscle devoid of myenteric plexus, neurotensin causes an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase. The neurotensin-induced inhibitory effect is mediated by activation of apamin-sensitive, calcium-dependent potassium channels (Christinck, Daniel and Fox-Threlkeld, 1992). In rat proximal colon, neurotensin causes a summation of excitatory and inhibitory effects, which are dependent on the influx of Ca2+ via L-type channels (Mule and Serio, 1997). Neurotensin contracts human colonic circular smooth muscle strips. The insensitivity of this contractile effect to atropine, levocablastine (a NTS2 receptor agonist) or TTX and the blockade with SR 48692 and SR 142948 (two selective NTS1 receptor antagonists) is consistent with the involvement of non-neuronal NTS1 receptors (Croci et al., 1999). Neurotensin infused intravascularly increases the frequency of peristaltic waves and the volume of fluid emptied per unit time in the guinea-pig ileum (Barthó et al., 1982a). However, atropine-resistant peristalsis is initially stimulated and then inhibited by neurotensin (Barthó et al., 1982a). These results were interpreted by the authors to represent the stimulatory effect of neurotensin on ACh and SP release and its subsequent direct inhibitory effect on smooth muscle. The non peptide NTS1 receptor antagonist SR 48692 abolished both effects. The inhibitory action of neurotensin apparently involves an apamin-sensitive mechanism (Ohashi et al., 1996). Electrophysiological studies demonstrated that neurotensin mimics the slow EPSP in 48% of S neurons and 26% of AH neurons in the guinea-pig myenteric plexus. Calciumdependent hyperpolarization was recorded in 18% of AH neurons but not in S neurons (Williams, Katayama and North, 1979). MOTILIN Motilin is a 22-aminoacid residue polypeptide, which is released at about 100-min intervals during the interdigestive state, while the presence of nutrients in the duodenum strongly suppresses its endogenous release. Although, strictly speaking, motilin is a gastrointestinal hormone rather than a neurotransmitter, it will be briefly dealt with in this chapter in light of the interesting developments on motilin agonists as gastrointestinal prokinetics (Peeters, 1993; Clark et al., 1999; Siani et al., 2000). For the history of the discovery of motilin and erythromycin derivatives as prokinetics, the reader is referred to the reviews by Itoh (1997) and Peeters (1993). In this context, it will suffice to remember that erythromycin and its derivatives, which are devoid of antibacterial activity such as EM574 (motilin-like macrolides or motilides), are strong motilin

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agonists and profoundly stimulate gut motility. This effect is brought about both through a direct action on smooth muscle motilin receptors (Depoortere, Peeters and Vantrappen, 1991; Hasler, Heldsinger and Owyang, 1992) and through neural receptors (Peeters, 1993; Van Assche, Depoortere and Peeters, 1995). Smooth muscle contraction is induced through a nifedipine-sensitive mechanism (which therefore involves entry of extracellular calcium), but there is evidence that the intracellular calcium pool may also have a role (Matthijs, Peeters and Vantrappen, 1989; Peeters, Matthijs and Vantrappen, 1991; Depoortere and Peeters, 1995; Farrugia et al., 1995). A neurally mediated effect of motilides in the gastrointestinal tract is indicated, at least in some models, by the observation that their effects are antagonized by atropine, hexamethonium or ondansetron (Peeters, 1993; Mizumoto et al., 1993; Fiorucci, Santucci and Morelli, 1993; Shiba et al., 1995; Van Assche, Depoortere and Peeters, 1995; Parkman, Pagano and Ryan, 1996; Boivin et al., 1997). Indeed, vagal and non-vagal cholinergic pathways as well as serotonergic pathways seem to play an important role in mediating the motor effect of motilides (Itoh, 1997; Inatomi et al., 1996). There are also data documenting the presence of motilin receptors in the central nervous system (Itoh, 1997). Inhibitory effects sometimes observed with motilides have been ascribed to stimulation of inhibitory neural pathways or Ca2+-channel blockade (Parkman, Pagano and Ryan, 1996; Minocha and Galligan, 1991; Furness et al., 1999). These effects, however, usually occur at high concentrations and are resistant to motilin receptor antagonists (Furness et al., 1999). GASTRIN-RELEASING PEPTIDE AND NEUROMEDIN B GRP is a 27-amino acid peptide which has been found in a variety of mammals along with the related peptide, neuromedin B (Spindel, 1986; Furness and Costa, 1987; Ohki-Hamazaki, 2000). Bombesin and alytesin are tetradecapeptides, isolated from amphibia, which share a similar sequence to the C-terminus of GRP (Ersparmer et al., 1972; Ersparmer and Melchiorri, 1980). Ranatensin is an endecapeptide which is also related to GRP (Ersparmer et al., 1972; Nakajima, 1981). GRP-immunoreactive neurons are found in the myenteric and submucous plexuses in the guinea-pig small and large intestine (Schultzberg et al., 1980; Hutchison, Dimaline and Dockray, 1981; Messenger, 1993). Myenteric neurons containing GRP include descending interneurons and long descending inhibitory motor neurons to the CM (Costa et al., 1984; Messenger, 1993). These neurons are also immunoreactive for VIP, NOS and dynorphin (Brookes, Steele and Costa, 1992). GRP has also been identified in LM-MP synaptosomes by radioimmunoassay (McDonald et al., 1988). In the guinea-pig gastric fundus, GRP is present in ascending excitatory muscle motor neurons and in neurons involved in the regulation of mucosal functions (Pfannkuche et al., 2000). Neuromedin B and GRP receptor mRNAs have been detected in human and rabbit colonic smooth muscle cells (Bitar and Zhu, 1993). In the cat, GRP and neuromedin B evoke concentration-dependent contractions in circular strips of esophagus and fundus (Milusheva et al., 1998). Similar responses have been detected in longitudinal strips of the duodenum and ileum longitudinal muscles (Kortezova et al., 1994; Milusheva et al., 1998).


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Bombesin, GRP and neuromedin B produce TTX-sensitive, tonic contractions of the guinea-pig ileum LM-MP preparation (Zetler, 1980; Pfannkuche, 1992). The contractions are inhibited by atropine, suggesting that these peptides stimulate the release of ACh from LM motor neurons (Ersparmer et al., 1972; Zetler, 1980; Garzón et al., 1985). Bombesin also stimulates SP release from LM-MP preparations in a TTX-sensitive manner (Holzer, 1984). Bombesin-, GRP- and neuromedin B-induced contractions are not altered by mepyramine, nicotine or methysergide (Ersparmer et al., 1972; Pfannkuche, 1992), suggesting that the peptides do not stimulate ACh release from cholinergic interneurons and that the contraction is independent of histamine and 5-HT. Bombesin increases the tone of guinea-pig ileum CM and induces rhythmic contractions (Holzer, Lembeck and Donnerer, 1980) in a TTX-sensitive way. However, this effect is not inhibited by tropicamide or hexamethonium (Holzer, Lembeck and Donnerer, 1980), suggesting that bombesin does not stimulate cholinergic CM motor neurons or interneurons. GRP modulates the mechanical activity of the human ileocaecal region in vitro. Longitudinal strips show a concentration-dependent increase in the rhythmic activity, whereas the circular strips react with a small decrease in tone. These effects are not affected by TTX, suggesting a direct action of GRP on smooth muscle probably mediated by distinct receptors (Vadokas et al., 1997). A study by Barthó et al. (1982a) demonstrated that intra-arterial infusion of bombesin in vitro stimulates peristalsis. This effect has not been investigated further. However, it is possible that the stimulation of peristalsis is a result of slow depolarization of excitatory CM motor neurons, causing increased excitability and increased release of a non-cholinergic transmitter. Bombesin and GRP mimic slow EPSPs in AH neurons. Depolarization is associated with an increase in input resistance which may be due to the closing of a calcium-dependent potassium channel (Zafirov et al., 1985). GALANIN Galanin is a 29-amino acid peptide originally isolated from porcine intestine (Tatemoto et al., 1983). Galanin immunoreactivity in guinea-pig ileal myenteric neurons is localized in specific subpopulations: neurons projecting to the mucosa, descending inhibitory motor neurons to the CM, descending interneurons and LM motor neurons (Melander et al., 1985; Furness et al., 1987). Galanin has also been identified by radioimmunoassay and high pressure liquid chromatography in LM-MP synaptosomes (McDonald et al., 1988). Galanin is present in anally projecting pathways in the guinea-pig colon (Messenger, 1993) and colocalizes with NOS and VIP in canine antrum, small bowel and colon (Wang et al., 1998). Galanin does not alter basal activity of the LM of guinea-pig ileum (Kuwahara, Ozaki and Tanaihara, 1989) and has no effect on neurally-evoked twitch responses (Kuwahara, Ozaki and Tanaihara, 1989, 1990). More recently, however, galanin was found to possess a marked inhibitory effect on twitch responses, probably via activation of GAL1 receptors (Sternini et al., 1999b), and on tritiated ACh release (Mulholland, Schoeneich and Flowe, 1992). Galanin does not alter the basal activity of the CM of guinea-pig ileum, but inhibits TTX-sensitive, electrically evoked contractions (Kuwahara, Ozaki and Tanaihara, 1989,

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1990). Galanin inhibits both the hyoscine-sensitive and -resistant components of the CM twitch (Kuwahara, Ozaki and Tanaihara, 1989), suggesting that it may act by inhibiting release of ACh and a tachykinin from excitatory CM motor neurons. Yau, Dorsett and Youther (1986a) demonstrated that galanin inhibits the electrically stimulated release of [3H]ACh from the myenteric plexus. It has no effect on ACh-induced contractions in the presence of TTX (Kuwahara, Ozaki and Tanaihara, 1989). In agreement with this, Grider and Makhlouf (1988a) found that galanin alone had no contractile or relaxant effect on isolated circular smooth muscle cells. However, galanin potentiated the relaxant response to VIP, isoproteronol and dibutyryl cAMP. This effect was blocked by apamin and by tetraethylammonium at high concentrations although apamin and tetraethylammonium did not alter the response to VIP, isoprenaline or dibutyryl cyclic AMP alone (Grider and Makhlouf, 1988a). Thus galanin appears to act by opening potassium channels in the smooth muscle which in turn enhances the relaxation produced by agents increasing cyclicAMP levels. Galanin may contract ileal circular muscle of various species (pig, guineapig, rat and rabbit) and relaxes dog ileum by a direct myogenic effect (Botella et al., 1992). Recently, out of the three galanin receptors so far characterized, a single receptor subtype (i.e. the GAL2 receptor) was recognized in the rat intestine (Waters and Krause, 2000). Galanin hyperpolarizes myenteric neurons, decreases input resistance and suppresses excitability. These effects mimic slow IPSPs in myenteric neurons (Palmer et al., 1986b). Galanin also inhibits fast EPSPs in S neurons. However, this does not appear to be due to an action at a presynaptic inhibitory receptor, since galanin also inhibits the depolarization evoked by exogenous ACh (Tamura, Palmer and Wood, 1987). NEUROMEDIN U Neuromedin U was originally isolated from porcine spinal cord (Minamino, Kangawa and Matsuo, 1985), but it has also been extracted from the guinea-pig ileum (Murphy et al., 1990), and identified by radioimmunoassay and high pressure liquid chromatography (Augood, Keast and Emsom, 1988). Neuromedin U immunoreactivity is localized in Dogiel type II, calbindin-positive and -negative myenteric neurons, anally projecting myenteric neurons and myenteric neurons projecting to the submucous plexus (Furness et al., 1989a). It is not found in motor neurons. In the rat gastric fundus CM and in turtle small intestine LM, neuromedin U evokes a concentration-dependent contraction (Bockman et al., 1989; Maggi et al., 1990b; Benito-Orfila et al., 1991). Since these responses are unaffected by atropine or TTX (Bockman et al., 1989), they are likely to be due to a direct effect of neuromedin U on the muscle. However CM from frog stomach and LM from rat and frog small intestine are insensitive to neuromedin U. Similarly, neuromedin U does not have any significant effect on LM from the guinea-pig ileum (Minamino, Kangawa and Matsuo, 1985; Bockman et al., 1989). Human neuromedin U cDNA has been cloned and characterized (Austin et al., 1995). Neuromedin U mRNA is expressed in human small intestine and stomach (Hedrick et al., 2000). Recently, in the human and rat intestine, a receptor for neuromedin U has been identified, which probably corresponds to the orphan G protein-coupled receptor FM3 (Raddatz et al., 2000; Szerekes et al., 2000; Fujii et al., 2000).


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BRADYKININ Bradykinin receptors of the B2 but not B1 subtype are located on the LM and CM but not the myenteric plexus of guinea-pig ileum (Manning et al., 1982; Haasemann et al., 1991; Tousignant et al., 1991; Ransom et al., 1992), and on colonic CM (Hasler et al., 1995). The B2 receptor has been cloned and expressed in Xenopus oocytes; it is a G proteincoupled receptor that appears to stimulate a chloride current (McEachern et al., 1991). Bradykinin induces contraction of LM-MP preparations from the guinea-pig ileum (Garzón et al., 1985) which is largely unaffected by atropine (Khairallah and Page, 1961, 1963; Garzón et al., 1987). However, bradykinin reverses the inhibitory effects of opioids on the cholinergic twitch in a hyoscine-sensitive manner (Goldstein et al., 1983). Thus bradykinin appears to stimulate ACh release from cholinergic motor neurons to a variable degree. The non-cholinergic component of the contraction may be mediated by the release of a non-cholinergic transmitter or by a direct action of bradykinin on the smooth muscle. Bradykinin produces a biphasic response in the guinea-pig ileum CM, i.e. contraction followed by relaxation (Calixto and Medeiros, 1991). Neither response is mimicked by a B1 receptor agonist. Both the contraction and relaxation are unaffected by atropine, prazosin, yohimbine, propranolol, pyrilamine, [Leu8]BK1–8, TTX, glibenclamide or phorbol ester (Calixto and Medeiros, 1991). Thus neither response involves the action of endogenous transmitters at muscarinic, α1-adrenoceptor, α2-adrenoceptor, β-adrenoceptor, H1 or B1 receptors. Since the effect of bradykinin is TTX-insensitive, it is likely to be a direct effect on the smooth muscle. Furthermore, bradykinin does not open ATP-sensitive potassium channels or act via stimulation of protein kinase C activity. Bradykinin induces colonic smooth muscle contraction via B2 receptors, with phosphoinositide turnover activation and adenylate cyclase inhibition (Hasler et al., 1995). In this preparation, the bradykinin-induced contraction is mediated by influx of extracellular Ca2+ via non-selective cation channels and, in part, by the release of Ca2+ from loosely bound internal store (Zagorodnyuk, Santicioli and Maggi, 1998). In the taenia caeci, bradykinin produces a relaxation followed by a contraction. This response is inhibited by the B2 receptor antagonist Hoe 140. Since both phases of the response are TTXinsensitive, it is likely that they represent a direct effect on smooth muscle (Field et al., 1994). In the opossum lower oesophageal sphincter, bradykinin induces relaxation which is apamin-sensitive, followed by a contraction (Saha, Sengupta and Goyal, 1991). The contraction is inhibited by nifedipine, but not TTX, suggesting a direct effect of bradykinin on the muscle. In the rat isolated duodenum, bradykinin induces a biphasic response (relaxation followed by contraction), which is mediated by activation of B2 receptors (Rhaleb and Carretero, 1994). Previous evidence demonstrated that the B2 receptor-mediated relaxation was apamin sensitive (Hall and Morton, 1991; Greisbacher, 1992). Longitudinal strips of postmortem human ileum display a strong contractile response to bradykinin. This effect is mediated by constitutive B2 receptors and inducible B1 receptors (Zuzack et al., 1996).

ANGIOTENSIN Immunohistochemical studies suggest that the octapeptide angiotensin is present in enteric neurons of the guinea-pig ileum, although angiotensin has not been extracted from the external musculature (Furness and Costa, 1982).

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Angiotensin contracts the LM of the guinea-pig ileum. This effect was originally believed to be due to, in part to, stimulation of ACh release from LM motor neurons for several reasons. The contraction was reduced by 60–85% by atropine and enhanced by anticholinesterase inhibitors (Khairallah and Page, 1961; Robertson and Rubin, 1962; Khairallah and Page, 1963; Blair-West and McKenzie, 1966). Hexamethonium does not inhibit the angiotensin-evoked contraction (Khairallah and Page, 1961; Robertson and Rubin, 1962; Khairallah and Page, 1963). However Paiva et al. (1976) demonstrated that the concentrations of atropine used in previous studies resulted in non-specific inhibition of contraction produced by a variety of agonists including histamine and bradykinin, both of which are believed to act directly on the muscle. The mechanism whereby angiotensin contracts the LM has therefore yet to be elucidated, but a direct effect on smooth muscle cells (as also observed in CM: Shimuta et al., 1999) is likely (Smith, Taylor and Whiting, 1994). In addition, angiotensin was found to produce a concentration-dependent inhibitory effect on twitch responses in LM-MP preparations (Smith, Taylor and Whiting, 1994). By contrast, in rat colonic preparations, angiotensin was found to facilitate ACh release at high concentrations (Voderholzer, Allescher and Muller-Lissner, 1995). The pharmacological and electrophysiological effects of angiotensin on myenteric neurons are unknown. ENDOTHELIN-LIKE PEPTIDES The endothelin (ET) family of peptides was originally isolated from cultured porcine aortic endothelial cells (Yanagisawa et al., 1988; Saida, Mitsui and Ishida, 1989). However, endothelins have also been found in human intestine and in human and porcine spinal cord neurons (Giaid et al., 1989; Shinmi et al., 1989; Egidy et al., 2000). ET-1, ET-2, ET-3 and vasoactive intestinal contractor, a related peptide, produce contraction of the isolated guinea-pig longitudinal smooth muscle cells through an action on the ETB receptor subtype. This contraction is dependent on intracellular and extracellular calcium (Yoshinaga et al., 1992). Apart from a direct effect on the smooth muscle, endothelins inhibit nerve-evoked LM contractions and ACh release (Maggi et al., 1989; Wiklund et al., 1989). This effect may be mediated by the ETA receptor subtype (Maggi et al., 1989). Endothelin receptors are present in enteric neurons and seem to play an important role in the development of the enteric nervous system (Gershon, 1997). ETA receptor activation stimulates, whereas ETB receptor activation inhibits peristalsis in guinea-pig isolated small intestine. The ability of BQ-788, a selective ETB receptor antagonist, to facilitate peristalsis per se, points to a physiological role of ETB receptors in peristaltic motor regulation (Shahbazian and Holzer, 2000).

CONCLUSIONS The intestine is capable of complex behaviours which allow mixing, propulsion, digestion and absorption of food. The mechanism whereby the intrinsic neurons of the enteric nervous system and the circular smooth muscle coordinate their activities to produce propagated contractions has been the subject of intensive investigations for almost a century. Peristalsis was originally described in 1899 and studied by a few investigators


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at the turn of the century. A burst of research activity occurred in the 1950s and 1960s. Since the 1980s interest has been focussed on the characterization of neuronal circuitries and mechanisms underlying peristalsis. An integrated approach to study a variety of parameters of intestinal function has shed some light on the factors which determine the threshold distension for triggering emptying, the strength of the contraction, the mechanism whereby the contraction propagates anally and the speed of propagation. The intestine contains a large number of neuroactive substances and an equally large number of receptors for a variety of chemicals. As a consequence, the LM-MP preparation of the guinea-pig ileum has been widely used by pharmacologists to test new drugs. We therefore have quite detailed information about the effect of drugs on LM motor neurons and the LM. Conversely, drugs are tested less frequently for their effects on CM motor neurons or CM activity. However, the irony is that the role of the LM in intestinal motor function is less understood than that of the CM. Furthermore, although we have detailed information on the effects of exogenous agents on a layer of muscle which appears to have a scarce role in peristalsis, we know little of the role of most endogenous chemicals. It is clear therefore, that future studies need to address this imbalance. The development of selective antagonists to receptors for neuropeptides, in particular, will greatly assist the ultimate goal of understanding the neuronal basis of peristalsis.

ACKNOWLEDGMENTS The authors wish to thank Dr. Amalia Di Nucci and Dr. Cristina Giaroni for their helpful suggestions during drawing up of the chapter, and Ms. Elena Giovanetti for assistance in preparing the reference list.

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7 Neuroeffector Transmission in the Intestine Charles H.V. Hoyle1, Pam Milner2 and Geoffrey Burnstock2 1

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK 2 Autonomic Neuroscience Institute, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK This chapter focuses on neuromuscular and neuroepithelial transmission in the intestine. There seems to be an ever increasing number of putative neurotransmitters in the enteric nervous system, but relatively few have a defined role in either neuromuscular or neuroepithelial transmission even though their chemical coding may have been established. In addition to covering the “old” classic transmitters, acetylcholine and noradrenaline, some more novel candidate transmitters such as endothelin and pituitary adenylate cyclase-activating polypeptide are discussed, as well as the “modern” classical transmitters ATP and vasoactive intestinal polypeptide (VIP), and the notso-classical nitric oxide. Co-transmission is a widespread phenomenon in the enteric nervous system and particularly with regard to inhibitory transmission, it is difficult to discuss one transmitter, such as ATP, in isolation from others such as nitric oxide and VIP. An area of current research is the plasticity of the enteric nervous system, and how expression of neurotransmitters changes in response to ageing, trauma, surgery or chronic drug treatment, and what the mechanisms underlying these changes are. Some pathological conditions are considered, namely diabetes mellitus, idiopathic chronic constipation, Hirschsprung’s disease and ulcerative colitis, in all of which intestinal neuromuscular transmission may be affected. KEY WORDS: intestinal neuromuscular transmission; intestinal neuroepithelial transmission; enteric nervous system; co-transmission; neuroplasticity.

INTRODUCTION HISTORICAL CONCEPTS Studies of neurotransmission in the enteric nervous system have played an important role in revealing the diversity of the autonomic nervous system as a whole and in developing new concepts in neurotransmission that are now being fully realised. The first clear evidence for non-adrenergic, non-cholinergic (NANC) neurotransmission came from two studies in 1963, one an electrophysiological study on guinea-pig intestine and 295

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the other a pharmacological study on cat stomach. These studies showed recordings of inhibitory junction potentials (IJPs) in intestinal smooth muscle during stimulation of enteric nerves in the presence of adrenergic and cholinergic blocking agents (Burnstock et al., 1963) and relaxation of stomach following vagal stimulation with adrenergic and cholinergic blockade (Martinson and Muran, 1963). They challenged the classical view of antagonistic actions of noradrenaline and acetylcholine causing either constriction or relaxation. In 1970, the purine nucleotide, adenosine 5′-triphosphate (ATP), was proposed as a principal transmitter in NANC neurotransmission (Burnstock et al., 1970; Burnstock, 1972). The recent molecular biological approaches to identify the molecular structures for purine receptor subtypes has greatly reinforced the concept of purinergic neurotransmission (Burnstock, 1997). MULTIPLICITY OF NEUROTRANSMITTERS IN THE ENTERIC NERVOUS SYSTEM Hints that there are in fact several different neurotransmitters in autonomic nerves came from ultrastructural studies of the enteric nervous system. At least 9 distinguishable types of axon profile were described (Cook and Burnstock, 1976). Subsequently, using newly available immunohistochemical techniques, several biologically active peptides were localised in neural elements of the gut (Furness and Costa, 1987). In addition to many polypeptides, 5-hydroxytryptamine (5-HT), dopamine and γ-aminobutyric acid (GABA) were proposed as autonomic neurotransmitters (see Gershon, Mawe and Branchek, 1989; Hills and Jessen, 1992). More recently, nitric oxide (NO) has been added to the list of neurotransmitters in the gastrointestinal tract (for reviews see Rand, 1992; Saffery et al., 1992; Sanders and Ward, 1992; Lefebvre, 1995; Lincoln, Hoyle and Burnstock, 1997). The rapid expansion of the number of proposed enteric neurotransmitters in recent years, including endothelin (Inagaki et al., 1991; Lin and Lee, 1992), vasoactive intestinal contractor (Saida, Mitsui and Ishida, 1989), secretoneurin (Schmid et al., 1995; Schürman et al., 1995; Dun et al., 1997), glutamate (Burns and Stephens, 1995) and carbon monoxide (Rattan and Chakder, 1993; Verma et al., 1993; Werkström et al., 1997), makes it likely that the list is still incomplete (Table 7.1). CHEMICAL CODING, CO-TRANSMISSION AND NEUROMODULATION The concept of co-transmission, that some nerves release more than one neurotransmitter (Burnstock, 1976) is well illustrated in the enteric nervous system where several different neuropeptides have been localised in a single neurone. Many of these substances act as neuromodulators, enhancing or diminishing the release or actions of primary transmitters. The precise combinations of substances contained in individual enteric neurones and their projections and central connections, termed “chemical coding”, have been defined in an elaborate series of surgical manipulations of the guinea-pig intestine (for reviews see Furness and Costa, 1987; Furness et al., 1992). Fourteen separate classes of neurones, accounting for more than 90% of the myenteric neurones of the guinea-pig ileum, have now been identified (Costa et al., 1996). Whilst there are some inter-species and interregional variabilities, some aspects appear to be constant: the primary transmitters are

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TABLE 7.1 Proposed neurotransmitters/neuromodulators in the enteric nervous system. Acetylcholine Adenosine 5′-triphosphate Angiotensin Calcitonin gene-related peptide Carbon monoxide Cholecystokinin Dopamine Dynorphins Endorphins Endothelin Enkephalins Galanin γ-Aminobutyric acid Gastrin Gastrin releasing peptide/bombesin Helodermin Helospectin 5-Hydroxytryptamine MERGL

Neo-endorphin Neurokinin A, B Neuromedin B, C, U Neuropeptide Y Neurophysin Neurotensin Nitric oxide Noradrenaline Oxytocin Peptide histidine isoleucine Peptide histidine methionine Pituitary adenylate cyclase-activating peptide Secretoneurin Somatostatin Substance P Thyrotropin-releasing hormone Vasoactive intestinal peptide Vasoactive intestinal contractor

generally well preserved in functionally equivalent neurones but their co-transmitters and neuromodulators may differ between species and regions of the gastrointestinal tract (Furness et al., 1994). Use of high affinity antagonists for many peptide receptors and recent advances in cloning genes encoding peptide receptor subtypes have enabled a detailed description of the molecular events involved in enteric peptidergic transmission (Dockray, 1994). Although there are many different transmitter substances in the gut, most are involved in neurotransmission or neuromodulation at the ganglion level and/or may have a trophic role. The number involved in neuromuscular transmission is more limited.

AUTONOMY OF THE ENTERIC NERVOUS SYSTEM The ability of the enteric nervous system to sustain local reflex activity independent of the central nervous system has been recognised for many years since the original studies of Bayliss and Starling (1899). This functional autonomy is attributed to the integrated intrinsic ganglionated plexuses which innervate the gastrointestinal tract. Scanning and electronmicroscopic studies have shown that the organisation of these ganglia is closer to that of the central nervous system than that of the sympathetic or parasympathetic ganglia (Gabella, 1972; Jessen and Burnstock, 1982; Gabella, 1990). Similarities with the central nervous system include the compact organisation of neural and glial cells with a paucity of extracellular space, the lack of penetration of either connective tissue or blood vessels into the ganglia and the limited access of intravascular molecules i.e. a blood-ganglion barrier analogous to the blood-brain barrier. For these reasons, enteric tissues have been proposed

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as grafts into the central nervous system as prospective treatment of neurodegenerative diseases. Enteric neurones and associated cells implanted together with the surrounding smooth muscle are able to survive in the corpus striatum for up to a year and stimulate axonal sprouting in the central nervous system (Tew, Anderson and Burnstock, 1992; Tew et al., 1996).

INTRINSIC GANGLIONATED PLEXUSES Of the two main interconnecting ganglionated plexuses of the enteric nervous system, the myenteric plexus, which lies between the external longitudinal and circular muscle coats, and the submucous plexus, which lies between the circular muscle and muscularis mucosae, the myenteric plexus contains most of the intrinsic neurones of the gut and is the main source of neuromuscular innervation of the gut wall (Furness and Costa, 1987). Using guinea-pig small intestine, it has been shown that the motor neurones for the circular muscle are located entirely in the myenteric plexus whereas most of those for the mucosa are located in the submucosal ganglia. Intrinsic myenteric neurones also synapse with cells in the same or other ganglia, running both orally and anally, and project to submucosal ganglia. They also connect with autonomic ganglia outside the walls of the gastrointestinal tract and send afferents to the central nervous system. Some enteric neurones project from the intestine to innervate the mesenteric arteries and arterioles of the colon (Holzer, Gamse and Lembeck, 1980). Videomicroscopic analyses have revealed that intrinsic submucosal neurones, by regulating neurogenic vasodilatation of submucosal arterioles, are involved in local physiological control of mucosal blood flow (Neild, Shen and Surprenant, 1990). Intestinal secretion is largely under reflex neural control by the enteric nervous system. Endocrine cells of the intestinal epithelium are receptive to stimulants that evoke secretion of peptides and amines from their basal aspect, and activate the neurones that run close to the epithelium. These neurones run to the myenteric plexus and from there, via interneurones, to the submucous plexus (Jodal, 1990).

EXTRINSIC INNERVATION Post-ganglionic sympathetic nerve fibres from the coeliac and superior and inferior mesenteric ganglia and pre-ganglionic parasympathetic fibres running in the vagus and pelvic nerves innervate all parts of the gut, including the nerve plexuses, muscle layers, blood vessels and epithelium. Many sympathetic nerves are associated with blood vessels and are related to vasomotor action. The vasoconstrictor innervation to the arteriolar network in the submucous plexus is mediated solely by extrinsic sympathetic nerves that release primarily ATP to act on P2X receptors on the arteriolar smooth muscle, with noradrenaline acting as a prejunctional modulator via α2-adrenoceptors causing depression of neurotransmission (Evans and Surprenant, 1992). Sensory-motor nerves with their cell bodies in dorsal root ganglia also project to the gut and blood vessels in the submucosa (Szurszewski and King, 1989). Submucosal arteriolar vasodilatation is mediated by extrinsic sensory reflex pathways which are likely to be activated during inflammatory conditions (Vanner and Surprenant, 1996).


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NEUROMUSCULAR TRANSMISSION CONTROL OF INTESTINAL SMOOTH MUSCLE CONTRACTILITY The extrinsic and intrinsic control of gut smooth muscle operates in concert with the hormonal and mechanical control mechanisms stimulated by gut contents. The dominant role of the intrinsic innervation is apparent after sympathetic and parasympathetic denervation. Intrinsic neurones are either excitatory or inhibitory. Localised distension of the intestine, mechanical deformation of the mucosa or application of acid to the mucosa elicits both ascending excitatory and descending inhibitory reflexes to the circular muscle (Smith, Bornstein and Furness, 1990). A descending excitatory reflex to the circular muscle has also been reported (Kow, Brookes and Costa, 1993). These polarised events involve a chain of neurones, sensory interneurones and motor neurones. In the guinea-pig small intestine, enteric motility reflexes can be initiated through entirely intrinsic mechanisms (Furness et al., 1995, 1998). Definitive identification of intrinsic primary afferent neurones has recently been established by intracellular recordings in response to chemical and mechanical stimuli of the gut (Furness et al., 1998).

AUTONOMIC NEUROMUSCULAR JUNCTION The junction between autonomic nerve terminals and smooth muscle is not a well-defined structure and lacks both the pre- and postjunctional specialisations found at a skeletal muscle motor end plate (Gabella, 1972, 1995; Burnstock, 1986). Autonomic nerves do not release transmitter solely from terminals per se, but rather from varicosities that occur at intervals of 5–15 µm along axons. In autonomically innervated organs the distance of the cleft between the varicosity and the smooth muscle, a neuromuscular junction, is between 20 nm (as in the vas deferens or sphincter pupillae) and 1–2 µm (in large elastic arteries). Short, thickened regions of the membranes of varicosities are associated with aggregation of vesicles, and these membranes may represent sites of transmitter release, but post-junctional thickenings are not seen on the smooth muscle membrane. One of the essential features of autonomic neuromuscular transmission is that transmitter is released in passage from varicosities during conduction of an impulse along an autonomic axon. Furthermore, it is possible that a given impulse will evoke release from only some of the varicosities that it encounters (Blakeley and Cunnane, 1979; Blakeley, Cunnane and Petersen, 1982; Brock and Cunnane, 1995). The effector is a muscle bundle rather than a single cell; individual smooth muscle cells are connected by low-resistance bridges that allow electrotonic spread of activity within the effector bundle. Morphologically the sites of electrotonic coupling are represented by gap junctions (or nexuses). The size of these gap junctions varies from punctate junctions to junctional areas of 1 µm2 in diameter. Little is known about the quantity and arrangement of gap junctions in effector bundles relative to the density of autonomic innervation. Circular muscle layers tend to be more densely innervated than longitudinal layers. In small animals such as rats, mice and guinea-pigs, innervation of the longitudinal layer of the intestine is very sparse, with no nerve bundles penetrating the muscle coat. In the guinea-pig taenia coli, nerve bundles containing only 3–5 axons are found in muscle bundles. In the circular

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muscle there may be 25–78 close neuromuscular junctions per 1,000 muscle cells in the toad intestine, and in the guinea-pig small intestine there are claimed to be 10–50 axons of inhibitory junction potential-producing neurones per functional unit of circular muscle (for reviews see Hoyle and Burnstock, 1989; Gabella, 1995). Autonomic neuromuscular junctions are probably labile structures with varicosities that are able to move along axons; the lack of postjunctional specialisation is consistent with this view. Another aspect of this type of junction is that it is particularly accessible at both pre- and postjunctional sites for neuromodulatory influences, where local agents may enhance or exacerbate release of neurotransmitter or alter the extent or time course of neurotransmitter action. The possible role of the interstitial cells of Cajal in neuromuscular transmission in the gut has been debated for many years (Rogers and Burnstock, 1966; Bortoff, 1976; Komuro, 1982). More recently, evidence has been presented that interstitial cells may play a role as pacemaker cells, having intimate contact with neurones and muscle cells (Torihashi et al., 1993). They are themselves contractile and they influence the rate of contraction of the muscle and may modulate neural transmission to the muscle (Thuneberg, 1982; Sanders, 1996; Shuttleworth and Sanders, 1996). The importance of these cells in normal and abnormal gastrointestinal motility is gaining recognition.

EXCITATORY NEUROMUSCULAR TRANSMISSION Acetylcholine Excitatory enteric neurones that project to intestinal smooth muscle utilise tachykinins and acetylcholine as neurotransmitters. Acetylcholine, either applied or released from neurones, produces a rapid depolarisation, or excitatory junction potential (EJP), via muscarinic receptors. In response to low-strength stimulation, perhaps a single electrical pulse, the EJP is transient, usually lasting less than 1 s, with a latency of the order of 100 ms. At higher strengths of stimulation the EJP may be accompanied by an action potential (Brock and Cunnane, 1995). Cholinergic EJPs have been recorded from several gut muscles (Hoyle and Burnstock, 1989; Brock and Cunnane, 1995). During repetitive stimulation of cholinergic nerves, EJPs may facilitate and develop faster rise times and larger amplitudes or depression may occur, in which case these parameters reduce. Although increasing either sodium or calcium conductance in a resting membrane will result in a depolarisation, the excitatory effects of acetylcholine acting on muscarinic receptors involve increases in permeabilities to these ions as well as to potassium and perhaps chloride ions. The reversal potential for muscarinic action of around –5 mV is indicative of simultaneous depolarising and hyperpolarising ion fluxes. In the guinea-pig ileum longitudinal muscle and rabbit jejunum and rectum longitudinal muscles, the cholinergically induced EJP reverses between –5 and +5 mV, while the non-adrenergic, non-cholinergic EJP in the guinea-pig ileum reverses at –27 mV. Although this might suggest an involvement of chloride ions, it could also reflect a simultaneous increase in potassium conductance along with sodium or calcium conductance (Hoyle and Burnstock, 1989; Brock and Cunnane, 1995). Single-cell voltage-clamp studies of rabbit ileum longitudinal muscle show that muscarinic receptors open channels that are permeable to sodium


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and potassium, slightly permeable to calcium, and impermeable to chloride (Bolton and Lim, 1991). Muscarinic receptors are not ligand-gated ion channels: all muscarinic receptors that have been cloned are G protein-coupled receptors. In smooth muscle, excitatory effects are predominantly mediated via G proteins coupled to phosphatidylinositol metabolism and subsequent calcium mobilisation (Gq/11), or to cation channels (Bolton and Lim, 1991; Zholos and Bolton, 1994; Prestwich and Bolton, 1995). Additionally, muscarinic receptors in intestinal smooth muscle may be coupled to a pertussis toxin-sensitive G protein (Gi/o) that is involved in inhibition of calcium-dependent potassium channels (Cole and Sanders, 1989; Prestwich and Bolton, 1995). Pharmacologically, the predominant muscarinic receptor on intestinal smooth muscle is selectively antagonised by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and is of the M3 subtype (Grider, Bitar and Makhlouf, 1987; Lucchesi et al., 1989; De Vos, 1993). M2 receptors are also present, although they form a smaller population (Lucchesi et al., 1989; De Vos, 1993). Substance P Within the gut, substance P-containing neurones do not appear to project for long distances; in transected and re-anastomosed ileum no change in substance P immunoreactivity could be found 2–4 mm away from the injury sites (Keast, Furness and Costa, 1982), implying that the substance P neurones are confined to segments or arcades of the gut. Many excitatory motor neurones contain both acetylcholine and tachykinins whilst fewer contain either acetylcholine or tachykinins (Brookes, Steele and Costa, 1991). Excitatory enteric neurones innervating circular muscle project either locally or orally (Bornstein, Furness and Kunze, 1994) whilst those innervating longitudinal muscle normally have short local projections. Opioid peptides (dynorphin, enkephalin and opioid-related peptides) co-localised in many excitatory enteric neurones do not appear to be primary neurotransmitters but rather have a neuromodulatory role, generally inhibiting motor neurotransmission (Tonini et al., 1992). Calretinin localised in some excitatory neurones projecting to longitudinal muscle is not found in excitatory neurones projecting to circular muscle. Substance P, produces slower responses than does acetylcholine, and its actions are mediated via neurokinin (NK) receptors (Maggi et al., 1990, 1997; Hoyle, 1996a). Tachykinins are preferentially released during high frequency neuronal firing but are probably also released with acetylcholine at lower frequencies (Holzer, Schulet and Maggi, 1993). Non-cholinergic contractile response to high frequency stimulation is effectively blocked by tachyphylaxis to substance P, and by selective NK receptor antagonists. In the superfused ileum in vitro, substance P is released during field stimulation in a manner that is calcium-, temperature-, and frequency-dependent, and tetrodotoxin- (TTX) sensitive; the amount released is enough to produce a physiological response (Baron, Jaffé and Gintzler, 1983). Similarly, high potassium-stimulated substance P release from isolated longitudinal muscle-myenteric plexus preparations is calcium-dependent, whereas field stimulationevoked release of substance P is TTX-sensitive. Raised intraluminal pressure in a segment of guinea-pig ileum, which will initiate peristalsis, is accompanied by substance P release. The substance P antagonist (D-Pro2,D-Trp7,9 )-substance P (DPDTSP) also inhibits atropineresistant peristalsis, as does substance P tachyphylaxis and hexamethonium indicating that

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Figure 7.1 NK1 receptor immunoreactivity in nerve cells in various regions of the guinea-pig gastrointestinal tract. (A) Wholemount preparation of a myenteric ganglion in the corpus of the stomach showing NK1 receptor immunoreactive nerve cell bodies. Cells show Dogiel type 1 morphology and immunoreactivity is largely confined to the cell surface membrane. (B, C) Wholemount preparations of myenteric ganglia in the antrum of the stomach. Staining appears particularly punctate in this region with large aggregations of immunoreactivity dotting the cell surface membrane. (D) Wholemount preparation of a myenteric ganglion in the duodenum illustrating immunoreactive nerve cells (arrow heads) and interstitial cells of Cajal (small arrows) at the level of the myenteric ganglia. The nerve cell bodies exhibit Dogiel type 1 morphology with flattened lamellar dendrites and a prominent axon. (E) Wholemount preparation of the myenteric ganglia of the distal colon showing three NK1 receptor immunoreactive neurones. Scale bars A, B, C, E = 20 µm; D = 50 µm. (Reproduced from Portbury et al., 1996, with permission from Wiley-Liss, Inc., a subsidiary of John Wiley and Sons Inc.).

substance P in intrinsic neurones has an active role in coordination of peristalsis (Barthó et al., 1982). Reflex contractions of circular muscle oral to sites of distension stimuli normally have a low threshold but in the presence of muscarinic blockade they have a high threshold. This high-threshold stimulation is sensitive to the substance P antagonists. Hence substance P neurones are final in a noncholinergic reflex pathway to the circular muscle and perhaps interneuronal, within the myenteric plexus, in a cholinergic reflex pathway (Costa et al., 1985). A recent study of the distribution of NK1 receptors in guinea-pig intestine showing lack of immunoreactivity to these receptors in the smooth muscle but strong immunoreactivity on the interstitial cells of Cajal at the inner surface of the circular muscle supports pharmacological evidence that tachykinin receptors on longitudinal muscle are a subtype of NK1 receptors (Chassaing et al., 1992) and suggests that circular muscle may be indirectly excited via interstitial cells of Cajal (Figure 7.1) (Portbury et al., 1996): these cells are linked to the muscle by gap junctions and are considered to be responsible for pacemaker activity in the gut (Vogalis, Ward and Sanders, 1991; Sanders, 1996). NK2 receptors are localised on circular and longitudinal muscle cells (Grady et al., 1996).


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In the guinea-pig ileum, substance P stimulates only those smooth muscle cells that have an excitatory neural input (Bauer and Kuriyama, 1982). During continuous application of substance P (0.5 µM), the primary phase of neurogenic depolarisation is abolished, and this depolarisation is inhibited by substance P antagonists, indicating that substance P is the transmitter. There appears to be a functional interaction between acetylcholine and substance P, perhaps released from the same neurones, in that there is a synergistic pro-kinetic action that involves muscarinic and NK2 receptors (Holzer and Maggi, 1994). Substance P and neurokinin A (NKA, a selective NK2 receptor agonist) evoke depolarisation of colonic smooth muscle cells, by opening non-selective cation channels, similar to those opened by acetylcholine, with a reversal potential close to 0 mV (Lee, Shuttleworth and Sanders, 1995). Noradrenaline Many adrenergic nerves in the muscle layers of the gastrointestinal tract are associated with blood vessels and are probably related to vasomotor action. In the peripheral nervous system, nearly all adrenergic cell bodies are confined to sympathetic ganglia that have their terminals in the myenteric plexus, submucous plexus, mucosa, and associated blood vessels. Adrenergic cell bodies are rarely seen, if at all, in the enteric plexuses (Furness and Costa, 1987). This distribution appears to be similar in many species, such as in the rat intestine, guinea-pig ileum, pig ileum, human ileum and cat ileum (Hoyle and Burnstock, 1989). However, intrinsic adrenergic cell bodies are found in the myenteric plexus of the guinea-pig proximal colon, and these account for 50–60% of the adrenergic fibres in the myenteric plexus; the remaining 40–50% and all the adrenergic fibres in the submucous plexus are extrinsic in origin (Furness and Costa, 1971a,b). Microsurgical interruption of the myenteric plexus shows that there are no ascending or descending adrenergic pathways because the adrenergic fibres are confined to arcades of the gut supplied by blood vessels and neurones entering via the mesentery (Furness, Costa and Llewellyn-Smith, 1981). Noradrenergic transmission is often excitatory in sphincteric muscles. For example, in the cat ileocolonic sphincter, peripheral splanchnic nerve stimulation is excitatory, is mimicked by phenylephrine, and is blocked by phentolamine (Rubin et al., 1980). The cat internal anal sphincter behaves similarly, and electromyograph records show that hypogastric nerve stimulation evokes EJPs that are mediated via α-adrenoceptors (Bouvier and Gonella, 1981). Endothelin Endothelin was originally identified as a 21 amino acid peptide with pressor activity, synthesised by endothelial cells. During identification of genes producing the family of endothelins in mouse intestine (endothelin-1, - 2 and - 3), a gene encoding a peptide closely related to endothelin-2 was identified. This peptide which differs from endothelin-2 by one amino acid residue was originally found only in the intestine, and not in endothelial cells, and although it possessed pressor activity it was a potent spamsogen in guinea-pig ileum, hence its name: vasoactive intestinal constrictor (VIC) (Ishida et al., 1989; Saida, Mitsui and Ishida, 1989; Saida et al., 1996). Immunoreactivity for endothelin-1 is found in

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human colon myenteric and submucous plexuses, where it is co-localised with vasoactive intestinal polypeptide (VIP), and endothelin receptors are mostly neuronal, with smaller populations on smooth muscle cells or epithelial cells (Inagaki et al., 1991; Escrig et al., 1992). Rat myenteric neurones in culture display endothelin-1 immunoreactivty and high levels of the appropriate mRNA (Eaker et al., 1995). Interestingly, myenteric neurones do not contain mRNA for endothelin-2 (de la Monte et al., 1995). Several molecular forms of endothelin exist (Hoyle, 1996a); endothelin-1, endothelin-2, endothelin-3, VIC and the related peptides, sarafotoxin-a, sarafotoxin-b and sarafotoxin-c all evoke concentration-dependent contraction of the ileum (Wiklund et al., 1991; Wollberg et al., 1991; Yoshinaga et al., 1992; Kan, Niwa and Taniyama, 1994). In addition to contracting ileal muscle, endothelin-1 inhibits release of acetylcholine while potentiating contractions evoked by exogenous acetylcholine (Wiklund et al., 1991). In contrast, VIC evokes release of acetylcholine, via non-endothelin receptors, but it also acts on endothelin receptors to evoke contractions (Kan, Niwa and Taniyama, 1994). Endothelins may also cause relaxation of guinea-pig ileal longitudinal muscle, mediated via apamin-sensitive Ca2+-dependent K+-channels (Lin and Lee, 1992). Whether endothelin-1 (or any other endothelin-related neuropeptide) is a neuromuscular transmitter, or is involved in ganglionic transmission or neuromodulation, remains to be established. INHIBITORY NEUROMUSCULAR TRANSMISSION Most examples of inhibitory neuromuscular transmission in the intestine can be accounted for by the troika of ATP, nitric oxide (NO) and VIP. Although there is no direct evidence that these neurochemicals are in fact co-transmitters, it is very likely that they are (see Hoyle, 1996b). In recent years, the discovery of NO as an inhibitory neurotransmitter has commanded a lot of attention, and related evidence has been reviewed many times (for example Rand, 1992; Sanders and Ward, 1992; Lefebvre, 1995; Lincoln, Hoyle and Burnstock, 1995, 1997). In some regions of the gastrointestinal tract, interstitial cells of Cajal are involved in nitrergic inhibitory transmission, acting as intermediary amplifiers between the inhibitory nerves and the smooth muscle (for reviews see Sanders, 1996; Shuttleworth and Sanders, 1996). The current that causes the hyperpolarisation of the IJP in response to nerve stimulation seems to be solely due to potassium efflux. The reversal potential of the non-adrenergic, non-cholinergic IJP has been determined; it lies very close to the potassium equilibrium potential (Hoyle and Burnstock, 1989). Also, in several studies, the dependency of IJP amplitude on membrane potential has been shown to be linear, again suggesting the involvement of only the one ion. Pharmacological investigations have also shown that potassium ion efflux is responsible for IJPs. The polypeptide toxin apamin, which is extracted from bee venom, blocks calcium-dependent potassium ion channels and abolishes IJPs by blocking this ion channel in the guinea-pig taenia coli (Maas et al., 1980). ATP, nitric oxide and vasoactive intestinal peptide The evidence that ATP acts as a neuromuscular transmitter in the enteric nervous system is now quite convincing, and has been reviewed extensively (Hoyle, 1992, 1996b; Bauer,


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1993; Itoh et al., 1995; Burnstock, 1996, 2001). Although there is little evidence from histochemical studies for the co-localisation of ATP with VIP or nitric oxide synthase (NOS) in inhibitory motoneurones, there are many examples of regions of the gastrointestinal tract in which neuromuscular transmission is mediated by more than one transmitter, and generally inhibitory transmission is carried out by members of this trio (Hoyle, 1996b). Although desensitisation of P2X receptors by α,β-methylene ATP or antagonism by arylazidoaminopropionyl ATP (ANAPP3) has effectively demonstrated that ATP is a neurotransmitter in nerves supplying autonomically innervated structures such as the vas deferens, seminal vesicles, urinary bladder, heart, and some blood vessels (Hoyle, 1992, 1994, 1996b; Sneddon, McLaren and Kennedy, 1996; Burnstock, 1997), neither of these agents successfully antagonises inhibitory P2 receptors in the gut, which are typically subclass P2Y. In the guinea-pig taenia coli, ANAPP3 does antagonise low concentrations of ATP (5 Hz, whereas non-adrenergic inhibitory responses are evokable by single pulses of electrical stimulation. In non-sphincteric muscles, where adrenergic nerve fibres are concentrated in the myenteric and submucous plexuses, it has been suggested that the relaxation of the smooth muscle may not be due to a direct action of noradrenaline released from sympathetic nerves but instead is due to an inhibition of a tonic excitatory pathway, most likely cholinergic, and as such represents a neuromodulation rather than neuromuscular transmission. Noradrenaline may act directly on the smooth muscle, especially when the adrenergic

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neurones are stimulated at high frequency and the neurotransmitter can overspill to act on muscle receptors (Gillespie and Maxwell, 1971). Exogenous noradrenaline is a potent inhibitor of acetylcholine release and neurotransmission in the myenteric plexus, and because neither exogenous noradrenaline nor sympathetic nerve stimulation affects the membrane potential of ganglion cell bodies in the myenteric plexus, the site of adrenergic action is probably remote from the cell body, i.e. on the axon terminals. Reciprocal axoaxonic synapses between adrenergic and cholinergic nerves in the enteric plexus have been observed, and acetylcholine and noradrenaline are able to inhibit their own and each other’s release (see Hoyle and Burnstock, 1989). In addition to modulating release of acetylcholine, noradrenaline can inhibit release of other neurotransmitters. In the guinea-pig taenia coli, noradrenaline reduces the relaxant effect of non-adrenergic inhibitory nerve stimulation (Sakato, Shimo and Bando, 1972); in the guinea-pig duodenum (Ohkawa, 1983) and caecum circular muscle (Reilly, Hoyle and Burnstock, 1987), and rat caecum circular muscle (Hoyle et al., 1988) the non-adrenergic IJP in response to transmural stimulation is reduced in amplitude by adrenoceptor agonists and is enhanced by guanethidine (Ohkawa, 1983), implying the existence of a tonic neuronal adrenergic modulation of the IJP. In the rabbit colon, where preganglionic vagal stimulation can evoke EJPs or IJPs, simultaneous splanchnic nerve stimulation blocks the vagal excitatory, but not inhibitory, effect; pelvic nerve stimulation excitation is very effectively blocked by lumbar colonic nerve stimulation (Gillespie and Khoyi, 1977). In this case the presynaptic inhibition is mediated via α-adrenoceptors, whereas direct inhibition observed at high stimulation frequencies is mediated via β-adrenoceptors.

NEUROEPITHELIAL TRANSMISSION IN THE INTESTINE Ion transport across the intestinal epithelium plays a role of paramount importance in body fluid homeostasis. The flux of ions, principally chloride, bicarbonate, sodium and potassium, through intestinal epithelial cells generates osmotic gradients across the epithelium which govern the rate and direction of passage of water to and from gut lumen and vascular system. They are innervated on their basal side, principally by nerves that have their cell bodies in the submucous plexus, but also by nerves originating in the myenteric plexus or in sympathetic ganglia, and by extrinsic sensory neurones. The epithelial cells have tonic activity but are under humoral and neural control. Secretomotor neurones from the submucous plexus transmit with acetylcholine and VIP, and NPY appears to be the main antisecretory transmitter. In general, the enterocytes on villi in the small intestine, and lining the lumen of the large intestine are absorptive, one of their primary features being that they transport chloride ions from the gut lumen across their apical membrane via a chloride-bicarbonate exchange. The epithelial cells that line the crypts of the small and large intestine are secretory, and a major feature is that these cells lack a chloridebicarbonate exchanger but have chloride ion channels in their apical membrane, which provide a route for chloride ions to flow from inside the cell, across the apical membrane, into the gut lumen.


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SECRETORY TRANSMITTERS Acetylcholine Choline acetyltransferase, the enzyme that catalyses production of acetylcholine from acetyl-CoA and choline, is found in myenteric and submucosal neurones projecting to the epithelium. These neurones may also contain cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin, and possibly galanin (Keast, Furness and Costa, 1982; Bornstein, Furness and Costa, 1987; Furness et al., 1987; Bornstein and Furness, 1988; Cooke, 1989; Porter et al., 1996). Thus it is likely that acetylcholine is a co-transmitter with these other substances. Choline acetyltransferase is also found in submucosal interneurones, in a population that does not appear to contain any neuropeptide. These nerves synapse in submucosal ganglia with secretomotor neurones (Bornstein, Furness and Costa, 1987). A third population of nerve fibres, which contains choline acetyltransferase, is sensory, projecting to the epithelium, and additionally contain substance P (Bornstein, Furness and Costa, 1987; Cooke, 1989). Stimulation of intramural neurones in the intestine evokes secretion that is partially mediated by acetylcholine (Wu, Kisslinger and Gaginella, 1982; Carey et al., 1987; Kuwahara et al., 1987a; Carey and Cooke, 1989; Chandan et al., 1991a; Javed and Cooke, 1992; Hogan et al., 1993). Acetylcholine promotes secretion from epithelial cells by acting on muscarinic receptors, however it has been argued that it would be incorrect to regard the cholinergic nerves as “secretomotor” because some of the neuropeptides that they contain, particularly NPY and somatostatin, would evoke absorption. In intestinal mucosa, the primary site for secretion appears to be in the crypts (Browning et al., 1978). There is probably not a homogeneous population of muscarinic receptors on the epithelial cell because the pharmacological profile of muscarinic receptor antagonists is not consistent with a single known subtype. The subtypes that are present appear to be M1 and M3, with M3 being preponderant (Carey et al., 1987; Kuwahara et al., 1987b; Chandan et al., 1991a,b; O’Malley et al., 1995). The main effect of acetylcholine is to inhibit sodium and chloride absorption from the lumen, across the apical membrane, to stimulate chloride secretion across the basolateral membrane and, in some tissues, to stimulate bicarbonate transport across the apical membrane (Cooke, 1989; Jodal, 1990; Chandan, O’Grady and Brown, 1991; Hogan et al., 1993). In porcine small intestine, muscarinic receptors involved in mucosal transport are found not only on epithelial cells but also on neurones. Activation of the epithelial receptor promotes chloride secretion, while activation of the neuronal receptor inhibits chloride secretion, presumably by stimulating release of an anti-secretory transmitter or by inhibiting tonic release of a pro-secretory transmitter (Chandan et al., 1991b). Vasoactive intestinal peptide and related peptides Vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) are colocalised with dynorphin and galanin in guinea-pig ileum submucosal neurones (Furness et al., 1987), and in the rat VIP is co-localised in submucosal neurones with NPY (Cox, Rudolph and Gschmeissner, 1994). These neurones project to the mucosa, forming an aganglionic villar plexus. They receive excitatory and inhibitory input from myenteric neurones, excitatory input from submucosal neurones, and inhibitory input from sympathetic

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Figure 7.7 Effects of vasoactive intestinal polypeptide (38 nM) on baseline and neurally evoked secretory responses in guinea-pig ileum. (A) Short-circuit current response to stimulation of enteric neurones (a, fast cholinergic phase; b, sustained phase). (B) Effect of VIP on baseline short-circuit current and on neurally evoked response. Stimulus parameters were: 0.5 ms, 10 Hz, 10 V (3.3 mA), applied as indicated by bar. Note that VIP increases the short-circuit current and enhances the response to nerve stimulation, especially the initial cholinergic phase. (Reproduced from Cooke et al., 1987, with permission from Harcourt).

neurones (Bornstein, Furness and Costa, 1987; Bornstein and Furness, 1988). Release of VIP from guinea-pig ileal and colonic mucosa has been measured, and is evoked by nicotinic stimulation or depolarisation by potassium chloride (Okuno et al., 1988; Reddix et al., 1994). VIP is a powerful secretagogue, with greater potency than its evolutionary homologues (see Hoyle, 1998), PHI, helodermin and PACAP-28 (Cox and Cuthbert, 1989b; Reddix et al., 1994). The mechanism of action of VIP involves an increase in intraepithelial cell production of cAMP (Laburthe et al., 1979; Beubler, 1980; Broyart et al., 1981; Prieto et al., 1981; Reimer et al., 1996), and subsequent activation of a cAMP-dependent protein kinase (Cohn, 1987). The relationship between intracellular levels of cAMP and stimulation of secretion by VIP is not entirely clear, and secretion may be evoked by concentrations of VIP that do not induce measurable changes in cAMP (Beubler, 1980), and increasing concentrations of VIP can evoke increasing rates of secretion even though intracellular levels of cAMP have been increased maximally (Laburthe et al., 1979; Broyart et al., 1981). Further, the increase in cAMP by VIP is thought to contribute to potentiation of cholinergicallyevoked secretion, as shown in Figure 7.7. The agonist potency of VIP and its evolutionary homologues, at evoking cAMP production, correlate well with the ability of the homologues to displace epithelial binding of radiolabelled VIP (Salomon et al., 1993) as well as their ability to evoke secretion (Cox and Cuthbert, 1989b; Reddix et al., 1994). The predominant site of activity of VIP is likely to be in crypt cells rather than villar cells, because, in response to VIP, enterocytes lining the crypt produce more cAMP than enterocytes on the villi , although the potency of VIP is the same in the two regions (Amelsberg et al., 1996). Homologues of VIP, for example PHI and the reptilian helodermin, also promote secretion in the small intestine. In the rat ileum, these substances cross-desensitise one another, which implies that they act on the same types of receptor (Cox and Cuthbert, 1989b). The actual receptor subtype remains to be elucidated, but it appears to be resistant to


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antagonists formed from VIP fragments such as VIP(10–28), [Lys1,Pro2,5,Arg3,4,Tyr6]VIP or [4Cl-D-Phe6,Leu17]VIP, or those derived from growth hormone releasing factor (GRF), which is an evolutionary relative of VIP (Hoyle, 1998), such as [AcTyr1,D-Phe2] GRF(1–29)-NH2 and [AcTyr1]hGRF-(1–40)-OH (Cox and Cuthbert, 1989b; Burleigh and Kirkham, 1993). In Ussing chamber experiments, application of PACAP to the basolateral surface of human jejunal and colonic mucosa stimulates chloride secretion (Fuchs et al., 1996). Both PACAP-27 and PACAP-38 are potent secretogogues in rat ileal mucosa (Cox, 1992). PACAP-27, but not PACAP-38 or VIP, at low, subnanomolar, concentrations causes secretion that is sensitive to TTX, desensitisation to substance P, and also capsaicinisation, and is likely to be mediated via stimulation of sensory nerve terminals. At higher, nanomolar, concentrations both PACAP-27 and PACAP-38 induce secretion, which like VIP, is insensitive to TTX (Cox, 1992). In guinea-pig colon, the direct effects of VIP and responses to non-cholinergic nerve stimulation are attenuated by the nonadecapeptide VIP fragment VIP(10–28) (Reddix et al., 1994), providing further evidence that VIP is a neurotransmitter in this tissue. VIP(10–28) may not always be a useful tool, because in rat colon, high concentrations of VIP(10–28) (1 and 3 µM) evoke low amplitude short circuit currents, but do not affect responses to applied VIP (Burleigh and Kirkham, 1993). Chymotrypsin, an enzyme that degrades VIP, and anti-VIP antisera have been used with extremely limited success to demonstrate a transmitter role of VIP in neuroepithelial transmission (Hubel, 1984; Cooke et al., 1987). Binding studies have shown that in the human small intestine epithelium, PACAP and VIP bind the same receptor, which has a higher concentration in villar cells than in crypt cells (Salomon et al., 1993), and is probably a PACAP-VIP type II receptor. However, in rodents it is likely that there is more than one type of receptor present in the mucosa. For example, in rat small intestine, low concentrations of PACAP-27 evoke transient secretory responses that are sensitive to TTX (Figure 7.8), desensitisation by substance P or pre-treatment with capsaicin, indicating an action mediated via stimulation of sensory nerve terminals (Cox, 1992). This action of PACAP-27 is not shared by VIP or PACAP-38, and is indicative of a separate receptor. In guinea-pig large intestine VIP, PACAP-27, PACAP-38, PHI and helodermin evoke responses that are inhibited, but not abolished by atropine. Additionally, TTX abolishes responses to PACAP-27 and PACAP-38, but only attenuates responses to VIP, PHI and helodermin, and then only responses evoked by high concentrations (Kuwahara et al., 1993). These results also indicate the presence of at least two receptors, one on nerve terminals and one on epithelial cells, with PACAP recognising only the neural receptor. Substance P Substance P is localised in neurones that have their cell bodies in the myenteric plexus in guinea-pig small intestine, and which do not receive a synaptic input (Bornstein, Furness and Costa, 1987). These neurones also contain choline acetyltransferase (Bornstein and Furness, 1988; Cooke, 1989). It is unclear whether there is a true physiological role for the action of substance P released from these nerves acting on the epithelial cells. In small and large intestine, across a range of species that includes rat, mouse, guinea-pig and dog,

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Figure 7.8 Effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27) on short-circuit current in rat small intestinal mucosa. PACAP-27 was applied cumulatively (↑) in the absence (upper trace) and presence (lower trace) of tetrodotoxin (TTX, 100 nM, ▲). Vasoactive intestinal polypeptide (VIP, 100 nM, ∆) was added at the end of the traces in order to define the maximum secretory response. The inset shows the response to a single, 1 nM, concentration of PACAP-27. Baseline currents are indicated at the start of the recordings. All doses were applied to the basolateral side of the epithelium. (Modified from Cox, 1992, with permission from the Nature Publishing Group).

responses to substance P are qualitatively similar in that substance P acts partly directly on the epithelial cells and partly indirectly on mucosal nerve terminals or submucosal neurones (Keast, Furness and Costa, 1985a; Fox et al., 1986; Perdue, Galbraith and Davison, 1987; Rangachari, Prior and McWade, 1990; Burleigh and Yull, 1992; Parsons et al., 1992; Wang et al., 1995). When cholinergic transmission is inhibited by atropine in guinea-pig small intestine, the residual non-cholinergic secretory response is markedly attenuated by either desensitisation to substance P or by anti-substance P antisera (Perdue, Galbraith and Davison, 1987). However, if the substance P is present only in sensory nerves, then under these experimental conditions, the substance P would be released from peripheral terminals due to antidromic stimulation of these nerves. This situation could occur under physiological conditions because it has been shown that lightly stroking guinea-pig colonic mucosa induces chloride secretion that is inhibited by the selective NK1 receptor antagonist GR-82334 (Cooke et al., 1997). In rat colon, it is unlikely that substance P is involved in neuroepithelial transmission because it has been shown that application of a neurokinin receptor antagonist, while blocking secretory responses to applied substance P, does not block responses to non-cholinergic nerve stimulation (Burleigh and Yull, 1992). Likewise, in guinea-pig ileum,


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NK1 receptor antagonists that block responses to applied substance P do not significantly affect neurogenic secretion (Reddix and Cooke, 1992). The receptor activated by substance P may be species-dependent. In guinea-pig small intestine two populations of neurokinin receptors have been identified, one on cholinergic and non-cholinergic nerve terminals in the mucosa, and one on the epithelial cell (Keast, Furness and Costa, 1985a; Perdue, Galbraith and Davison, 1987). In contrast, another group has found that the response to substance P is mediated via NK1 receptors, with no evidence for NK2 or NK3 receptors, in guinea-pig small intestine (Reddix and Cooke, 1992). In these experiments, responses to substance P were markedly attenuated by TTX, but not atropine, implying that the receptor is on non-cholinergic neurones, an action that had been observed previously (Kachur et al., 1982). However, the mRNA for NK1 receptors has been located in isolated colonic epithelial cells and in colonic crypts (Cooke et al., 1997), indicating that epithelial cells of the large intestine probably express NK1 receptors. In rat colon, it is likely that NK1, NK2 and NK3 receptors are present. When the activity of a series of tachykinins was examined (Cox et al., 1993), including substance P and its evolutionary homologues, neurokinin A, neurokinin B and neuropeptide Y (Hoyle, 1998), all were found to evoke secretion. The responses to some tachykinins were abolished by TTX, while some were reduced and some were unaffected. The use of selective agonists and antagonists led to the conclusion that NK1 receptors are both neuronal and epithelial, NK2 receptors are predominantly epithelial with a comparatively small functional neuronal population (on non-cholinergic nerves), and NK3 receptors are neuronal (Cox et al., 1993). Responses to substance P may involve an interaction with mast cells. In mouse small intestine, substance P evokes increases in short circuit current (chloride secretion) via NK1 receptors on enteric nerves and epithelial cells (Wang et al., 1995). H1 and H2 receptor antagonists reduce the secretory responses to non-cholinergic nerve stimulation and applied substance P. In the genetically mast cell-deficient WBB6F1 W/W(V) mouse, responses to substance P are weaker than in control mice, and are unaffected by H1 and H2 antagonists. Thus, in control mice, responses to substance P are augmented by histamine released from mucosal mast cells (Wang et al., 1995). In some pathological conditions, substance P pathways may become involved. For example, in rats infected with an intestinal parasite, the helminth Nippostrongylus brasiliensis, there is a marked increase in the density of innervation of the mucosa by substance P-containing nerves, in rat small intestine (Masson et al., 1996). Furthermore, responses to substance P become reduced to approximately 25% of their control value, at a time when cholinergic neuroepithelial transmission is abolished (Masson et al., 1996). ATP Exogenous ATP stimulates active ion transport across intestinal epithelial mucosa (Kohn, Newey and Smyth, 1970; Korman et al., 1982; Cuthbert and Hickman, 1985; Richards et al., 1987). The ability of ATP to act from the serosal surface of the intestinal epithelium to increase short circuit current, raises the possibility that release of ATP from enteric nerves may be a mechanism for causing intestinal secretion (Korman et al., 1982). In isolated crypts of rat distal colon, basolaterally applied ATP increases [Ca2+]i and acts as a secretagogue via a P2Y receptor, probably P2Y1. Furthermore, P2Y receptor agonist-induced

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[Ca2+]i elevations are most marked at the crypt base, which is the secretory part of the colonic crypt. These responses are not mediated by adenosine (Leipziger et al., 1997). There is at present no conclusive evidence that ATP is involved in neuroepithelial transmission and in addition to a potential neural source, ATP released from neighbouring cells such as epithelial cells and fibroblasts may be available to mucosal sites. Purine receptors, notably UTP-sensitive receptors including P2Y2 (P2U), are also localised to the apical domain of secretory cells (Inoue et al., 1997; Leipziger et al., 1997). ATP may act via degradation to adenosine and subsequently elicit chloride secretion by occupation of adenosine receptors localised on epithelial cells (Dho, Stewart and Foskett, 1992; Stutts et al., 1995). An indirect action of ATP has been proposed following observations of partial TTX-inhibition of exogenous ATP-induced chloride secretion in rat colonic epithelium: this suggests that ATP acts predominantly on neuronal elements in the lamina propria (Cuthbert and Hickman, 1985).

ANTI-SECRETORY TRANSMITTERS Noradrenaline In vivo, intra-arterial injection of noradrenaline promotes water absorption from the small intestine. This effect is mediated via α2-adrenoceptors, since it is mimicked by UK 14,304 but not phenylephrine, and is blocked by yohimbine (Liu and Coupar, 1997). More specifically, the receptor in rat intestine is likely to be the α2A or α2D subtype, as evidenced by the antagonistic effect of BRL 44408 (Liu and Coupar, 1997). There is tonic sympathetic activity, shown by α2-adrenoceptor antagonists decreasing basal rates of absorption, and chemical sympathectomy with 6-hydroxydopamine resulting in inhibited absorption. In vitro, xylazine, which is another α2-agonist, induces a yohimbine-sensitive reduction in short-circuit current in voltage-clamped small intestinal mucosa (Cox and Cuthbert, 1989a). However, the antisecretory activity of xylazine is markedly attenuated by piroxicam, indicating that endogenous eicosanoid formation is involved (Cox and Cuthbert, 1989a). The human colonic epithelial cell line, Colony-1, also possesses α2-adrenoceptors that are functionally antisecretory (Holliday, Tough and Cox, 1997).

Neuropeptide Y and related peptides Nerve fibres containing NPY are distributed throughout the wall of the intestine, belonging to myenteric, submucosal and extrinsic neurones (Keast, Furness and Costa, 1985b). At an ultrastructural level, NPY-containing nerves lie in close apposition to mucosal epithelial cells (Feher and Burnstock, 1986). Furthermore, NPY is co-stored with VIP in a population of neurones that have their cell bodies in the submucous plexus and innervate mucosal epithelial cells (Cox, Rudolph and Gschmeissner, 1994). Neuropeptide Y is an antisecretory neuropeptide, which in vitro inhibits chloride secretion from intestinal mucosa (Friel, Miller and Walker, 1986; Hubel and Renquist, 1986; Cox and Cuthbert, 1988; Cox et al., 1988). Piroxicam and indomethacin, both cyclooxygenase inhibitors, block responses to applied NPY, indicating that production of


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prostaglandins is involved (Cox and Cuthbert, 1988; Cox et al., 1988). The NPY receptor is located preponderantly on the basolateral rather than apical aspect of the epithelial cells. The subtype of receptor may be species-dependent: for example, in the human epithelial cell line, Colony-6, Y1 receptors are present, in rat and rabbit intestinal mucosa there are Y2 receptors, and in dog intestinal epithelium there are Y4; at all these receptors PYY is more potent than or equipotent with NPY (Playford and Cox, 1996). However, in Colony-6, the pharmacological profile indicates an heterogeneous population of receptors (Cox and Tough, 1995). In rat jejunum, transport and ligand-binding studies also indicate an heterogeneous population of Y1 and Y2 receptors, together with a third, pancreatic polypeptideselective receptor (Souli et al., 1997). In humans, intravenous infusion of NPY increases jejunal net absorption of water, sodium, potassium and chloride ions under basal conditions, and markedly reduces the secretion stimulated by intraluminal instillation of prostaglandin E2 (PGE2) (Holzer-Petsche et al., 1991). In guinea-pig large intestinal mucosa, NPY has little or no effect on basal secretion, but was able to inhibit secretion evoked by either nerve stimulation, VIP or the cholinomimetic, bethanechol, (McCulloch et al., 1987). Similarly, in pig distal colon, NPY inhibits secretion induced by stimulating nerves electrically or with leukotriene C4 (Traynor, Brown and O’Grady, 1995). In rat small intestine, close arterial injection of NPY has no effect on basal secretion, but inhibits that evoked by PGE2 (Saria and Beubler, 1985). Peptide YY is an evolutionary analogue of NPY (Hoyle, 1998), but is found in enterochromaffin cells rather than enteric nerves. It also has antisecretory effects, mediated via Y receptors (Playford and Cox, 1996). Y receptors are G protein-coupled receptors that mediate inhibition of adenylate cyclase (Cox and Krstenansky, 1991; Mannon, Mervin and Sheriff-Carter, 1994; Playford and Cox, 1996). The human colonic epithelial cell line, Colony-1, is unresponsive to NPY and PYY, but in these cells, pancreatic polypeptide (PP) is a potent inhibitor of VIP-stimulated secretion. Responses to PP are unaffected by preincubation with PYY, implying that this cell line bears Y4 receptors (Holliday and Cox, 1996).

PLASTICITY OF EXPRESSION OF NEUROTRANSMITTERS PLASTICITY IN THE AUTONOMIC NERVOUS SYSTEM The particular combination and quantity of neuroactive substances expressed by neurones is partly pre-programmed and partly determined by the influence of trophic factors that trigger the expression or suppression of the appropriate genetic machinery. The plasticity of the autonomic nervous system during ageing, following trauma, surgery, after chronic exposure to drugs and in disease is well documented (Burnstock, 1990; Milner and Burnstock, 1994). The enteric nervous system is no exception to this. For example, whilst the total number of myenteric neurones in the intestine declines with age (Gabella, 1990), an increasing proportion remaining contain NOS (Santer, 1994; Belai, Cooper and Burnstock, 1995; Belai and Burnstock, 1999). During mammalian hibernation, when there are extended periods of gastrointestinal inactivity, there is a selective increase in the number of substance P and CGRP-containing enteric neurones (Shochina et al., 1997). There are

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also many examples of plasticity of enteric neurones during chronic denervation and in colonic disease which are given below. Effects of chronic extrinsic denervation Surgical extrinsic denervation of the gut results in an altered innervation profile of submucosal arteries in guinea-pig ileum. Normally, submucosal arteries are innervated by extrinsic sensory nerves, which contain both substance P and CGRP, and sympathetic nerves, which contain noradrenaline, ATP and NPY. Six weeks after denervation, substance P- (without CGRP) and VIP-containing fibres are abundant in these vessels, possibly as a result of sprouting of axons of intrinsic origin (Galligan, Costa and Furness, 1988). Myenteric neurones appear to be essential for normal, sympathetic reinnervation of gastrointestinal circular smooth muscle. After surgical extrinsic denervation of the jejunum combined with chemical myenteric denervation with benzyldimethyltetradecylammonium chloride, the pattern of sympathetic nerves reinnervating the gut is altered and there is evidence for innervation of the circular muscle by the submucous plexus, probably induced by the increased production of neurotrophic factors from the hyperplastic smooth muscle (Luck et al., 1993). These changes may represent a part of the adaptive response of the enteric nervous system which permits normal intestinal function in the absence of extrinsic neuronal inputs. In some species, VIP levels in the small intestine are increased following long-term extrinsic denervation (Nelson, Sarr and Go, 1991), probably as a result of enhanced transcriptional regulation in the intrinsic ganglia, as occurs following extrinsic denervation and transection of the intestine (Stadelmann et al., 1996). Surgical coeliac ganglionectomy does not affect innervation of the intestine by substance P-containing nerve fibres or neurokinin receptor-binding density (Ouyang et al., 1996). Effects of chronic sympathectomy Long-term chemical sympathectomy leads to altered peptide expression in the gut wall that differs from the responses to surgical sympathectomy. Five months after guanethidine sympathectomy of neonatal rats, levels of CGRP and substance P increase in the myenteric plexus and surrounding smooth muscle of the ileum while NPY levels are unchanged. More surprisingly, noradrenaline levels, which are depleted six weeks after treatment, return to normal (Figure 7.9). This suggests that enteric neurones may turn on synthesis of noradrenaline and specific neuropeptides when the extrinsic sympathetic innervation is irreversibly destroyed (Milner et al., 1995). The functional implications of these changes are awaited. Guanethidine sympathectomy of adult rats leads to transitory increases in VIP and neurotensin levels in the colon one week after cessation of treatment, which normalise five weeks later (Nelson et al., 1988). Immunosympathectomy by neonatal administration of antiserum to nerve growth factor (NGF) leads to an increase in VIP, galanin, and substance P in nerves of the myenteric plexus of the ileum of the rat at four and eight weeks of age, but has no effect on noradrenaline, CGRP- and NPY-containing nerves (Belai, Aberdeen and Burnstock, 1992). Thus NGF availability may be an important regulatory agent in the post-natal expression of at least some enteric neuropeptides.


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Figure 7.9 Noradrenaline levels in the external muscle layers containing the myenteric plexus of ileum from neonatal guanethidine-treated rats at 6, 12 and 20 weeks of age (dotted bars) compared with age-matched controls (clear bars). ** P < 0.001. Results are expressed as mean ± S.E.M (µg/g tissue). The number of animals per group is given below the horizontal axis. (Reproduced from Milner et al., 1995, with permission from Elsevier Science).

NEUROEFFECTOR DYSFUNCTION IN PATHOLOGICAL CONDITIONS Diabetes mellitus Gastrointestinal dysfunction is frequently reported in diabetes mellitus, with either diarrhoea or constipation. No causal relationship between altered motility and changes in innervation in the gut has been established, but autonomic neuropathy has been implicated in reduced peristalsis, dilation of the oesophagus, gastric retention and disordered small intestinal movement and colonic atony or megacolon. Studies on the streptozotocin-treated diabetic rat, which has been used as a model of diabetes mellitus, have shown that during the course of the progression of diabetes, there are differential changes in the expression of neurotransmitters/neuromodulators in nerves supplying the bowel. Whilst there are degenerative changes in VIP- and noradrenalinecontaining nerves early on in the development of the disease, the expression of 5-HT, substance P and CGRP in nerve fibres is altered later on. The lack of release of VIP and CGRP from enteric nerves of early-diabetic rats during transmural electrical stimulation was reversed by acute application of insulin (Burnstock, Mirsky and Belai, 1988). Rigorous control of glycaemia in streptozotocin-induced diabetes prevents the increased expression of VIP and galanin in the myenteric plexus (Belai et al., 1996). NPY-immunoreactive

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nerve fibres appear comparatively resistant to change in diabetes although increased NPY levels in ileal myenteric neurones have been reported after a long diabetic period . In comparison with the ileum and proximal colon, the distal colon, appears to be relatively resistant to neurodegenerative changes due to diabetes (Figure 7.10). This may be explained by functional differences along the intestine or by the difference in origin of the sympathetic nerves to the ileum and proximal colon and to the distal colon (Belai et al., 1991). A reduction of the expression of NOS in the myenteric plexus of the antrum of the diabetic rat is not apparent in the ileum and colon (Takahashi et al., 1997; Wrzos et al., 1997). Idiopathic chronic constipation Patients with idiopathic constipation with normal bowel diameter have an increased whole-gut transit time. This may be related to an imbalance of enteric transmitter release, since VIP levels are reduced in the myenteric plexus and muscle layers of patients with this colonic motility disorder while levels of substance P and NPY are normal (Koch et al., 1988; Milner et al., 1990). In view of its role as an inhibitory neurotransmitter in peristalsis, an abnormally low concentration of VIP would possibly correlate with excess segmenting and reduced propulsion in constipation. Disturbances in the function of cholinergic innervation of the taenia coli of the colon have also been reported (Burleigh, 1988). 5-HT levels are elevated in circular smooth muscle and mucosa in this disorder, but unchanged in preparations containing the plexuses (Lincoln et al., 1990). Reduced numbers of substance P and VIP-immunoreactive nerve fibres in colonic circular muscle have been reported in biopsy samples taken from children with severe intractable constipation (Hutson, Chow and Borg, 1996). Reduced levels of substance P have also been reported in the mucosa (Goldin et al., 1989) and in submucosa isolated from rectal biopsies from patients with slow transit constipation showing normal levels of VIP and somatostatin (Tzavella et al., 1996). It appears then that disturbances or imbalances of both excitatory and inhibitory elements of intestinal innervation may contribute to bowel motility disorders associated with chronic constipation. In constipation associated with idiopathic megacolon or megarectum, abnormalities in inhibitory systems may contribute to abnormal gut function and subsequent bowel dilatation; in the rectum of these patients the density of innervation by nerves containing VIP and NADPH diaphorase (a marker for NO) is increased in the muscularis mucosae and lamina propria but decreased in the longitudinal muscle layer (Gattuso et al., 1996). Hirschsprung’s disease Hirschsprung’s disease is a congenital disorder of the gut characterised by the absence of enteric ganglia in variable lengths of the terminal colon. The aganglionic gut, in the absence of inhibitory neurones, is constricted and there is formation of megacolon on the oral side. There is a striking hyperinnervation of the colonic smooth muscle by extrinsic nerves while intrinsic neurones do not appear to enter the aganglionic segment (Hamada et al., 1987). In the absence of postganglionic cells, cholinergic fibres appear to seek alternative targets, in particular the smooth muscle; because of this, cholinergic nerve stimulation can cause colonic spasticity. In adjacent segments of ganglionic bowel, the extrinsic innervation appears


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Figure 7.10 Immunofluorescence micrographs showing VIP-immunoreactive nerve fibres in the myenteric plexus of (Column A) ileum and (Column B) distal colon from control (C) and 8-week (D8), 16-week (D16) and 25-week (D25) streptozotocin-diabetic rats. Note that in the ileum, after 8-weeks of diabetes there was an increase in fluorescence intensity and density of VIP-immunoreactive nerve fibres compared to controls, followed by a progressive decrease by 16 and 25 weeks. In the distal colon, the pattern of change was different, with no difference in VIP-immunoreactivity after 8 weeks of diabetes but increased fluorescence intensity of VIP-immunoreactive nerve fibres mainly around the unstained ganglion cells of the plexus which was restored to control levels by 25 weeks of diabetes. Calibration bar = 30 µm. (Reproduced from Belai et al., 1991, with permission from Harcourt).

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normal and comparatively sparse (Gannon, Noblet and Burnstock, 1969). Nerve fibres containing NPY appear to proliferate in the aganglionic region (Larsson, 1994), probably because they have their cell body oustide the gut wall. In the aganglionic sections there is a depletion of neuronal substance P and VIP (Bishop et al., 1981; Larsson, 1994), enkephalin, galanin, PACAP and CGRP (Larsson, Malmfors and Sundler, 1983; Shen et al., 1992; Larsson, 1994). Several authors have reported an absence of neuronal NOS from aganglionic segments, although it may be found within extrinsic nerve fasciculi (Larsson, 1994; O’Kelly et al., 1994; Larsson et al., 1995; Guo et al., 1997). Neuronal NOS mRNA is expressed in aganglionic segments of Hirschsprung’s disease only at very low levels (Kusafuka and Puri, 1997). There is no presence of inhibitory transmission in the aganglionic segment, either at a mechanical or electrophysiological level (Kubota, Ito and Ikeda, 1983; Tomita et al., 1995). In the lethal spotted mouse (or piebald lethal mouse), a mutant that develops foetal megacolon subsequent to colonic aganglionosis, although inhibitory transmission in affected large intestine is lost, the smooth muscle still relaxes in response to VIP or the NO generator, sodium nitroprusside (Chakder, McHugh and Rattan, 1997). In tissue from patients with long aganglionic regions, there may be small relaxant neurogenic responses in smooth muscle lying adjacent to the ganglionic region (Kamimura, Kubota and Suita, 1997); these are probably due to nerve fibres penetrating from the ganglionic region, or could be due to NOS contained in extrinsic fasciculi or rami. Intracellular recordings have shown that there are no differences in resting membrane potentials in the smooth muscle cells from ganglionic and aganglionic segments, but whereas ganglionic segments support regular slow-wave activity, the activity in aganglionic segments is very irregular (Kubota, Ito and Ikeda, 1983). Single pulses of electrical field stimulation evoke non-adrenergic relaxation in ganglionic but not aganglionic segments, whereas EJPs and IJPs can be recorded in ganglionic but not aganglionic segments (Kubota, Ito and Ikeda, 1983). The lethal spotted mouse also demonstrates a lack of junction potentials in the constricted aganglionic segment (Okasora and Okamoto, 1986). The lack of IJPs in either the human condition or the animal model may indicate that the spasticity in the colon is due to the lack of postganglionic, non-adrenergic inhibitory neurones, in addition to the proliferation of cholinergic neurones. The lack of these neurones is responsible for the non-propagation of migrating contractile complexes from ganglionic to aganglionic segments of the colon. Three susceptible genes have been identified in this disorder, the RET proto-oncogene, the endothelin B receptor gene, and the endothelin 3 gene (Edery et al., 1994; Amiel et al., 1996; Hirata, 1996). Ulcerative colitis In ulcerative colitis, in which the mucosa becomes inflamed, there are changes in the gut musculature, and probably its innervation, although the inflammation does not usually extend beyond the submucosa. The haustration of the colon is destroyed due to shortening of the longitudinal muscles (taenia coli) and relaxation of the circular muscles. This results in a decrease in the distal resistance to faecal flow, decreased transit time, diarrhoea, and compromised water absorption.


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There are changes in kallikrein and kininogen activity in the muscle layers (Zeitlin and Smith, 1973) in ulcerative colitis. The increased activity of kinins in the muscle layers may be responsible, at least in part, for the loss of haustration in the colon, because in the human taenia coli, bradykinin has been shown to cause an increase in tone, and in the circular muscle it causes a loss of tone (Fishlock, 1966). It is also likely that the kinins modulate neuronal activity directly, although they could also modulate it indirectly by affecting the postjunctional muscle membrane. Decreased colonic tissue levels of VIP in the mucosal/submucosal layers in ulcerative colitis together with increased VIP mRNA expression supports the suggestion of axonal degeneration and increased expression of neuronal VIP (Schülte-Bockholt et al., 1995). Greater responses of the colonic smooth muscle to electrical field stimulation in the presence of adrenergic and cholinergic blockade in tissues from patients with ulcerative colitis suggest that NANC inhibitory nerves play an important role in the observed impaired motility (Tomita, Munakata and Tanjoh, 1998).

SUMMARY AND FUTURE DIRECTIONS Most information on neurotransmission in the enteric nervous system is on animal gut, with still comparatively little information available in man. The recent advances in identifying and localising an increasing number of putative neurotransmitters and neuromodulators in the enteric nervous system and molecular biological approaches to identify their receptor subtypes will expand the search for selective agonists and antagonists as tools to unravel further the mechanisms of intestinal neurotransmission. These will have potential use to alleviate disorders of motility or secretion. There is growing recognition of the plasticity of enteric nerves, some of which may be adaptive to allow normal functioning of the gut. This opens up the possibility of manipulation of these events to facilitate beneficial compensatory changes and offers new therapeutic strategies to improve gastrointestinal function.

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8 Neural Control of Intestinal Vessels Neela Kotecha Department of Physiology, Monash University, Clayton, Victoria-3800, Australia Intestinal circulation is important in its own right due to its immense capacity for demand-related regulation of blood flow. As such, intestinal blood flow is influenced by well-developed neural regulatory mechanisms involving extrinsic nerves (sympathetic and sensory) as well as the intrinsic submucosal plexus of the enteric nervous system. The submucosal nerves subserve a vasodilator role as illustrated by studies on postprandial hyperaemia of the small intestine. However, the activity of the intrinsic nerves can be modified by extrinsic sympathetic nerves which act as a “brake” and serve to divert blood away from the intestine, when required. Although the mechanisms underlying sympathetic nerve transmission in the intestinal vessels have been a focus of study for the past two decades, it is only in the last decade that our understanding of mechanisms underlying responses to stimulation of sensory and intrinsic nerves has increased. It is clear from recent work that endothelial paracrine factors, the electrical connectivity between endothelium and vascular smooth muscle, a direct action on the vascular smooth muscle plus the interaction between vasomotor pathways are all involved in the neural regulation of vascular tone of the intestinal vessels. Despite substantial progress, we still do not know which particular subclasses of nerves are involved in normal physiological responses, nor do we have much understanding of changes in neural control resulting from diseased states such as diabetes, which is associated with widespread neuropathy and dysfunction of the enteric nervous system. Evaluating the roles of intrinsic and extrinsic nerves is an essential step towards understanding the neural regulation of intestinal arterioles in normal and pathophysiological states. The next decade promises to be both exciting and challenging as we piece together the jigsaw of neural control of intestinal vessels. KEY WORDS: autonomic nervous system; intestinal microcirculation; enteric nervous system; sensory nerves.

INTRODUCTION The splanchnic circulation is the largest strictly regional systemic circulation in the body as it receives at least one-fourth of the cardiac output. Its total capacity is as great as the entire blood volume. About a third of the splanchnic circulation passes through the hepatic circulation to the liver and the bulk of the remainder is distributed to the stomach and the small and large intestines through the mesenteric circulation. The intestinal circulation is interesting not only because it receives a large proportion of the cardiac output but also because of its impressive potential for demand related up- or downregulation of the blood 341

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flow (Mitchell and Blomqvist, 1971). In the resting mammal, 10–15% of the cardiac output is distributed to the small intestine and 2–3% to the colon (Lundgren, 1984). The two together total nearly 20% of the cardiac output which is equal to blood flow in either the renal or the cerebral circulation. The vascular beds of the small and large intestine are the major circuits supplied by the coeliac artery and the superior and inferior mesenteric vessels. The superior mesenteric artery conveys the lion’s share of the splanchnic blood flow and perfuses the small intestine and the proximal bowel whereas the distal bowel is supplied mainly by the inferior mesenteric artery.

ANATOMICAL ARRANGEMENT OF BLOOD VESSELS IN THE INTESTINE The superior mesenteric artery arises from the anterior surface of the aorta at the level of the first lumbar vertebral body. It enters the mesentery and ends there by forming an arch with one of its own branches, the ileal branch of the ileocoelic artery, forming the superior mesenteric loop. The superior mesenteric veins lie to the right of the superior mesenteric artery in its course through the mesentery. The mesenteric arteries divide and anastomose forming a series of arterial arcades that divide into two branches as they reach the intestine and run longitudinally along the mesenteric border in the aboral and oral directions. Small vessels are derived from the first series of arcades and these contribute to a second series. As the vessels progress further into the mesentery, more complex patterns are formed and three or even four tiers of arcades are present. The terminal series of arcades give off a pair of small vessels, which pass around the small intestine on either side of the mesenteric border, penetrating into the intestinal layers and forming the arteriolar tree. The arteriolar tree lies in a sheet of connective tissue, along with the submucous plexus, between the overlying circular muscle layer and the underlying mucosa. The submucosal arterioles have been implicated as the major resistance vessels within the intestine and therefore the main determinant of blood flow to a particular region and the site of the local and remote control systems that continually regulate the rate of blood flow by changing vascular smooth muscle tone (Lundgren, 1984). However recent work suggests that the arcade small arteries and the submucosal arterioles contribute roughly equally to the mesenteric resistance and point to a major role for small arteries in resistance regulation in this vascular bed (Fenger-Gron, Mulvany and Christensen, 1995). The capillaries arising from the arterioles feed the mucosa. In the villi of the mucosa, the inflow capillary and the outflow capillary are close together, separated by perhaps just 10 µm. There are no arterio-venous shunts or if they exist, these are of minor functional importance in the gastrointestinal vascular bed, (Lundgren, 1984) and the extensive network in the mucosa cannot be bypassed. The short distance between the inflow and outflow capillaries, i.e. the countercurrent flow arrangement, permits diffusion of lipid soluble substances to cross the capillary endothelium thereby short-circuiting their passage through the capillary bed in the tip of the villus (Jodal and Lundgren, 1986). Oxygen meets this requirement and at normal or high blood pressure only a small portion of the oxygen carried by the feeder vessels escapes the long trip to the villus tip. In low flow states, however, the proportion of oxygen diffusing directly from inflow to outflow

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vessels is much greater. In other words, from base to tip of the villus there is an oxygen gradient; this may be a factor in cell death at the tip – ordinarily a normal process of mucosal cell turnover. The collecting venules converge to form individual mesenteric veins leaving the intestine near the entry points of the arteries of the mesenteric border eventually leading the blood flow into the hepatic circulation. Overall, the intestinal microcirculation consists of three major vessel types each subserving a different primary function. Arterioles offer resistance to the flow of blood, thus allowing these vessels to control blood pressure and flow to the intestine. Capillaries permit exchange of substances from blood to cells and vice versa. Lastly, venules store and release blood as needed by the rest of the body.

FACTORS THAT INFLUENCE VASCULAR TONE The blood flow to the intestine is normally highly variable, responding to the need for fluid secretion and absorption and the metabolic demands of the mucosa and muscle. Vascular tone is a balance between vasoconstrictor and vasodilator influences that are prevalent. These factors include circulating vasoactive agents, such as secretin, gastrin, and cholecystokinin (CCK), substances released from the endothelium, such as endotheliumderived relaxing factor (EDRF), endothelium-derived hyperpolarizing factor (EDHF) and endothelin, local metabolic products and neurogenic mediators released from both vasoconstrictor and vasodilator nerves that innervate the blood vessels. (See Table 8.1 for a list of abbreviations used in this review). These factors are not mutually exclusive; one can modulate the influence or release of another. Overall, these mechanisms work in order to maintain an adequate blood flow to meet the requirements of the target organ. TABLE 8.1 List of abbreviations. ACh ATP cAMP CCK CGRP ChAT 4-DAMP DYN EJP GAL EDHF EDRF ENS 5-HT NA NADPH NO NPY

Acetylcholine Adenosine 5′-triphosphate Cyclic adenosine monophosphate Cholecystokinin Calcitonin gene-related peptide Choline acetyl transferase 4-diphenylacetoxy methylpiperidine methiodide Dynorphin Excitatory junction potential Galanin Endothelium-derived hyperpolarizing factor Endothelium-derived relaxing factor Enteric nervous system 5-Hydroxytryptamine Noradrenaline Nicotinamide adenine dinucleotide phosphate Nitric oxide Neuropeptide Y

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In this review, the focus will be on the neurogenic influences that control vessel calibre of the intestinal blood vessels. It has long been known that intestinal blood flow is influenced by well developed neural regulatory mechanisms within the enteric nervous system (ENS) of the intestine itself, as well as by extrinsic nerves. Relaxation of vascular smooth muscle induced by changes in neurogenic activity is achieved by two mechanisms acting either independently or simultaneously. The first of these mechanisms involves withdrawal of vasoconstrictor activity of sympathetic nerves. The second mechanism involves the release of substance/s which promotes vascular smooth muscle relaxation. The function of large arteries is to conduct blood to the distributing vessels. The smaller arteries and arterioles form the resistance vessels that are primarily involved in controlling blood flow to the organ. It is here that neural control, receptors for circulating hormones, and local factors have the greatest influence on organ blood flow because small changes in diameter will alter blood flow resistance to the greatest extent (resistance ∝ 1/radius4). Hence, I will focus my discussion mainly to neural mechanisms involved in the control of tone in arterioles of the small intestine as this is the most thoroughly investigated area of the gastrointestinal tract.

INNERVATION OF INTESTINAL BLOOD VESSELS The intestinal mucosa is richly supplied with nerve endings that are involved with enhancement of blood flow in the intestine. The control of intestinal blood vessels is complex, involving cholinergic inhibitory, adrenergic excitatory, and non-adrenergic, non-cholinergic inhibitory nerves of the autonomic nervous system. Intestinal blood vessels are specialised in that they are controlled by both extrinsic and intrinsic nerves. Extrinsic nerves include the postganglionic vasoconstrictor sympathetics arising from the prevertebral sympathetic ganglia and the sensory nerves arising from the dorsal root ganglia. No parasympathetic vasodilator innervation of the arterioles of the small intestine has been demonstrated, however the parasympathetic innervation of the colonic circulation arises from two main sources; the vagal and the pelvic nerves. Electrical stimulation of the vagal fibres to the colon does not elicit any flow changes apart from those caused by increased motility, whereas on pelvic nerve stimulation, blood flow increases three-fold (Hultén, Jodal and Lundgren, 1969). The mucosal lining of the gastrointestinal tract is provided with an extensive nervous supply, the ENS. Although known for more than a century, its function has been studied in detail only in the past two decades. Apart from controlling motility of the gut, the intrinsic nerves (all unmyelinated) of the ENS appear to form a dilator nerve supply to the arterioles in the intestine. There appears to be a paucity of innervation of the collecting venules of the guinea-pig small intestine and the rat mesentery (Furness, 1973; Furness and Costa, 1987). EXTRINSIC VASOCONSTRICTOR NERVES These arise from the sympathetic ganglia to supply the intestinal blood vessels where they form a meshwork of perivascular nerves. The nerves that supply the arterioles are restricted to the adventitial surface; there are many varicosities from which transmitter


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release may occur along the surface of the arteriole and ultrastructural studies indicate that virtually all of the varicosities form close appositions with arteriolar smooth muscle (Luff, McLachlan and Hirst, 1987). Sympathetic nerves are also prominent around myenteric and submucous ganglia (Furness and Costa, 1987). The morphological simplicity of the submucous arteriolar preparation makes it ideal for the study of vasomotor neurotransmission (Hirst, 1977; Hirst and Neild, 1978). The function of the extrinsic sympathetic nerves is primarily to reduce blood flow to the intestine. The sympathetic nerves supplying the gastrointestinal blood vessels are tonically active and they participate in cardiovascular homeostasis by altering intestinal vascular resistance to meet regional and whole body demands. Stimulation of the sympathetic nerves to the submucous arterioles causes a transient depolarisation of the arteriolar smooth muscle membrane potential, which is referred to as an excitatory junction potential (EJP). That these EJPs result from nerve stimulation has been demonstrated by their abolition by tetrodotoxin (TTX), which prevents propagation of action potentials by blocking Na+ channels, and by their absence when the arterioles have been extrinsically denervated (Hirst, 1977). During trains of stimuli, the EJPs summate to exceed a threshold, triggering an action potential which leads to constriction of the arteriole (Hirst, 1977). This increases the vascular resistance and decreases blood flow to the intestine. There is now plenty of evidence to suggest that noradrenaline (NA) and ATP act as co-transmitters, being released from sympathetic nerves in variable proportions depending on the tissue, the species and on the parameters of stimulation (for a review see Burnstock and Ralevic, 1994). Considerable variation exists in the ratio of NA:ATP released in different vessels. ATP appears to be the major component of sympathetic co-transmission (via P2X purinoceptors) in the intestinal arterioles of the guinea-pig and the function of NA is to act prejunctionally on α2 receptors to decrease the amount of transmitter released (Evans and Surprenant, 1992) or to cause a slow depolarisation accompanied by a slow contraction, via its action on extrajunctional α1 receptors. Additionally, ATP has a direct action on P2Y purinoceptors on the endothelium to cause the release of NO, thus effecting vasodilatation (Ralevic and Burnstock, 1988). Neuropeptide Y (NPY) is also found in most sympathetic nerves, including all sympathetic nerves supplying vascular smooth muscle (Uddman et al., 1985; McLachlan and Llewellyn, 1986; Morris et al., 1986). NPY has no vasoconstrictor effect on the submucous arterioles unlike other vascular beds (Neild and Kotecha, 1990). Normally NPY potentiates the actions of neurogenic or exogenously applied vasoconstrictors to the arterioles via its actions on the Y1 receptors (Xia, Neild and Kotecha, 1992). Alternatively, NPY can cause inhibition via the Y2 receptors localised on the smooth muscle membrane (Neild and Lewis, 1995). NPY can also modulate vasodilator neurotransmission by decreasing release from intrinsic vasodilator nerves via prejunctional Y2 receptors (Kotecha, 1998). Also both NA (via α2 receptors) and NPY can modulate release from sensory nerves (Kawasaki et al., 1990b, 1991). Hence, there is great scope for intercommunication between the sympathetic, sensory and intrinsic vasodilator nerves, in that NPY released during sympathetic nerve stimulation will not only potentiate the effects of constrictors that are co-released but also attenuate vasodilator neurotransmission, thus enhancing the effects the constrictors. On the other hand, direct effect of NPY on the muscle Y2 receptors would cause inhibition of the vascular

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Figure 8.1 Schematic representation of putative interactions between sympathetic, sensory and intrinsic nerves and their possible effects on the vascular smooth muscle and endothelium of submucosal arterioles.

smooth muscle. This latter role could be fulfilled by release of NPY from the NPY neurones in the submucosal vasodilator ganglia. These neurones project to the arterioles and NPY released by them could also act prejunctionally on the sympathetic nerve terminals to decrease the size of the excitatory junction potentials evoked by sympathetic stimulation (Kotecha, 1998; Neild and Kotecha, 1990). Figure 8.1 shows an overview of putative interactions between the extrinsic sympathetic and sensory nerves with the intrinsic nerves, and their effect on submucosal arterioles. EXTRINSIC SENSORY NERVES Primary afferent sensory nerves originating from the dorsal root ganglia project along the mesenteric arteries to enter the intestine and provides a perivascular network of fibres around the arterial system. The neuropeptides substance P (SP) and calcitonin generelated peptide (CGRP), potent vasodilators in many systems, are the principal transmitters in primary afferent nerves and have been shown to co-exist in the same perivascular terminals (Gibbins et al., 1985). Apart from the control exhibited by intrinsic nerves and extrinsic sympathetic nerves, extrinsic sensory nerves can also influence arteriolar tone. The majority of these fibres continue to run along the arterioles of the submucous plexus, ultimately sending branches which terminate in the mucosa and around some neurones in


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the ganglia of the submucous plexus. Several groups (Lundgren, Svanvik and Jivegard, 1989; Rozsa and Jacobson, 1989; Hottenstein et al., 1991) have proposed a role for extrinsic sensory nerves in the regulation of intestinal blood flow in vivo. For example, Kawasaki et al. (1991) have shown that, in the rat, stimulation of these nerves produces a substantial vasodilatation in the mesenteric arteries which can be antagonised by a fragment of CGRP, implying that CGRP is a vasodilator neurotransmitter. Further evidence for the participation of sensory nerves in the control of arteriolar tone in the submucosa comes from the study of Vanner (1993) who used capsaicin to excite the sensory nerves. Vanner (1993) found that preconstricted guinea-pig submucosal arterioles relaxed when exposed to capsaicin, suggesting that extrinsic sensory nerves provide an additional neural pathway within the submucosal plexus for modulation of blood flow. The exact mechanism via which capsaicin causes its effects is unclear but integrity of sensory nerves is a prerequisite for its actions as arterioles that have been extrinsically denervated do not respond to capsaicin (Vanner, 1993). In the submucous arteriolar preparation, small constrictions elicited by perivascular nerve stimulation are augmented in the presence of human CGRP(8–37) (unpublished personal observations), suggesting that CGRP is released from the sensory nerves running along the arteriole to inhibit vasoconstriction. Deactivation of the sensory nerves using capsaicin also results in augmentation of neurogenic constrictions; this potentiation is accompanied by an increase in the amplitude of the evoked EJPs suggesting that there is an increase in the amount of transmitter released from the sympathetic nerves (Coffa and Kotecha, 1999; Kotecha and Neild, 1995b). The corollary of this is that in in vitro experiments, sensory nerves are normally stimulated along with sympathetic nerves in the perivascular nerve bundle, to inhibit transmitter release from the sympathetic nerves. Although both the sensory neuropeptides (i.e. SP and CGRP) have been implicated in the modulation of arteriole tone in the intestinal microcirculation (Vanner, 1994), their mode of action is different. It is unlikely that neurally released SP, which is known to be a totally endotheliumdependent vasodilator, can reach the endothelium in sufficient quantity to effect inhibition of the vascular smooth muscle. The most likely role of neurogenic SP is to inhibit transmitter release from the sympathetic nerves, whilst the postjunctional actions of sensory nerves are mediated by CGRP (Coffa and Kotecha, 1999). Several mechanisms underlying the control of blood flow by sensory nerves have been implicated and may differ between species and different vascular beds of the same species. These mechanisms include postsynaptic action on the smooth muscle membrane to cause inhibition (Han, Naes and Westfall, 1990; Kawasaki et al., 1990a; Remak, Hottenstein and Jacobson, 1990), excitatory effect on intrinsic vasodilator nerves (Vanner and MacNaughton, 1995), inhibitory effect on the sympathetic nerves (Coffa and Kotecha, 1999), effect on the endothelium to release NO (Hill and Gould, 1995) or antidromic excitation of motor collaterals of sensory nerves (axon reflex; Lundgren, Svanvik and Jivegard, 1984; Rozsa and Jacobson, 1989). All of these mechanisms may be involved in the control of intestinal vessels and the particular neural circuits involved may depend on the stimulus. For example, Meehan, Hottenstein and Kreulen (1991) have shown that electrical stimulation of sensory nerves produces an inhibitory junction potential, which was sensitive to capsaicin and TTX, in guanethidine-treated mesenteric arterial smooth muscle, suggesting a postjunctional site of action. Sensory nerve mediated recovery of

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blood flow after sustained sympathetic nerve stimulation has been demonstrated in the rat superior mesenteric artery (Remak, Hottenstein and Jacobson, 1990) and may involve an endothelium-dependent mechanism similar to that seen in the rat irideal arterioles (Hill and Gould, 1995). Immunological studies have shown that extrinsic sensory nerve fibres project not only to the arterioles but also to the submucosal and myenteric plexuses (Gibbins, Furness and Costa, 1987). Thus, there is scope for extrinsic sensory nerves to excite submucosal vasodilator neurones that project to the arterioles. Vanner (1993) ruled out the possibility that sensory neurones could excite intrinsic cholinergic neurones on the basis that capsaicin-induced vasodilatations could not be prevented by the muscarinic antagonist, 4-diphenylacetoxy methylpiperidine (4-DAMP). However, Kotecha and Neild (1995a) have shown that although intrinsic cholinergic neurones are the main candidates for vasodilator effects of submucosal ganglion stimulation, there is clearly a role for other neurones. In fact, Rozsa and Jacobson (1989) have demonstrated that bile-oleate-induced intestinal vasodilatation involves primary afferent nerve fibres of the gut that release vasoactive intestinal peptide (VIP), and this release is not via nicotinic cholinergic synapses. Also sensory nerves are subject to modulation by NA and NPY released from the sympathetic nerve terminal (Kawasaki et al., 1990b; 1991). In turn, sensory nerves can modulate transmitter release from sympathetic nerves (Coffa and Kotecha, 1999). Whatever the mechanism by which sensory nerves have their effect, this neural pathway may contribute to responses evoked by different stimuli that transit through the intestinal lumen. INTRINSIC NERVES The arterioles of the small intestine are supplied with nerves that originate in the submucosal plexus of the ENS. In 1975, Hirst and McKirdy recorded the first synaptic potentials from the neurones of the guinea-pig submucous plexus. The presence of putative vasodilator transmitters in these neurones of the submucous ganglia (Furness and Costa, 1987) prompted Neild, Shen and Surprenant (1990) to investigate the effects of stimulating these ganglia on the arterioles of the submucosa. The anatomy of the ENS has been studied extensively but only in the guinea-pig. The submucous ganglia of the guinea-pig intestine contain at least three types of neurones which project to the submucous arterioles (Galligan, Costa and Furness, 1988; Brookes, Steele and Costa, 1991). They can be distinguished by their characteristic pattern of immunoreactivity for a variety of peptides. One type shows immunoreactivity for VIP, galanin (GAL) and dynorphin (DYN), and makes up 45% of the neurones in the plexus. Another shows immunoreactivity for SP and choline acetyl transferease (ChAT) and makes up 11% of the total neurone count, and the third shows immunoreactivity for calretinin and ChAT and makes up 12% of the total (Brookes, Steele and Costa, 1991). Recent work done in our laboratory (unpublished) has found that the neurones containing NPY also appear to project to the submucous arterioles. In addition to NPY, these neurones, which are known to project to the mucosa, contain ChAT, CCK, CGRP, DYN(1–8), somatostatin and possibly GAL (Song et al., 1992). All neurones are distributed throughout the submucous ganglia and will be referred to as VIP, SP, NPY and calretinin neurones. The vasodilator effects of these neurones can be demonstrated in isolated preparations of the submucosal plexus of the guinea-pig (Neild, Shen and Surprenant, 1990; Kotecha and Neild, 1995a). Many of the substances


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found in the neurones that project to the arterioles can cause vasodilatation in these and other arteries. However, the presence of multiple messengers in neurones does not imply that all of them subserve a transmitter role and it is not possible to decide from histochemical investigation alone if the innervation is efferent or afferent. Acetylcholine (ACh) appears to be the main mediator of this vasodilatation but others are clearly involved (Kotecha and Neild, 1995a); vasodilatation to 48% of the ganglia tested were completely abolished by the muscarinic antagonist, pirenzepine, 31% were partly abolished and 21% were not affected; as the individual ganglia contains from one to ten neurones (Furness and Costa, 1987), it was not surprising that some ganglia did not contain a cholinergic neurone. Vanner and Surprenant (1991) have also found cholinergic and non-cholinergic components, attributed to SP and VIP, of vasodilatation (in response to submucosal ganglionic stimulation) in the submucous arterioles of the guinea-pig colon. The neurones responsible for the cholinergic component of vasodilatation in the submucous arterioles of the ileum, could be the calretinin-containing neurones (Brookes, Steele and Costa, 1991) or the putative NPY neurones mentioned above. The only other neurone type in the ganglia known to contain ChAT are the SP neurones but these are unlikely to be involved as they cannot be stimulated at a rate greater than 1 Hz (Bornstein, Furness and Costa, 1989) but the vasodilator effect produced by ganglion stimulation was graded with stimulus frequency up to 10 Hz (Neild, Shen and Surprenant, 1990). Additionally, there appears to be no evidence for the involvement of SP in the dilator response in normal preparations, although it could be demonstrated in arterioles that had been extrinsically denervated for more than 30 days (Galligan et al., 1990) and the source of this neurally released SP appears to be the myenteric neurones (Jiang and Surprenant, 1992). Brunsson et al. (1995) have shown that the effects of SP in feline small intestine were via release of VIP. CHOLINERGIC TRANSMISSION Cholinergic neurones have several important roles. Cholinergic secretomotor neurones augment water and electrolyte secretion. They supply excitatory input to gastric acid secreting cells and to the external muscle and some function as interneurons. As mentioned before, ACh is the main mediator of arteriolar vasodilatation to submucosal ganglionic stimulation (Neild, Shen and Surprenant, 1990; Kotecha and Neild, 1995a). In the rat skin, ACh activates sensory nerves to cause its effect (Ralevic et al., 1992). However, Neild, Shen and Surprenant (1990) did not see a block of response to ACh when the submucosal arterioles were extrinsically denervated to get rid of the sensory nerves, suggesting that the cholinergic response in the submucous arterioles is not mediated via the sensory nerves. The endothelium plays a crucial role in mediating the vasodilator response to ACh and it has been shown that neurogenic ACh released from submucosal ganglia reaches the endothelium to mediate its effect via the release of nitric oxide (NO) (Andriantsitohaina and Surprenant, 1992; Kotecha and Coffa, 1999). Ach-induced vasodilatation is accompanied by a rapidly developing hyperpolarization of the smooth muscle (Kotecha and Neild, 1995a) similar to the hyperpolarization caused by NO in other arteries (Tare et al., 1990). Whilst a NO-independent hyperpolarization (Hashitani and Suzuki, 1997) and a direct inhibitory effect on the intestinal arteriole smooth muscle (Kotecha, 1999) can be

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demonstrated with exogenous ACh, it is clear that all of the inhibitory effects of neurogenically-released ACh on the intestinal arterioles are mediated via the release of NO from the vascular endothelium (Kotecha and Coffa, 1999). Additionally, the role of AChinduced hyperpolarization in modulating tone is also not clear since NO appears to be almost exclusively implicated in the dilator response of these arterioles to ACh when constricted using exogenously applied U46619 (Andriantsitohaina and Surprenant, 1992) or phenylephrine (Coffa and Kotecha, 1999). Recently Plane and Garland (1996) have demonstrated that vasodilator responses can be changed dramatically from NO to EDHF just by changing the mode of induction of tone. Contribution of Ca2+ from internal stores in response to different constrictor agents may vary in the different vascular beds (Low et al., 1996). As such, it may be envisaged that EDHF might be more effective against a contractile agonist that relies on voltagedependent Ca2+ influx, such as ATP (Reilly and Hirst, 1996). Although the ACh induced hyperpolarization (in conjunction with release of endothelial NO) may underlie the inhibition of applied ATP constrictions, these factors fail to account for the inhibition of neurogenic ATP constrictions (Kotecha, 1999). There is clearly another powerful inhibitory mechanism involved. Therefore, it seems that although endothelial factors can account for inhibition of constrictions mediated via exogenously applied constrictors, there appears to be a limited role for the physiological effectiveness of the endothelium in situations where the contractile state of the arteriolar muscle is determined by the level of sympathetic nerve activity. Hence, apart from a direct postjunctional effect of vasodilator nerves, an additional mechanism that needs to be considered is the neural cross talk between the vasodilator nerves and the extrinsic sympathetic nerves. Vasodilator nerves can regulate sympathetic activity by influencing the release of transmitter from the sympathetic nerves, as shown by a decrease in the amplitude of EJP (Kotecha and Neild, 1995b) probably by an action of ACh on the prejunctional M2 receptors on the sympathetic nerve terminals (Kotecha and Neild, 1993). The structure of the sympathetic nerves around the guinea-pig submucosal arterioles has been examined in detail (Luff, McLachlan and Hirst, 1987). No specialised junctions between nerves have been seen that would account for the interaction between vasodilator and sympathetic nerves, although close approaches between nerve axons in paravascular nerve bundles are common (Luff, McLachlan and Hirst, 1987). The putative vasodilator nerves that run in these bundles are immunoreactive for ChAT (Brookes, Steele and Costa, 1991). Recent work (Kotecha, 1999) has confirmed that exogenously applied ACh, at low concentration, can significantly reduce the amplitude of the EJPs evoked by stimulation of the perivascular nerves, without significantly altering the time constant of decay of EJPs. This confirms ACh to be at least one of the mediators involved in reduction of transmitter release from sympathetic nerves. Decrease in sympathetic outflow mediated via prejunctional muscarinic receptors has been known for some time (Eglen and Whiting, 1990; Fernandes et al., 1991; Komori and Suzuki, 1987; Kotecha and Neild, 1993). ACh-mediated inhibition of neurogenic constrictions have been compared with exogenous ATP constrictions, in the presence of agents that prevent the actions of NO and prostanoids. ACh causes much more potent inhibiton of neurogenic constrictions than ATP-constrictions, suggesting that ACh-induced hyperpolarization is not accountable. This implicates a prejunctionally mediated action of ACh on the sympathetic


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transmission (Kotecha, 1999). Hence, there appears to be a very powerful role of prejunctional inhibitory mechanism and the one that appears to be physiologically important. In conclusion, although release of endothelial factors in response to ACh can be demonstrated, we must be cautious about its importance and question its physiological role in the vascular bed that is being investigated. It is also clear that prejunctional mechanisms can be very powerful and it is possible that in this vascular bed only prejunctional effects modulate neurogenic constriction whereas endothelial factors may be important when looking at circulating constrictor agents. NON-CHOLINERGIC TRANSMITTERS Vasodilatation of the arteriole that was not sensitive to the muscarinic antagonist pirenzepine could be divided into two types; one that was not accompanied by hyperpolarization (attributable to VIP) and the other that was associated with a hyperpolarization (Kotecha and Neild, 1995a). A few experiments have been conducted in which dilatation has been obtained by intracellular stimulation of a single neurone, rather than a whole ganglion (Bornstein and Neild, unpublished; Surprenant, unpublished). The neurone has been marked with dye and later processed for immunohistochemical identification of peptides. The one clear result that has emerged from these few experiments is that VIP cells can cause vasodilatation. Also, stimulation of the whole ganglion can release VIP to cause vasodilatation as deduced from comparison of the smooth muscle membrane potential profile obtained during vasodilatation obtained by exogenous application of VIP and that obtained when the ganglion is stimulated. These dilatations are not associated with hyperpolarization of the smooth muscle membrane and are insensitive to muscarinic receptor antagonist, pirenzepine (Kotecha and Neild, 1995a). Itoh et al. (1985) also found that VIP caused no change in membrane potential in the rabbit mesenteric artery, although Standen et al. (1989) found that VIP opened potassium channels in isolated cells from the rat and rabbit mesenteric arteries. However, in both rabbit mesenteric arteries (Itoh et al., 1985) and cat cerebral arteries (Edvinsson et al., 1985) VIP caused an increase in cyclic adenosine monophosphate (cAMP) production which could cause vasodilatation without a change in membrane potential. VIP axons are associated with small blood vessels, mostly in the submucosa and the mucosa, throughout the gastrointestinal tract. VIP is a potent dilator of intestinal blood vessels (Furness and Costa, 1987). VIP is also involved in the mediation of vasodilator response of submucous arterioles in the guinea-pig colon to ganglion stimulation (Vanner and Surprenant, 1991). Vasodilator responses associated with pirenzepine-insensitive hyperpolarization were presumably mediated by GAL, DYN or some other unidentified substance whose vasodilator effects and effects on membrane potential have not been characterised (Kotecha and Neild, 1995a). Both GAL and DYN cause hyperpolarization of a similar magnitude and time course and cannot be readily distinguished from each other (Kotecha and Neild, 1995a). GAL has been reported to cause a small increase in K+ conductance in mudpuppy parasympathetic (Parsons and Konopka, 1991) and cultured RINm5f cells (an insulin-secreting pancreatic cell line; Homaidan, Sharp and Nowak, 1991), but in both these tissues its major action was to reduce the current flowing through voltage-sensitive

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Ca++ channels. This may also be the basis of its vasodilator action in the submucous arterioles. Among other substances found in the putative NPY neurones that project to the arteriole, CCK is capable of producing vasodilatation. Somatostatin increases absorptive fluxes in small and large intestine and reduces secretion that has been enhanced by theophylline, prostaglandin, 5-hydroxytryptamine (5-HT) or VIP (Furness and Costa, 1987). The roles of CCK and somatostatin in mediating responses to the submucous neurone stimulation have not been studied. There is also a possibility that NO nerves, originating in the enteric plexuses, mediate control of intestinal vessels. Yu, Li and Deng (1993) suggested that the effect of bradykinin in perfused rat mesentery may be mediated via NO released from nerves. More recently, Gyoda et al. (1995) have shown the presence of NADPH-diaphorase positive nerves and demonstrated that electrical field stimulation of the guinea-pig superior mesenteric artery induced a vasodilator response that was mediated by NO and CGRP. It is not clear at this stage whether such nerves are present on the small arteries and arterioles of the intestine although several workers have reported the presence of NO nerves in the myenteric and submucosal plexus and around submucosal arterioles of the monkey and human digestive system. NO-nerves have also been described in the ganglia of the two plexuses and within the blood vessels throughout the guinea-pig small and large intestines but, as yet, there is no indication of perivascular NO nerve fibres (Nichols, Krantis and Staines, 1992; McConalogue and Furness, 1993; De Giorgio et al., 1994). Release of NO from autonomic nerves and myenteric ganglia of the guinea-pig has also been demonstrated (Grider and Jin, 1993; Wiklund et al., 1993). It is not clear at this stage what role neurogenic NO plays in the regulation of intestinal blood flow, although NO is an important mediator for other endogenous vasodilator substances which act on the endothelium. Table 8.2 summarises the actions of the various transmitters involved in the control of intestinal blood vessels and the receptor types, where known.

TABLE 8.2 Summary of putative transmitters of intestinal nerves and their actions. Transmitter Sympathetic neurones ATP NA

Receptor

Putative actions

P2X P2Y α1

Mediator of arteriolar constriction Release of NO from endothelium Mediator of slow arteriolar constriction May be involved in autoregulatory escape Prejunctional inhibition of transmitter from sympathetic and sensory nerves Potentiator of vasoconstrictors Inhibition of arteriolar smooth muscle Prejunctional inhibition of submucosal and sensory nerves

α2 NPY

Sensory neurones SP CGRP and SP

Y1 Y2

NK1

Inhibition of transmitter release from sympathetics Mediators of dilatation in arterioles


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TABLE 8.2 Continued Transmitter

Receptor

Putative Actions Mediators of axon reflex Prejunctional excitation of submucous neurones

Submucous neurones ACh

Dynorphin Galanin NPY VIP

M3 M2

Y2

Release of NO from endothelium Inhibition of transmitter release from sympathetics Mediator of dilatation in arterioles Mediator of dilatation in arterioles Mediator of dilatation in arterioles Inhibition of transmitter release from sympathetics Mediator of dilatation in arterioles

NORMAL CONTROL OF INTESTINAL VESSELS The neural control of blood flow to the intestine is targeted towards maintaining mucosal integrity and meeting the needs for secretion and absorption. AUTOREGULATORY ESCAPE Increase in the resistance of a vascular bed, in the presence of a continuous vasoconstrictor stimulus, is often not maintained. Spontaneous relaxation (i.e. “autoregulatory escape”) appears to result from redistribution of blood flow within the intramural vessels. The decreased resistance is due to a partial dilatation of the small arteries and arterioles (Ross, 1971; Marshall, 1982). The vasoconstrictor stimulus may be neural (Folkow, Öberg and Rubinstein, 1964; Greenway, 1984) due to exposure to a vasoconstrictor substance (Ross, 1971). The presence of an autoregulatory escape from the influence of vasoconstrictor stimuli has been demonstrated in several species and appears to be a property of small arteries and arterioles (Fara, 1971; Duling et al., 1981). Several mechanisms (metabolic, myogenic, tissue-pressure) probably underlie the autoregulatory escape mechanisms (Lundgren, 1984) and it is not possible to quantify the contribution of these mechanisms. However, it is of interest to survey the evidence for a neural contribution. The relaxation of the submucous arteriole during an escape is preceded by repolarisation of the membrane potential (Neild and Kotecha, 1989). Neild and Kotecha (1989) proposed that NA (via α receptors) activated adenylate cyclase, leading to a rise in cAMP, favouring Ca2+ uptake into internal stores. Intracellular Ca2+ levels would then fall, thus closing calcium-dependent chloride channels, leading to hyperpolarization and vasodilatation. Additionally, ATP could act on P2Y purinoceptors on the endothelium to release NO, thus mediating vasodilatation (Ralevic and Burnstock, 1988). It is also possible that sensory nerves may be involved in this autoregulation since they appear to have a role in recovery of blood flow after sustained constriction by perivascular nerve stimulation in rat superior mesenteric artery (Remak, Hottenstein and Jacobson, 1990). Demonstration of Y2 receptors

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on the arteriolar muscle of the submucosa (Neild and Lewis, 1995) opens up another possibility for autoregulatory escape mechanism, since NPY, released from sympathetic nerves, could act on these Y2 receptors to cause inhibition. Escape may be physiologically important mechanism for avoiding tissue ischaemia during extreme vasoconstrictor stimuli and it would not be surprising if various complex neural control mechanisms are in place to fulfil this need. POSTPRANDIAL REFLEX VASODILATATION IN THE SMALL INTESTINE Digestion is an energy-consuming process that increases the oxygen demands of the small intestine. The entry of partly digested food and bile into the small intestine provokes dilatation of mucosal arterioles that can double blood flow to the mucosa (see Lundgren, 1984). This effect has been ascribed to local reflexes, hormones, and vasoactive metabolic products as it can be demonstrated in segments of intestine that have been acutely extrinsically denervated. The ENS aims at adjusting blood flow according to the metabolic demands of the tissue and therefore it seems logical to presume that the ENS would, at least in part, mediate change in the intestinal haemodynamics postprandially. There is plenty of evidence now to suggest that local neural reflex mechanisms may be responsible for the postprandial increase in blood flow in the ileum and the proximal colon, whereas the reflex vasodilatation evoked in the distal colon is mediated via the pelvic nerves (Biber, Lundgren and Svanvik, 1971; Fasth et al., 1977). Local neural reflex mechanisms would require all the neural elements, i.e. sensory, inter and motor neurones, to be present in an isolated piece of intestine. These reflexes can be divided into two types depending on the sensory neurones that mediate the signal transduction and the type of stimulus that evokes the reflex (i.e. chemical or mechanical). Both vasodilator reflexes are probably involved in normal digestive processes and although distinct populations of sensory neurones may be activated, these probably converge on the same subset of enteric motor neurones effecting vasodilatation presumably via release of VIP. LOCAL INTRINSIC VASODILATOR REFLEX-ELICITED BY MECHANICAL STIMULATION All the elements necessary for a reflex are wholly intrinsic to the intestine and removal or destruction of extrinsic nerves has no bearing on the reflex response to the right stimulus. The intrinsic sensory neurone is likely to be the proposed SP, calbindin immunoreactive neurone which is located in the submucosal plexus (Kirchgessner, Tamir and Gershon, 1992). Mechanical stimulation of the mucosa of an extrinsically denervated intestinal segment can increase blood flow in the small bowel, often more than two-fold; this increase is sensitive to TTX, lidocaine (a local anaesthetic), and 5-HT receptor blocking agents suggesting that the vascular response was elicited via an intramural nervous reflex arch involving 5-HT receptors (Biber, Lundgren and Svanvik, 1971; Biber, Fara, and Lundgren, 1974). In addition, Biber, Fara and Lundgren (1973) showed that transmural electrical field stimulation gave a TTX-sensitive increase in intestinal blood flow which again was indicative of


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an intramural vasodilator reflex. Sensitivity of this reflex to 5-HT receptor antagonists has led many workers to suggest that 5-HT released from enterochromaffin cell is involved in mediating it (Lundgren, Svanvik and Jivegard, 1989; Vanner, Jiang and Surprenant, 1993). Both VIP and ACh have been implicated as neurotransmitters at the vascular smooth muscle effecting vasodilatation (Lundgren, Svanvik and Jivegard, 1989; Vanner, Jiang and Surprenant, 1993). AXON REFLEX-ELICITED BY CHEMICAL STIMULI In this type of reflex, the activated sensory neurones are extrinsic, with cell bodies in the dorsal root ganglia and axonal projections to the motor limb of the reflex. These reflexes still occur in a freshly isolated piece of intestine where the sensory terminals and the collaterals that these nerves send out to the motor limb of the reflex are functional. This reflex is sensitive to capsaicin, a sensory neurotoxin (Holzer, 1991), or to prior denervation of the sensory afferents if time is allowed for the sensory afferents to degenerate before testing vasodilator reflexes in an isolated preparation of ileum. Rozsa and Jacobson (1989) have showed that the vasodilator response in rats, to a bile/oleate mixture in the intestinal lumen, was almost abolished by capsaicin treatment, indicating a role for the dorsal roots sensory neurones. It has long been known that the stimulation of the same nerves results in release of significant quantities of VIP from the intestine, and this could be responsible for some vasodilatation. This prompted Rozsa and Jacobson (1989) to apply VIP antiserum to the small intestine and this caused a dose-dependent reduction in the vasodilator response to the bile/oleate mixture. This response was insensitive to hexamethonium, implying that the connection was not by a cholinergic synapse. As immunoreactivity for VIP has not been demonstrated in primary sensory fibres in most species, it appears that there must be a connection from sensory fibres to the VIP-containing neurones of the ENS via a non-nicotinic interneurone. However, there is a possibility that the motor collaterals of sensory afferents may release VIP, in view of reports by Maggi et al. (1989, 1990) that VIP might be present in sensory nerves of human isolated ileum. The action of cholera toxin on the small intestine also involves intestinal vasodilator neurones (Cassuto et al., 1983; Jiang et al., 1993) and direct involvement of VIP neurones is supported by the work of Jiang et al. (1993) who have proposed that VIP neurones are activated by the B subunit of cholera toxin to effect vasodilatation.

INTESTINAL DISORDERS Intestinal ischaemia results in hypoxic injury and death of cells or tissue layers and often kills the patient. Usually death is a result of irreversible shock and multisystem organ failure but severe intestinal ischaemia is the central pathophysiological event. A study by Jrvinen et al. (1994) has revealed that 82% of the 214 patients with acute intestinal ischaemia died over a period of 30 days after diagnosis; 86% of these patients had a prior history of cardiovascular disease. Since many of the intestinal ischaemic conditions are secondary to other life-threatening disorders, usually cardiovascular, there is often a delay in the diagnosis of this condition, which may be fatal. As most of the small intestine is

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supplied by a single artery (i.e. the superior mesenteric artery), intestinal ischaemia results if blood flow to this artery is impaired, making the mucosa of the intestine susceptible to injury. As we have already seen, the intestinal vessels are supplied with a complex system of extrinsic and intrinsic neural pathways that work towards controlling the blood flow to meet the need for fluid secretion and absorption and metabolic demands of the muscle and mucosa. An aberration in any of these pathways may result in vascular insufficiency leading to undesirable clinical problems. For example, over-activity of the sympathetic component seen in some forms of hypertension will culminate in constriction of mesenteric blood vessels and sharply reduced blood flow. An early victim of ischaemic injury is the endothelial cell; this results in impaired control of vascular tone by neurogenic mechanisms that act via release of vasorelaxants from the endothelium, thus adding to the ischaemic insult. Decreased sensory nerve function in ageing animals may contribute to age-related alterations in circulatory haemodynamics in the rat mesentery (Li and Duckles, 1993) and this may be a contributory factor in age related intestinal disorders. Alterations in neurally mediated responses may also be the underlying cause of intestinal problems associated with some diseases. In diabetes, gastrointestinal neuropathy can involve virtually the entire length of the gastrointestinal tract. Clinical symptoms such as constipation and diarrhoea (Ross, 1993), which may be a result of a motility dysfunction may also be aggravated by abnormal fluid secretion or absorption and may indicate widespread abnormalities in extrinsic and intrinsic neural function. For example, Ralevic, Belai and Burnstock (1993) have demonstrated that vasodilator function of sensory-motor nerves in the mesenteric arterial bed of streptozotocin-induced diabetic rats is severely impaired. Since sensory nerves appear to form an integral part of the neural control of intestinal vessels, a deficit in these nerves could be expected to lead to many problems related to vascular insufficiency in the intestine. Thus, deficits in sensory nerve-mediated responses, including autoregulation, may culminate in ischaemia and tissue hypoxia. Changes in reflex vasodilatation in response to food, may underlie some of the gastrointestinal disorders associated with diabetes. It is interesting to draw parallels between disorders associated with neuropathy with results obtained in experiments where denervation has been part of the procedure. Neild, Shen and Surprenant (1990) showed that vasodilator response, in guinea-pig submucous arterioles, to submucosal ganglion stimulation was bigger after the arterioles had been extrinsically denervated. Galligan et al. (1990) showed that this neurogenically-mediated vasodilator response (after extrinsic denervation) was mediated by SP although SP was not involved in the neurogenic vasodilator response normally. Jiang and Surprenant (1992) later showed that this neurogenic SP response was mediated by myenteric neurones that had re-innervated the arterioles. In essence, this extrinsic denervation mimics diabetic neuropathy in which the sensory nerves are substantially affected. This speculation is supported by the work of Belai and Burnstock (1990) who showed that there was an increase in SP-like immunoreactive nerve fibres in the ileal myenteric plexus of 16 week streptozotocin-diabetic rats. Changes in the content of other neuropeptides have also been shown in both the submucosal and myenteric plexuses of diabetic rat ileum (Belai and Burnstock, 1990; Belai et al., 1993).


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It is tempting to speculate that in face of natural denervation, extrinsic and intrinsic, the tissue responds by developing pathways to compensate for the deficiency and this may be a normal remedial mechanism in diseased states, like diabetes, at least in the early stages of the disease. Pronounced changes in gut neuropeptide content and innervation patterns have also been observed in inflamed intestine of patients with inflammatory bowel disease but it is not clear whether these changes are due to altered synthesis and release from intrinsic and/ or extrinsic neurones and nerve fibres (Eysselein and Nast, 1991). Mayer, Raybould and Koelbel (1988) have suggested that neurogenic inflammation, elicited by activation of and release of mediators from primary afferent nerves, may be an important factor in inflammatory bowel disease. There is an ever-growing number of reports devoted to the study of neural impairment in various intestinal disorders (Hirschsprung’s disease, Crohn’s disease, ulcerative colitis) but how this relates to neural dysfunction in intestinal vascular tone has not been the focus of these studies. It is not within the scope of this review to assess how this might affect the control of tone in the intestinal vessels; however it stands to reason that altered haemodynamics in the intestinal circulation, related to neural dysfunction, may contribute to the pathogenesis of these disorders.

SUMMARY The precise coordination of mucosal function and smooth muscle activity necessary to maintain normal gut function is the prime function of the ENS. For optimum functioning, the blood flow to the intestine needs to be controlled so as to cope with the ever-changing requirement of the intestinal muscle, mucosa and digestive processes. The normal smooth interactions between the overlapping complex neural pathways – both extrinsic and intrinsic – that are involved in the control of intestinal blood vessels are responsible for adequate intestinal perfusion. Dysfunction of the bowel often involves both secretion/ absorption and motility. For example, bacterial toxins may stimulate sensory neurones to the mucosa which excite both secretomotor neurones and distension-sensitive neurones to promote motility. Stimulation of the secretomotor reflexes would lead to secretion of water and electrolytes, which in turn may lead to further activation of stretch reflexes. Although our knowledge of the pathways from sensory elements to physiological responses is quite extensive for peristaltic reflexes, it is somewhat fragmented when it comes to vasodilator reflexes. This is despite a dramatic development in our understanding of some the neural elements involved in controlling intestinal blood flow in the past couple of decades. Also the invaluable neurophysiological understanding of the normal control of intestinal vascular tone falls somewhat short of providing information about the neural basis of vascular tone under pathophysiological conditions. Substances present in the nerves may function as neurotransmitters leading to vasoconstriction or vasodilatation. They may also modulate the effect or release of other transmitters thus forming a means of communication between nerves. Additionally they may be involved in forming a “backup” system to ensure optimal blood flow or to compensate for selective neural deficiencies seen in diseased states.

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Questions that need to be addressed include; Which substances are acting as neurotransmitters? Where are the sites of action of each of the transmitters involved? What are the roles of the various nerves in normal physiological and pathophysiological conditions? The next decade promises to be both exciting and challenging, as we piece together the jigsaw of neural control of intestinal blood vessels.

ACKNOWLEDGEMENTS I am deeply grateful to Dr Tim Neild and Professor Mollie Holman for providing useful criticism of the manuscript.

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Proceedings of the Australian Physiological and Pharmacological Society, 24, 193P. Kotecha, N. and Neild, T.O. (1995a). Vasodilatation and smooth muscle membrane potential changes in arterioles from the guinea-pig small intestine. Journal of Physiology, 482, 661–667. Kotecha, N. and Neild, T.O. (1995b). Actions of vasodilator nerves on arteriolar smooth muscle and neurotransmitter release from sympathetic nerves in the guinea-pig small intestine. Journal of Physiology, 489, 849–855. Li, Y. and Duckles, S.P. (1993). Effect of age on vascular content of calcitonin gene-related peptide and mesenteric vasodilator nerve activity in the rat. European Journal of Pharmacology, 236, 373–378. Low, A.M., Kotecha, N., Neild, T.O., Kwan, C.Y. and Daniel, E.E. (1996). Relative contributions of extracellular Ca2+ and Ca2+ stores to smooth muscle contraction in arteries and arterioles of rat, guinea-pig, dog and rabbit. Clinical and Experimental Pharmacology and Physiology, 23, 310–316. Luff, S.E., McLachlan, E.M. and Hirst, G.D.S. (1987). An ultrastructural analysis of the sympathetic neuromuscular junctions on arterioles of the submucosa of the guinea-pig ileum. Journal of Comparative Neurology, 257, 578–594. Lundgren, O. (1984). Microcirculation of the gastrointestinal tract and pancreas. In The Handbook of Physiology; section 2: The Cardiovascular System: volume IV. Microcirculation, part 2, pp. 799–863. Bethesda, MA: American Physiological Society. Lundgren, O., Svanvik, J. and Jivegard L. (1989). Enteric Nervous System. Physiology and pathophysiology of the intestinal tract. Digestive Diseases and Sciences, 34, 264–283. Maggi, C.A., Santicioli, P., Del Bianco, E., Geppetti, P., Barbanti, G., Turini, D. et al. (1989). Release of VIPbut not CGRP-like immunoreactivity by capsaicin from the human isolated small intestine. Neuroscience Letters, 98, 317–320. Maggi, C.A., Giuliani, S., Santicioli, P., Patacchini, R., Said, S.I., Theodorsson, E. et al. (1990). 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Partial depletion of neuropeptide Y from noradrenergic perivascular and cardiac axons by 6-hydroxydopamine and reserpine. Regulatory Peptides, 13, 147–162. Neild, T.O. and Kotecha, N. (1989). A study of the phasic response of arterioles of the guinea pig small intestine to prolonged exposure to norepinephrine. Microvascular Research, 38, 186–199. Neild, T.O. and Kotecha, N. (1990). Actions of neuropeptide Y on arterioles of the guinea-pig small intestine are not mediated by smooth muscle membrane depolarization. Journal of the Autonomic Nervous System, 30, 29–36. Neild, T.O. and Lewis, C.J. (1995). Reduction of vasoconstriction mediated by neuropeptide Y Y2 receptors in arterioles of the guinea-pig small intestine. British Journal of Pharmacology, 115, 220–221. Neild, T.O., Shen, K.-Z. and Surprenant, A. (1990). Vasodilatation of arterioles by acetylcholine released from single neurons in the guinea-pig submucosal plexus. Journal of Physiology, 420, 247–265. 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9 Enteric Neuro-Immunophysiology Jackie D. Wood Department of Physiology, College of Medicine, The Ohio State University, 300 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210, USA The intestine has an effective defence mechanism that purges the bowel of foreign threats through an integrated system of mast cells, the enteric nervous system (ENS) and effectors made-up of the musculature, secretory epithelium and blood vasculature. Enteric mast cells acquire and retain memory of antigenic threats as they appear and reappear in the digestive tract throughout a lifetime. Antibody-based memory enables mast cells to detect and respond to forbidden antigens whenever they appear. Insofar as mast cells send signals to the enteric nervous system, their function is analogous to sensory neurons. Like sensory neurons, mast cells detect and signal. They release chemical signals (e.g. histamine) that alert the ENS to the presence within the lumen of a potential threat. The ENS responds by calling-up from its library of programs a coordinated cascade of events that quickly purges the threat. The program starts by stimulating the secretion of H2O, electrolytes and mucous to flush the antigen from the cryptic depths of the mucosa and suspend it in solution in the lumen. This is followed by initiation of powerful propulsive motility that rapidly moves the secretions and suspended antigen toward the anal outlet. These defensive events are accompanied by the side effects of abdominal distress, urgency and diarrhoea. KEY WORDS: enteric nervous system; mast cells; histamine; neural programs; antigens.

INTRODUCTION The gastrointestinal tract exhibits patterns of behaviour that are characteristic for specific digestive and pathological states. Activity of the musculature, mucosal epithelium and blood vasculature are coordinated to produce these specific patterns. A neural program for each pattern is stored in the memory of a program library in the enteric nervous system (brain-in-the-gut). The programs are identified by their motility component. They are: (1) segmentation motility in the digestive state of the small intestine; (2) migrating motor complex in the interdigestive state of the small intestine; (3) orthograde power propulsion in the small and large intestine; (4) retrograde power propulsion in the small intestine during emesis; (5) haustral formation in the large intestine; (6) physiological ileus (Wood, 1995a,b). Selective secretory behaviour is organised by the program in concert with the motility pattern. 363

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Activation of the program for orthograde power propulsion is a significant outcome of immuno-neural communication. This can occur after sensitisation of the enteric immune system to foreign antigens. Sensitisation may be caused by foreign antigens in the form of foodstuffs, toxins or invading organisms. After sensitisation, a second exposure to the same antigen triggers predictable integrated behaviour of the intestinal effector systems (e.g. Harari, Russell and Castro, 1987; Baird and Cuthbert, 1987). Neurally coordinated activity of the musculature, mucosa and blood vasculature results in organised behaviour of the whole intestine that rapidly expels the antigenic threat from the lumen. Recognition of an antigen by the sensitised immuno-neural apparatus activates the power propulsion program, the motor component of which is coordinated with copious secretion of water, electrolytes and mucous into the intestinal lumen, (Castro, 1989; Wang, Palmer and Cooke, 1991; Cowles and Sarna, 1990, 1991; Sarna et al., 1991). Detection by the enteric immune system and signalling to the enteric nervous system initiates the adaptive behaviour. Power propulsion is a specialised form of intestinal motility that forcefully and rapidly propels any material in the lumen over long distances to effectively empty the lumen. Its occurrence is accompanied by abdominal distress and diarrhoea in animal models and humans (Sethi and Sarna, 1991; Phillips, 1995). Output of the power propulsion program reproduces the same stereotyped motor behaviour in response to radiation exposure, mucosal contact with noxious stimulants or antigenic detection by the sensitised enteric immune system (Sarna et al., 1991). The neural program for power propulsion incorporates connections between submucous and myenteric divisions of the enteric nervous system that coordinate mucosal secretion with motor behaviour (Cooke, Wang and Rogers, 1993). The program is organised to stimulate copious secretion that flushes the mucosa and holds intraluminal contents in liquid suspension in the receiving segment ahead of the powerful propulsive contractions, which in turn, empty the lumen. The overall benefit is rapid excretion of material recognised by the immune system as threatening. The side effects are symptoms of abdominal distress and diarrhoea. Aside from containing a second brain with as many neurons as the spinal cord, the digestive tract is recognised as the largest lymphoid organ in the body together with a unique compliment of mast cells. In its position at one of the dirtiest of interfaces between the body and outside world, the intestinal mucosal immune system continuously encounters dietary antigens, bacteria, viruses and toxins. Physical and chemical barriers at the epithelial interface are insufficient to exclude fully the large antigen load, thereby allowing chronic challenges to the mucosal immune system. Evidence suggestive of direct communication between the mucosal immune system and the intrinsic neural networks in the intestine is derived from electrophysiological recording from enteric neurons in antigen sensitised animal models. The communication is meaningful and results in adaptive behaviour of the bowel in response to circumstances within the lumen that are threatening to the functional integrity of the whole animal. Communication is chemical in nature (paracrine) and incorporates specialised sensing functions of intestinal mast cells for specific antigens, together with the capacity of the enteric nervous system for intelligent interpretation of the signals. Immuno-neural integration progresses sequentially, beginning with immune detection followed by signal transfer to enteric microcircuits followed by neural interpretation and then selection of a specific neural program of coordinated mucosal secretion and motor propulsion (power propulsion) that effectively clears

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the antigenic threat from the intestinal lumen. Investigation of immuno-neural interactions merges the disciplines of mucosal immunology and enteric neurophysiology and will be the focus of this chapter.

BRAIN-IN-THE-GUT CONCEPT The conceptual model for the enteric nervous system is the same as the brain and spinal cord (Figure 9.1). Like the vertebrate brain and spinal cord, the enteric nervous system is organised with the neural elements and integrated circuitry necessary for independent processing of sensory information and the programming of organised behaviour of effector systems in the control of the intraluminal environment of the bowel. The neural elements are sensory neurons, interneurons and motor neurons. Each kind of neuron has specialised regions (e.g. cell bodies, axons and dendrites) that are differentiated in expression of specific receptors, ionic channels and other functions that underlie basic mechanisms of neural signalling and information processing. The three kinds of neurons are synaptically connected

Figure 9.1 The conceptual model for enteric neuro-immunophysiology encompasses the brain, the enteric nervous system, neuro-immune communication and behaviour of the digestive effector systems. The enteric nervous system is an independent integrative nervous system. It processes information derived from sensory neurons central nervous inputs and immune/inflammatory cells (e.g. mast cells). The enteric neural networks contain reflex microcircuits and a library of gut behavioural programs. Interneuronal circuits determine the outflow of information in motor neurons to the intestinal effector systems. Coordinated activity of the effectors determines momentto-moment behaviour of the gut. Mast cells detect threatening antigens and alert the enteric nervous system to their presence. Mast cells signal the enteric nervous system by releasing paracrine mediators. The central nervous system signals the enteric nervous system through a brain to mast cell connection as well as direct neural pathways. (Reproduced with permission from Wood, 1995c).

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into integrated circuits that process sensory information and program the variety of digestive functions found in specialised regions of the digestive tract during ever changing demands of the ingestive/digestive cycles of the functioning gut. Enteric integrated circuits determine the distinctive patterns of motility found along the gastrointestinal tract. Microcircuits in the intestine incorporate polysynaptic reflex circuits analogous to those in the spinal cord and basic to all recognised forms of motility. The integrated circuits store the programs for organisation of the commonly occurring digestive and interdigestive motility patterns emphasised above, as well as a library with additional programs that determine less frequent behaviours such as retropulsion during emesis, in the small bowel. Microcircuits in the myenteric division of the enteric nervous system contain the cell bodies of the motor neurons to the gastrointestinal musculature. Like spinal motor neurons, enteric motor neurons represent the final common pathway to the effector systems. Enteric motor neurons are uniaxonal neurons with Dogiel Type I morphology. There are both excitatory and inhibitory motor neurons to the musculature with specific axonal projections for each (reviewed by Brookes and Costa, 1994). Acetylcholine and substance P are recognised as important neurotransmitters released by excitatory motor neurons at neuromuscular junctions; whereas, nitric oxide and vasoactive intestinal peptide are implicated as inhibitory neurotransmitters (reviewed in Murray et al., 1991; Sanders and Ward, 1992; Makhlouf and Grider, 1993; Maggi et al., 1994; Wood, 1994; Maggi, 1995). Microcircuits of the submucous division of the enteric nervous system contain the cell bodies of the secretomotor neurons to the secretory epithelium. Secretomotor neurons stimulate secretion of water, electrolytes and mucous by releasing acetylcholine and vasoactive intestinal peptide at neuroepithelial junctions (reviewed by Cooke, 1989). Axonal collaterals of submucous secretomotor neurons project to the submucous vasculature. When the neuron fires to stimulate secretion from the crypts, the axon collaterals simultaneously release acetylcholine at their junctions with the vasculature (Adriantsitohaina and Surprenant, 1992). Acetylcholine activates the vascular endothelium to release nitric oxide, relax the arteriolar muscle and thereby increase mucosal blood flow in support of the demands of stimulated secretion in the intestinal crypts (reviewed by Vanner and Surprenant, 1996). Integrative microcircuits formed by synaptic connections of interneurons determine the timing and strength of neural outflow in the motor neuronal pathways to the musculature and secretory epithelium. In addition to individual control of each of these effector systems, the internuncial synaptic circuits coordinate the activity of each of the systems for homeostatic behaviour at the level of the integrated organ system. The enteric nervous system can be perceived, justifiably, as a mini-brain with specialised programs in juxtaposition to the effector systems it controls (Figure 9.1). Like the central nervous system, the enteric brain continuously processes sensory code on the momentto-moment state of each segment of gut and uses mechanisms of set-point determination and negative feedback for automatic control of the moment-to-moment intraluminal and intramural states. This model assumes that the central nervous system monitors the activity in the peripheral networks and transmits commands to the distant minibrain as appropriate for adjustment of gastrointestinal function in maintenance of whole body homeostasis.


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ENTERIC IMMUNOLOGY The intestinal tract is colonised from birth with a complement of lymphoid and myeloid cells that fluctuates with luminal conditions and pathophysiological states (reviewed by McDonald, 1993). A variety of cell types including polymorphonuclear leukocytes, lymphocytes, macrophages, dendrocytes and mast cells are present in varying numbers in the intestinal mucosa and/or lamina propria and may be present in the muscle coats and connective tissue lamina between circular and longitudinal muscle coats. These are often found in close histo-anatomical association with the neuronal elements of the enteric nervous system and vagal nerve projections (Stead et al., 1987; Befus, 1994; Gottwald et al., 1995; Williams, Berthoud and Stead, 1995). In the normal bowel, both histo-anatomical and immunophysiological evidence suggest that elements of the enteric immune system are strategically positioned to cooperate with the enteric nervous system in establishing a first line of defense against foreign invasion at a vulnerable interface between the body and the outside environment. In pathophysiological states of inflammation (e.g. Crohn’s disease, ulcerative colitis and parasitic infection) close histo-anatomical proximity of lymphocytes and polymorphonuclear leukocytes to enteric nerve elements suggest the possibility for released cytokines and chemical mediators to reach and influence enteric nervous functions. Electrophysiological studies in enteric neurons described later in the chapter confirm that mediators of this nature do in fact alter electrical and synaptic behaviour of enteric neurons. ENTERIC MAST CELLS All of the cell types related to immunity and inflammation are putative sources of paracrine signals to the enteric nervous system. Nevertheless, most is known about signalling between mast cells and the neural elements of the local microcircuits of the enteric nervous system. The cytoplasm of mast cells is characterised by large numbers of electron-dense granules that are sites of storage for a variety of pre-formed chemical mediators. In addition to mediators that are preformed and stored, are substances that are newly synthesised in response to stimulation. Mast cells can be stimulated by antigens or secretagogues to secrete the mediators. Antigen stimulation involves receptors for antibodies on mast cells all of which have highaffinity receptors for IgE or other immunoglobulins (depending on animal species). When the receptors are occupied by antibodies to a sensitising antigen and cross-linking occurs by interaction of the sensitising antigen with the bound antibody, the mast cells release a melange of mediators. The list of mast cell mediators is long and only a partial list of those implicated in enteric immuno-neural communication by direct electrophysiological neural recording are listed in Table 9.1. Intestinal mast cells proliferate during infection of the intestine with nematode parasites such as Trichinella spiralis and Nippostrongylus brasiliensis. Animal models infected with these parasites as well as food allergy models utilising hyper-sensitivity to milk protein have proved informative in studies on mast cell involvement in enteric immuno-neural communication. In the sensitisation models, a second exposure to antigen isolated from the nematode, or to the milk protein, β-lactoglobulin results in predictable integrated behaviour of the intestinal

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TABLE 9.1 Mast cell mediators: a truncated list. Preformed and stored

Newly synthesised

Biogenic amines Histamine 5-Hydroxytryptamine

Platelet activating factor

Enzymes Proteases Peroxidase Superoxide dismutase Chemotactic factors for inflammatory cells

Nitric oxide

Cytokines Tissue necrosis factor-α Interleukins IL-4

Arachidonic acid metabolites Leukotrienes, e.g. LTB4, LTC3 Prostaglandins, e.g. PGD2 Cytokines Tissue necrosis factor-α Interleukins IL-1β, IL-6

effector systems (e.g. Harari, Russell and Castro, 1987; Baird and Cuthbert, 1987; McKay and Perdue, 1993a,b). Recognition of the antigen by antibodies bound to sensitised mast cells triggers degranulation and release of mediators. The mediators then become messengers to the brain-in-the-gut which responds by suppressing other programs in its library and running the program designed to eliminate the antigen from the lumen (i.e. power propulsion coupled with copious secretion). In this respect, intestinal mast cells are uniquely equipped and situated to recognise agents that threaten whole body integrity and then signal to the enteric nervous system to program an appropriate response. Mast cell function in immuno-neural communication is an immune analogue of sensory detection and information coding in the classic sense of the nervous system (Figure 9.2). Sensory neurons, are genetically programmed to express a detection mechanism for a specific stimulus energy that remains unchanged throughout the life of the individual. Mast cells, on the other hand, acquire specific detection capabilities through the flexibility of recognition functions inherent in antibody expression by the immune system. Detection specificity functions can be acquired throughout life due to formation of new antibodies that become associated with mast cells. The output signals from mast cells are analogous to those from sensory neurons. Both mast cells and sensory neurons ultimately code information on the sensed parameter as a chemical message that is decoded by internuncial information-processing circuits in the nervous system. Apart from local signalling to the enteric nervous system, central messages from the cephalic brain to the enteric nervous system may be transmitted to the enteric brain through the mast cells. This is a brain-gut interaction by which central psychological status may be linked to irritable states of the digestive tract. Functional evidence for a brain-to-mast cell connection is found in reports of Pavlovian conditioning of mast cell degranulation in the gastrointestinal tract (MacQueen et al., 1989). Release of mast cell protease into the systemic circulation is a marker for degranulation of enteric mucosal mast cells. This can be demonstrated as a conditioned response


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Figure 9.2 Detection and signalling functions of mast cells are analogous to sensory neurons. Receptor regions of sensory neurons (e.g. Pacinian corpuscles) detect changes in stimulus energy and transduce the changes into action potential code. The action potential code is transformed to a chemical code at the synapse of the sensory fibre with the second order neuron in the central neural processing circuitry for interpretation. Sensory neurons detect and code the same stimulus throughout its life. Intestinal mast cells detect sensitising antigens and use a chemical code to signal the presence of the antigen to the processing circuits of the enteric nervous system. The enteric nervous system interprets and responds to the mast cell signals with outputs that are adaptive for the animal. Mast cells detection is based on immune functions that enable the cells to learn to detect and remember antigenic substances and signal the presence of the allergen to the enteric nervous system whenever it reappears. Mast cell detection and signalling is updated for new allergens as they are encountered throughout life.

in laboratory animals to either light or auditory stimuli and in humans as a conditioned response to stress (Santos et al., 1998), suggestive of a brain-to-enteric-mast cell connection. Central nervous influence on mast cells in the upper gastrointestinal tract is suggested as well by work showing association between vagal afferents and enteric mast cells (Williams, Berthoud and Stead, 1995) and increased levels of histamine in intestinal mast cells in response to vagal nerve stimulation (Gottwald et al., 1995). Findings that stimulation of neurons in the brain stem by thyrotropin-releasing hormone evokes degranulation of mast cells in the rat small intestine are added evidence for brainmast cell interactions (Santos et al., 1996). Overall, the brain-mast cell connection is significant because the gastrointestinal symptoms associated with mast cell degranulation are expected to be the same whether the mast cells are stimulated by antigen-antibody crosslinking or neurotransmitters. Intracerebroventricular injection of thyrotropin-releasing hormone in the rat evokes the same kinds of inflammation and erosions in the stomach as cold-restraint stress. In the large intestine, restraint stress exacerbates nociceptive responses and these effects are associated with increased release of histamine from mast cells (Gue et al., 1997). Intracerebroventricular injection of corticotropin-releasing factor mimics the responses to stress. Intracerebroventricular injection of a corticotropin- releasing factor antagonist or pretreatment with mast cell stabilising drugs suppresses stress-induced lower gastrointestinal responses.

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Mast cell degranulation may release mediators that sensitise silent nociceptors in the large intestine. In animals, degranulation of enteric mast cells results in a reduced threshold for pain responses to intestinal distension that is prevented by treatment with mast cell stabilising drugs (Coelho, Fioramonti and Bueno, 1998). In addition to signalling the enteric minibrain, mast cells release messenger substances that attract leukocytes into the lamina propria from the vascular system. Introduction of purified subunits of Clostridium difficile toxin into intestinal loops stimulates influx of polymorphonuclear leukocytes as well as activating the neural program for coupled secretion and power propulsion. Blockade of enteric nerves by tetrodotoxin, treatment with tachykinin NK1 antagonists or mast cell stabilisation by the drug ketotifen all prevent the transmigration of polymorphonuclear cells into the lamina propria and the acute inflammatory response to the toxin (Rachmilewitz et al., 1992; Castagliuolo et al., 1994; Pothoulakis, Castagliuolo and LaMont, 1998). This suggests that enteric nervous input utilising substance P, which is a putative transmitter for enteric slow excitatory postsynaptic potentials and axonal reflexes mediated by primary afferent neurons (see below), participates in toxin-induced mast cell degranulation and the release of chemoattractant factors for inflammatory cells. Application of toxin-A to neurons in the enteric nervous system alters both the electrical behaviour of the neuronal cell bodies and inhibitory noradrenergic neurotransmission to secretomotor neurons in the submucous plexus (Xia et al., 1999). Altered electrical behaviour includes slow EPSP-like depolarisation and elevated excitability. Tetrodotoxin or a histamine H2 receptor antagonist does not affect the depolarisation evoked by toxin-A. Failure of the histamine antagonist to suppress the actions of toxin-A indicates that its neuronal actions are direct and not mediated by degranulation of intramural mast cells. Among the actions of toxin-A on neurotransmission is suppression of inhibitory postsynaptic potentials evoked in uniaxonal neurons in the submucous plexus by stimulation of sympathetic nerve fibres. The toxin acts presynaptically to suppress the release of noradrenaline from the sympathetic innervation of the enteric nervous system. This results in removal of sympathetic braking action from secretomotor neurons. Together with toxinevoked neuronal excitation, this may be an underlying factor in the diarrhoea associated with C. difficile overgrowth in the large intestine. Substance P is known to be a secretagogue for histamine release from mast cells (Cocchiara et al., 1999) but often in concentrations so high that the physiological relevance has been questioned (Shanahan et al., 1985). It was generally assumed that interactions of the hydrophobic N-terminal moieties of substance P with the lipid bilayer, not the basic amino acids believed to bind the receptor site, was responsible for degranulation of mast cells by the neuropeptide (Repke et al., 1987). The findings of Castagliuolo et al. (1994) with the C. difficile suggest that this may not be the case and that NK-1 receptors may mediate substance P action on the mast cells. Relative to these uncertainties, Befus (1994) points out the fact that mast cell populations are heterogenous, that there may be selective release of mast cell mediators by different physiological messengers and histamine release may not be the optimum end point for assaying effects of putative secretagogues. For example, patch clamp studies show that picomolar concentrations of substance P evokes changes in ion channel behaviour in peritoneal mast cells (Janiszewski, Bienenstock and Blennerhassett, 1994) and the IC50 for mast cell-mediated intestinal mucosal secretion is 100 nM for substance P and 1 nM for substance P(4–11) (Wang et al., 1995).


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SIGNALLING FROM MAST CELLS TO THE BRAIN-IN-THE-GUT Several mast cell-derived mediators share common neuropharmacological actions on electrical and synaptic behaviour of neurons in the enteric nervous system. These include histamine, 5-hydroxytryptamine, adenosine, interleukin-1β (IL-1β), interleukin-6 (IL-6), tumour necrosis factor α (TNFα), and platelet activating factor. HISTAMINE Histamine is not localised to any extent in enteric neurons and is not considered as a putative neurotransmitter in enteric microcircuits (Panula et al., 1985). The principal source of histamine in the intestine is the population of mast cells. Understanding of histaminergic actions on intestinal neurons is derived from results obtained in electrophysiological studies on single neurons of the small and large intestine of the guinea-pig. These studies were done with either extracellular electrodes that detect action potential discharge of individual neurons or intracellular microelectrodes that record changes in neuronal membrane potential, input resistance, action potential discharge and synaptic potentials. Detailed accounts of the methods, as well as a review of the cellular neurophysiology of enteric neurons can be found in Wood (1989, 1994). Mayer and Wood (1975) reported that application of histamine excited neurons in the myenteric plexus of cat small intestine. Subsequent work in the guinea-pig revealed two important actions of histamine on neuronal elements of the enteric microcircuits. One of the actions occurs at the neuronal cell body and consists of long-lasting excitation. The second is at nicotinic synapses, where histamine acts to suppress synaptic transmission. These actions are found in the circuitry of both the myenteric and submucous plexuses of the small and large intestine. Histamine mimics slow synaptic excitation The excitatory effects of histamine mimic slow synaptic excitation (Nemeth, Ort and Wood, 1984; Tamura and Wood, 1992; Frieling, Cooke and Wood, 1993). Slow synaptic excitation (slow EPSP) is reviewed in detail in Wood (1994). It is a response detected by intracellular microelectrodes when specific neurotransmitters are released from enteric axons at synapses on the cell bodies of the recorded neurons (Figure 9.3). Binding of slow EPSP mediators to their receptors activates transduction mechanisms that change conductance states of a variety of ionic channels to produce the characteristic alterations in electrical behaviour. The changes in electrical behaviour during slow EPSPs include depolarisation of the membrane potential, increase in the electrical resistance of the membrane and enhanced excitability reflected by spike discharge. In addition, hyperpolarising after-potentials in AH/Type 2 (after-hyperpolarisation/Dogiel Type II neurons) neurons are suppressed to permit repetitive spike discharge. AH/Type 2 neurons are a distinct class of enteric neurons characterised by low excitability and the presence of long-lasting hyperpolarising after-potentials. In the resting state

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Figure 9.3 Histamine and 5-hydroxytryptamine mimic slow synaptic excitation in AH/Type 2 enteric neurons. Slow synaptic excitation is characterised by depolarisation and a prolonged train of action potentials. The slow EPSP in the top trace was evoked by focal electrical stimulation of the axon responsible for synaptic input. Application of histamine (middle trace) or 5-hydroxytryptamine (bottom trace) by pressure microejection from a fine-tipped pipette mimics the depolarisation and spike train of the slow synaptic response.

and in the absence of slow synaptic mediators, these neurons either do not fire or fire only one or a few action potentials to a depolarising stimulus. The action potentials are followed by hyperpolarising after-potentials that last for several seconds. After-hyperpolarisation is a mechanism that prevents repetitive discharge when the neuron is not under the influence of a slow excitatory signal. Suppression of the after-hyperpolarisation enables the repetitive discharge of spikes that is seen to occur in prolonged trains during slow synaptic activation (Figure 9.3). Repetitive discharge is an important functional event because each action potential, after starting in the cell body of the multipolar neuron, will propagate to the terminals of each neurite where the neuron’s neurotransmitters will be released at synapses with other elements of the microcircuits. Signal transduction for slow EPSPs Transduction of slow synaptic signals involves activation of adenylate cyclase and second messenger function of adenosine 3′,5′-cyclic monophosphate (cAMP) (Palmer, Wood and Zafirov, 1986, 1987a). Excitatory receptors for slow synaptic excitation are coupled to adenylate cyclase through G proteins in enteric neuronal membranes (Tamura, Itoh and Wood, 1995).


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Histamine receptors for slow excitatory responses Histamine H2 receptors are the mediators of the slow excitatory response to histamine in cell bodies of enteric neurons in the guinea-pig (Nemeth, Ort and Wood, 1984; Tamura and Wood, 1992; Freiling, Cooke and Wood, 1993). The selective H2 agonist, dimaprit, mimics the excitatory actions of histamine and the H2 antagonist, cimetidine, blocks them. Exposure to histamine elevates levels of cAMP in myenteric ganglia and this action is also blocked by selective histamine H2 receptor antagonists and mimicked by selective agonists (Xia, Fertel and Wood, 1996). The hyperexcitability evoked by interaction of histamine with the H2 histamine receptor does not desensitise during prolonged exposure. All of the slow EPSP-like actions of histamine persist unabated for as long as histamine is present in the bathing media (Tamura and Wood, 1992). This is expected to continuously energise the microcircuits that control the behaviour of the intestinal effector systems and provide a long-lasting drive to maintain the immune “alarm” program in effect. Chronic release of histamine from mast cells is likely to do this in the living animal. Occurrence of this activity in the microcircuits that control the secretomotor neurons to the intestinal crypts is predicted to enhance intestinal secretion leading to diarrhoeal symptoms like those associated with infective agents or food allergies. Watery diarrhoea symptoms associated with mastocytosis and microscopic colitis in humans have been treated effectively with histaminergic blocking drugs (Baum, Paramjit and Miner, 1989).

Pattern generation evoked by histamine Pattern generators are networks of neurons that generate the timing and phasing cues for the rhythmic behaviours of effector systems. They are often called central pattern generators because the network is capable of generating the same basic activity pattern in experimental isolation from other regions of the nervous system. Behaviours driven by neural pattern generators occur in fixed action patterns that may be triggered by an external stimulus or by intrinsic command inputs. These fixed action patterns are similar to reflexes, but differ in important respects. Unlike reflexes, the duration, latency and intensity of effector activity of fixed action patterns are not determined by the stimulus alone. Behaviours driven by pattern generators are also unlike reflexes in that the fixed action pattern can occur in the absence of sensory input, whereas reflexes cannot. Although, sensory feedback often modifies ongoing motor activity, it is not essential for the generation or timing of a motor pattern. This is programmed by the pattern generator and communicated to the motor neurons for execution of the pattern. A common characteristic of pattern generating networks is the occurrence of rhythmic discharge of spike bursts in individual neurons of the network. This is the case for networks that determine fixed action patterns of movements in both vertebrate and invertebrate central nervous systems. Exposure of the neural networks in the intestinal submucous plexus to histamine evokes patterned bursts of spikes suggestive of a central pattern generator in a subpopulation of neurons in the network (Figure 9.4). At periodic intervals in the presence of histamine, the membrane potential depolarises spontaneously and this is accompanied by a crescendo of

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Figure 9.4 Pattern generation evoked by histamine in an AH/Type 2 submucous neuron of the guinea-pig colon. (A) Recurrent trains of action potentials evoked by exposure of the neuron to histamine. (B) Continuous traces of a single train of action potentials from A recorded on an expanded time base. Action potential discharge was preceded by spontaneous depolarisation of the membrane potential. Intracellular injection of constant current depolarising pulses did not evoke spikes prior to the train. The current pulses triggered spikes as the membrane potential depolarised and excitability increased, leading up to the spike train. Spikes continued to be evoked by the depolarising current pulses until the membrane potential repolarised and excitability was reduced to a silent period before the next spike train. (Reproduced with permission from Frieling, Cooke and Wood, 1993).

spike discharge that subsides to silence prior to the start of the next cycle. In most neurons, the recurrent spike bursts occur at intervals of 2–3 min. The patterned behaviour can be evoked by histamine H2 receptor agonists, but not by histamine H1 receptor agonists (Frieling, Cooke and Wood, 1993). The effects of histamine are blocked by histamine H2 but not by histamine H1 receptor antagonists. Patterned discharge evoked by histamine occurs only in the networks of the submucous division of the enteric nervous system; it is not found in the myenteric division (Tamura and Wood, 1992). The electrophysiological observations suggest that histamine acts like a neuromodulator to activate a central pattern generator in the enteric neural networks. Discussion below will show how the central generator drives rhythmic patterns of effector behaviour when the neural networks are overlaid with histamine, either from experimental application or from antigen-stimulated release from mucosal mast cells. As in other nervous systems, it may be that the enteric neural networks are not hard-wired groups of neurons that exist only in an active or inactive mode and generate only one pattern of behaviour when in the active mode. The enteric networks may be like other ensembles of neurons known to be capable of producing a variety of behaviours depending on the kind of neuromodulatory overlay. This is sometimes referred to as multiple task processing where different neuromodulators reconfigure different rhythmic outputs from a single neural network.


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Presynaptic inhibition by histamine Presynaptic inhibition is the suppression of release of neurotransmitters from release sites on axons. In the enteric nervous system, this occurs at both fast and slow excitatory synapses and at neuroeffector junctions. Presynaptic inhibition may involve axo-axonal transmission, whereby release of a neurotransmitter from one axon acts at receptors on another to suppress release of transmitter from the second axon or it can be mediated by substances released from mast cells or other non-neuronal cells into the milieu surrounding the synaptic circuits. Ten or more kinds of receptors for neurotransmitters/neuromodulators are known to be involved in presynaptic inhibition in the enteric nervous system (reviewed in Wood, 1994). Histamine is one of these mediators. It acts at presynaptic receptors on cholinergic axons to suppress fast EPSPs at nicotinic synapses in the enteric microcircuits (Tamura, Palmer and Wood, 1987). Histamine also acts on sympathetic nerve terminals in the submucous plexus to suppress the release of noradrenaline and the occurrence of slow inhibitory postsynaptic potentials that are mediated by noradrenaline in secretomotor neurons (Figure 9.5). This action of histamine is blocked by histamine H3 receptor antagonists and

Figure 9.5 Histamine suppresses slow inhibitory postsynaptic potentials (IPSPs) mediated by release of noradrenaline from sympathetic postganglionic neurons in the submucous plexus of guinea-pig small intestine. (A) Control IPSP evoked by focal electrical stimulation of sympathetic postganglionic fibres. (B) Suppression of the IPSP during exposure to histamine. (C) Return of the IPSP after washout of histamine.

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is mimicked by histamine H3 receptor agonists, suggesting that histamine H3 receptors are responsible for the presynaptic inhibitory action of histamine. Discussion later in the chapter will describe how histamine released during antigenic degranulation of intestinal mast cells acts in a like manner. 5-HYDROXYTRYPTAMINE 5-Hydroxytryptamine is another preformed mediator expected to be released during degranulation of intestinal mast cells in most mammals except the guinea-pig. In the guineapig and some other species, 5-hydroxytryptamine localisation is restricted to neurons and enterochromaffin cells. Two actions of 5-hydroxytryptamine on guinea-pig enteric neurons are essentially the same as those of histamine, whereas in other respects the actions differ from histamine (Wood and Mayer, 1979; Mawe, Branchek and Gershon, 1986; Wade and Wood, 1988; Frieling, Cooke and Wood, 1991; Tack et al., 1992). 5-Hydroxytryptamine acts at 5-HT1p receptors on the neuronal soma (Mawe, Branchek and Gershon, 1986) to produce long-lasting excitatory responses that mimic all aspects of slow synaptic excitation and the slowly activating excitatory response to histamine (Figure 9.3). The 5-HT1p receptor is a metabotropic receptor linked through G proteins to activate adenylate cyclase and elevate levels of cAMP as part of the transduction process in myenteric ganglia from the guinea-pig (Fiorica-Howells, Wade and Gershon, 1993; Xia, Fertel and Wood, 1994). 5-Hydroxytryptamine acts at presynaptic receptors on cholinergic axons to suppress fast EPSPs at nicotinic synapses in the enteric networks (North et al., 1980). This action is also the same as the presynaptic inhibitory action of histamine at nicotinic synapses. The presynaptic inhibitory receptors behave like the 5-HT4 receptor subtype (Frieling, Cooke and Wood, 1991). Unlike histamine, and contrary to an earlier report (Surprenant and Crist, 1988), we have not found presynaptic inhibition of slow inhibitory postsynaptic potentials in the guinea-pig submucous plexus. 5-Hydroxytryptamine also produces fast excitatory depolarising responses that are similar to fast nicotinic excitatory postsynaptic potentials in enteric neurons. These responses are mediated by 5-HT3 ionotropic receptors that function as part of ligandgated channel complexes (Derkach, Surprenant and North, 1989). Responses similar to the 5-HT3 receptor mediated responses have not been found for histamine in the enteric nervous system. 5-Hydroxytryptamine also differs from histamine in not evoking the rhythmic discharge seen for histamine in submucous neurons. This carries over to the motor output from the submucosal network which does not show the rhythmic oscillations of effector behaviour that will be discussed later in the chapter for histamine and antigen exposure in the sensitised intestine. The actions of 5-hydroxytryptamine, when compared with histamine, suggest a neuromodulatory function that may be associated with network reconfiguration that differs from the “alarm” program that is called into play by the neuromodulatory action of histamine. Whereas, 5-hydroxytryptamine is involved in both neural and paracrine transmission, histamine seems to act solely as an “alarm” message to the enteric nervous system signalling the presence of a threat in the intestinal lumen.


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ADENOSINE The extent to which adenosine is released from mast cells during antigen exposure or in response to secretagogues is unclear. If adenosine were released as a neuromodulatory overlay on the enteric networks, it is clear that the effect would be a braking action on excitatory processes within the circuits. All of the actions of adenosine that are mediated by the adenosine A1 receptor subtype on neural elements in the enteric microcircuits of the guinea-pig are inhibitory. Adenosine acts through A1 receptors to suppress both fast nicotinic excitatory postsynaptic potentials and slow excitatory transmission. It inhibits excitation in the cell bodies of AH/Type 2 neurons by opening K+ channels and hyperpolarising the membrane (Palmer, Wood and Zafirov, 1987a,b; Christofi and Wood, 1993, 1994). Suppression of slow excitatory transmission by adenosine seems to involve A1 receptors positioned both pre- and postsynaptically. Application of adenosine after the onset of stimulus-evoked slow EPSPs aborts the response, indicating a post-synaptic action. In release studies of putative neurotransmitters for slow synaptic excitation (e.g. tachykinins), adenosine A1 stimulation suppresses the release of putative neurotransmitters for slow synaptic excitation (e.g. tachykinins) in studies on release from myenteric plexus preparations (Christofi, McDonald and Cook, 1990; Broad et al., 1992). Part of the mechanism of adenosinergic inhibition at the cell somas of enteric neurons involves inhibition of adenylate cyclase and suppression of cAMP formation (Xia, Fertel and Wood, 1997). Adenosine acts through G protein-coupled adenosine A1 receptors to inhibit stimulation of adenylate cyclase by forskolin (Zafirov, Palmer and Wood, 1985) and by histamine (Palmer, Wood and Zafirov, 1987b; Xia, Fertel and Wood, 1997). In contrast to its inhibitory action on the slow excitatory actions of histamine and on histamine stimulation of adenylate cyclase, adenosine does not inhibit the slow excitatory actions of 5-hydroxytryptamine nor the stimulation of adenylate cyclase by 5-hydroxytryptamine (Palmer, Wood and Zafirov, 1987b; Xia, Fertel and Wood, 1997). This may be a reflection of subtlety of neuromodulatory events that reconfigure the neural networks for changes in program output. Presynaptic inhibition by adenosine at fast nicotinic synapses in the enteric microcircuits occurs at the A1 type of P1 purinoreceptor (Barajas-Lopez, Surprenant and North, 1991; Christofi, Tack and Wood, 1992; Christofi and Wood, 1994). This occurs also at noradrenergic inhibitory synapses on neurons in the submucous plexus.

CYTOKINES Investigation of actions of cytokines on the nervous elements of the enteric neural networks is preliminary. The cytokines investigated are TNFα, IL-1β and IL-6. These are of potential importance because they may be released in paracrine fashion from a variety of immune and inflammatory cells as well as mast cells. Actions of the three cytokines are essentially the same. Like histamine and 5-hydroxytryptamine, they produce excitatory responses that mimic slow synaptic excitation (Xia et al., 1995, 1999). The natural IL-1 receptor antagonist (nIL-1ra) prevents the excitatory action of IL-1β. All three cytokines also suppress the release of noradrenaline from

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sympathetic nerve terminals in the enteric nervous system and thereby block inhibitory neurotransmission to secretomotor neurons in the submucous plexus. This can be observed as suppression of noradrenergic inhibitory postsynaptic potentials in microelectrode recording from the neurons (Xia et al., 1995; 1999). Ruhl, Hurst and Collins (1994) reported that IL-1β suppressed the release of noradrenaline from intestinal preparations in vitro and that IL-6 acted synergistically with IL-1β in the suppression of noradrenaline release. Microelectrode recording from submucous neurons with Dogiel I morphology (presumably secretomotor neurons) revealed that application of either IL-1β or IL-6 dose-dependently suppressed the amplitudes of stimulusevoked noradrenergic inhibitory postsynaptic potentials (Xia et al., 1999). Application of IL-1β and IL-6 combined produced greater suppression of the inhibitory synaptic potentials than predicted from the additive effects of the two cytokines applied separately. Neither of the cytokines applied separately or in combination suppressed the amplitude of inhibitory responses to micropressure pulses of noradrenaline. Absence of reduction in responses to noradrenaline while noradrenergic neurotransmission was suppressed meets criteria for a presynaptic inhibitory action of the cytokines on release of noradrenaline. The results with the cytokines, like those with histamine, suggest that accumulation of these mediators, derived either from degranulation of mast cells or other cell types associated with inflammatory states, will lead to suppression of the physiological effects of activation of the sympathetic innervation to the bowel. The physiological actions of sympathetic activation are presynaptic inhibition at nicotinic synapses resulting in shut-down of enteric microcircuits and inhibition of excitatory secretomotor neurons to the intestinal crypts. This predicts neural network behaviour that reflects removal of the braking action of the sympathetic nervous input to the intestine resulting in hyper-activity of the circuits with further enhancement produced by the excitatory action of the cytokines on the neurons in the network. Inhibition of the sympathetic brake on secretomotor neurons in parallel with neuronal stimulation would result in enhanced secretion from the crypts and development of a potential for secretory diarrhoea. PLATELET ACTIVATING FACTOR, LEUKOTRIENES AND PROSTAGLANDIN D2 Similarities and differences are found for the actions of lipid membrane derived mediators. Platelet activating factor (PAF) has excitatory actions on enteric neurons that are essentially the same as for the other above mentioned immuno-physiological mediators that mimic slow synaptic excitation (Xia et al., 1996). It acts in concentration-dependent manner on the membranes of the neuronal cell bodies to depolarise the membrane potential, increase input resistance, increase action potential discharge during intraneuronal injection of depolarising current and to induce spontaneous spike discharge. Like the other mediators, it acts presynaptically to inhibit release of noradrenaline from sympathetic nerves in the submucous plexus and thereby remove the sympathetic brake from the secretomotor innervation of the intestinal crypts. Leukotrienes B4, E4, D4 and C4 (LTB4, LTE4, LTD4 and LTC4) also have excitatory actions that are essentially the same as described for other putative mediators in immunoneural communication (Frieling et al., 1995). Like histamine, but unlike the other mediators


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described above, LTE4, LTD4 and LTC4 evoke recurrent periodic discharge of trains of action potentials in submucous neurons. Prostaglandin D2 (PGD2) has excitatory actions on enteric neurons that are essentially the same as described above for PAF (Frieling et al., 1994b). Unlike the actions of histamine and 5-hydroxytryptamine, PGD2 does not suppress fast nicotinic neurotransmission in the submucous microcircuits, but does inhibit some forms of slow synaptic excitation. NITRIC OXIDE Although nitric oxide has important consequences in inflammatory responses and is formed and released by intestinal mast cells, it appears to have minimal effects on the neuronal elements of the enteric microcircuits. Release of nitric oxide from sodium nitroprusside does not affect resting membrane potentials in myenteric neurons of the guinea-pig intestine, except for an occasional decrease in input resistance attributable to suppression of excitatory neurotransmitter release (Tamura, Schemann and Wood, 1993). Release of nitric oxide does not alter fast nicotinic neurotransmission, but does suppress noncholinergic slow excitatory postsynaptic potentials. It does not affect the slow depolarising responses to 5-hydroxytryptamine or substance P, suggestive of a presynaptic site of action for suppression of stimulus-evoked slow synaptic excitation. The nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester, does not affect resting membrane excitability or excitatory synaptic events in myenteric neurons of the guinea-pig.

NEURAL NETWORKS AND EFFECTOR SYSTEMS BEHAVIOUR The mucosa, musculature and blood vasculature are the primary effector systems involved in overt intestinal responses to mast cell degranulation. Mast cell degranulation leads to stimulated secretion from the intestinal crypts in association with increased mucosal blood flow. Degranulation of mast cells is associated also with a specialised form of motility that was referred to as power propulsion earlier in the chapter. The migrating spike bursts recorded with serosal electrodes by Mathias et al. (1976) in response to enterotoxin and by Schanbacher et al. (1978) in response to infection by T. spiralis are reflections of the muscle behaviour during power propulsion. The giant migrating contractions recorded with serosal strain gauges in vivo in antigen-sensitised and other animal models also reflect the occurrence of power propulsion (Cowles and Sarna, 1990, 1991; Sarna et al., 1991). RESPONSES TO HISTAMINE AND LEUKOTRIENES In preparations of guinea-pig colonic mucosa in vitro, application of histamine evokes transient increases in secretion resulting from the direct action of histamine with histamine H1 receptors on the enterocytes and evoked release of prostaglandins and other unidentified mediators (Wang and Cooke, 1990). After the transient response to histamine subsides, cyclical bursts of electrolyte secretion appear and are seen as changes in short-circuit current

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Figure 9.6 Histamine evokes periodic cycles of secretion in the mucosa of the guinea-pig large intestine. The record shows short-circuit current indicative of electrogenic chloride secretion as investigated in an Ussing flux chamber during continuous exposure to histamine. (Modified with permission from Wood, 1991).

in Ussing chamber studies. They persist unchanged for periods of hours (Figure 9.6; Wang and Cooke, 1990). Application of the nerve-blocking agent tetrodotoxin abolishes the secretory cycles, indicating that they are neurally driven. The secretory cycles are prevented by drugs that block the histamine H2 receptor subtype. Selective histamine H2 receptor agonists reproduce the cyclical behaviour induced by histamine, whereas histamine H1 receptor agonists do not. Exposure to atropine or mecamylamine suppresses the recurrent secretory cycles. The action of atropine, taken together with what is known about cholinergic stimulation in mucosal preparations, suggests that the cycles are driven mainly by the release of acetylcholine from secretomotor neurons at neuroepithelial junctions. The blocking action of mecamylamine suggests that nicotinic synapses are active in the microcircuits responsible for generating the patterned output to the mucosa. Extended exposure to LTE4, LTD4 or LTC4 was also reported to evoke recurrent cycles of electrolyte secretion in the guinea-pig colon reminiscent of the cyclical behaviour induced by histamine (Frieling et al., 1995). The response to the leukotrienes, like that of histamine, was blocked by tetrodotoxin, atropine or a nicotinic blocker. Cooke, Wang and Rogers (1993) recorded short-circuit current and muscle contraction simultaneously during exposure to histamine in Ussing chamber studies. They found that phasic contractions recorded by strain gauges oriented along the circular muscle axis of the flat sheet preparations were synchronised with the secretory cycles. A contraction appeared to be coupled to each secretory cycle with each contraction lagging behind the secretory event. Coupling of the secretory cycles with phasic contractions required neural connections between the myenteric and submucous plexuses. Severing the connections prevented the contractile responses without affecting the secretory cycles. NEURAL PATTERN GENERATION The cyclic trains of action potentials described earlier in the chapter for submucous neurons during exposure to histamine are apparently the neurophysiological correlates of the cyclic secretory and contractile behaviour evoked by histamine in full-thickness preparations of intestine in Ussing chamber studies. Results of the neurophysiological and integrated system studies suggest that the cyclic behaviour is driven by a neural pattern generator that switches secretomotor neurons between active and inactive states to drive


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periodic bursts of secretion of water and electrolytes. Activity of the pattern generator is part of the specialised program stored in the program library of the enteric nervous system. An overlay of histamine on the neural network commands the program into action. The program includes mechanisms for activating the population of secretomotor neurons to the intestinal crypts and motor neurons to the musculature. It also incorporates timing functions that coordinate secretion with motility. The power propulsion pattern of muscular behaviour and associated hypersecretion triggered by noxious stimulation of the mucosa in vivo are postulated to be the integrated output of the immuno-neural “alarm” program. Patterned neural discharge and cyclic secretory behaviour and coupled contractions seen in neurophysiological and Ussing chamber studies in vitro represent fragmented operations of the program. The integrated output of the neural network in response to histamine can be attributed to two actions on single elements of the circuits. Excitation of the neuronal cell body by histamine H2 receptors and presynaptic inhibition mediated by histamine H3 receptors at nicotinic synapses in the circuits are the only definable actions at the single neuron level of organisation. The patterned output emerges from integration at higher order levels of circuit organisation. Like integrative neural networks in general, understanding of the integrated circuit level of organisation in the enteric nervous system is less advanced than understanding of actions at the level of the individual elements of the network. Two aspects of enteric integrated circuit function are especially interesting, but not well understood mechanistically. One is the emergent mechanism that accounts for the programming of the central pattern generator when exposed to histamine. The second is the mechanism by which a population of submucosal secretomotor neurons is recruited to fire simultaneously in supplying synchronous excitatory drive to the secretory epithelium. Slow synaptic excitation is thought to underlie the latter phenomenon, whereas there are few clues to the mechanism underlying pattern generation. SLOW SYNAPTIC EXCITATION Slow synaptic excitation is postulated to be the basic function in a mechanism for gating spread of excitation among the neurons in an intestinal microcircuit. Slow synaptic gating and feed forward excitation have been proposed as a synaptic mechanism in circuits that recruit synchronous discharge in neuronal pools consisting of hundreds of enteric neurons. This is necessary in neuronal populations responsible for generation of the same motor event simultaneously in the musculature around the circumference and for extended distances along the longitudinal axis of an intestinal segment. A similar mechanism can explain synchronous recruitment of secretomotor neurons to the intestinal epithelium to generate the cyclical secretory behaviour resulting from histamine action and immuno-neural communication in the sensitised intestine described later in the chapter. Slow synaptic excitation and slow paracrine excitation underlie a mechanism for longlasting activation or inhibition of gastrointestinal effector systems. The prolonged discharge of spikes during the slow excitatory event drives the release of neurotransmitter from the axon for the duration of the event measured in seconds or minutes (Figure 9.3). It results in prolonged inhibition or excitation at neuronal synapses and neuroeffector junctions.

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Contractile responses of the muscles and secretory responses of the epithelium are sluggish events that last for several seconds from start to completion. The train-like discharge of spikes during slow EPSPs is the candidate correlate of long-lasting responses of the effector systems in the functioning gastrointestinal tract. COORDINATED RECRUITMENT Slow excitatory events in cell somas of AH/Type 2 neurons emerge as an explanation for coordinated recruitment functions at the microcircuit level of organisation. Coordinated recruitment refers to the simultaneous conversion of a pool of several hundred AH/Type 2 neurons from the hypo- to hyper-excitable state with repetitive spike discharge being initiated in the cell somas of each of the individual members of the population. Coordinated firing within the AH/Type 2 interneuronal population ultimately drives the behaviour of motor neurons to the effector systems (Figure 9.7). Importance of coordinated recruitment is the requirement that populations of motor neurons be stimulated to activity in synchrony in order to evoke simultaneous responses of the musculature or secretory epithelium in a segment of digestive tract with several square centimetres of surface area. This is believed to be a function of “driver circuits” comprised of interconnected AH/Type 2 neurons (Wood, 1995a; Figure 9.7). Driver circuits synchronise motor and secretory events around the circumference of a defined length of bowel.

Figure 9.7 Driver circuits are formed by populations of AH/Type 2 neurons that are synaptically connected for feed forward synaptic excitation. Synchronous discharge in the driver circuit is the mechanism responsible for simultaneous activation of populations of motor neurons to gastrointestinal effectors. The cell somas of AH/ Dogiel II neurons in driver circuits behave like a somal gate that is closed when the soma is in its hypoexcitable state and opened when the somal membrane is made hyperexcitable by slow synaptic input. Open gates permit feed forward (positive feedback) excitation in the circuit; closed gates inactivate the feed forward function of the circuit.


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These circuits are thought to provide simultaneous synaptic drive to subpopulations of inhibitory or excitatory neurons to the muscle layers during operation of propulsive motor programs. On-off behaviour of the driver circuits is the most plausible function to account for the on-off cyclical behaviour of neurally driven electrolyte and water secretion described earlier in the chapter. The off-state of the driver circuit corresponds to the low excitability condition of the AH/Type 2 neurons that make-up the circuit; whereas, the on-state is the hyperexcitable condition of slow synaptic excitation or paracrine actions of histamine or other neuromodulators. Sixteen or more neuro/paracrine mediators have receptors on enteric neurons that mimic slow synaptic excitation when activated and an equivalent number of neuro/paracrine modulators inhibit slow synaptic excitation (reviewed by Wood, 1994). The AH/Type 2 neurons in the driver networks are assumed to be the multipolar Dogiel Type II neurons that have extensive ramifications of axonal projections in the circumferential axis of the intestine (Bornstein et al., 1991). They are thought to be interneurons that are synaptically interconnected for feed forward excitation. This is a kind of synaptic connectivity in which the neurons of the circuit make recurrent slow excitatory synaptic connections one with another (Kunze, Furness and Bornstein, 1993). It is a positive feedback system that leads to rapid amplification of excitation in the population of driver interneurons. It underlies a mechanism that ensures simultaneous activation of the entire network around the circumference of the segment of bowel (Thomas, Bertrand and Bornstein, 1999). Simultaneous activation is obviously important for effective application of the forces necessary for propulsion of the luminal contents and coordination of mucosal secretory events with behaviour of the musculature. GATING FUNCTIONS Slow excitatory events in cell somas of AH/Type 2 neurons emerge also as an explanation for a form of gating function at the microcircuit level of organisation. Control of feed forward excitation and coordinated recruitment of neuronal activity develops from a gating function of the cell body of AH/Type 2 neurons. Gating in this case refers to the control of propagation of action potentials between the neurites arising from opposite poles of the multipolar cell somas of AH/Type Dogiel Type II neurons. Slow excitatory events are an integral part of the gating mechanism. Figure 9.7 illustrates how the gating mechanism is thought to work in this kind of neuron. The membrane of the cell body of AH/Type 2 neurons can exist in an inexcitable state, hyper-excitable state or intermediate state of excitability. Inexcitability reflects the absence of slow excitatory mediators, hyper-excitability reflects exposure to elevated levels of one or more mediator and intermediate states reflect intermediate levels of one or the other of the sixteen or so putative excitatory mediators. When the cell body is in the inexcitable state, action potentials propagating toward the cell body in one of its neurites cannot fire the membrane of the cell soma and the gate is closed. In intermediate states of excitability where the somal membrane happens to be fired by an inbound spike, the action potential in the cell body will be followed by the characteristic after-hyperpolarisation and the prolonged refractory period will prevent firing of the somal membrane by any additional incoming spikes. In this state, the partially open somal gate fractionates the transfer of information

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across the cell body. In the hyper-excitable state, the probability that the cell body will be fired repetitively by inbound spikes is dramatically increased. This occurs because excitability of the membrane is enhanced, membrane resistance and space constant are increased, and after-spike hyperpolarising potentials are suppressed. The gate is wide-open in this state and each arriving action potential in any given neurite is conducted across the cell body to activate spike discharge in all neurites around the periphery of the multipolar neuron. When this occurs, output of the discharge in the cell body is distributed to multiple synaptic sites in the circuitry by the extensive ramifications of the Dogiel Type II neurites. PRESYNAPTIC INHIBITION A significant aspect of the importance of presynaptic inhibition emerged from work on electrolyte secretion in Ussing chamber studies. The amplitude of recurrent cycles of electrolyte secretion, seen in Ussing chamber studies on guinea-pig large intestinal mucosa, was found to be smaller when the cycles were evoked by histamine than when the selective histamine H2 receptor agonist, dimaprit, was applied to evoke the cycles (Wang and Cooke, 1990). This suggests that activation of a histamine receptor different from the histamine H2 receptor subtype is involved in suppression of the neurogenic secretory activity. Phamacological and electrophysiological evaluation indicates that the receptor is the histamine H3 receptor located at presynaptic terminals or motor nerve terminals at neuroeffector junctions. Use of the drug burimamide, which was mentioned earlier in the chapter as acting as an antagonist at presynaptic histamine receptors at nicotinic synapses, helps to clarify why dimaprit evokes larger amplitude secretory cycles than histamine. The amplitude of the histamine cycles grows to the size of the cycles evoked by dimaprit when burimamide is applied in the presence of histamine. Burimamide does not change dimaprit-evoked cycles. On the other hand, application of the selective histamine H3 receptor agonist, sodium methylhistamine, reduces the amplitude of the cycles evoked by dimaprit. These observations together with neurophysiological evidence for presynaptic inhibitory histamine H3 receptors (Tamura, Palmer and Wood, 1987) point to activation of the presynaptic inhibitory receptors as the factor responsible for the blunted amplitude of the recurrent secretory cycles when evoked by histamine. The cycles are larger when evoked by dimaprit because this selective histamine H2 receptor agonist spares the presynaptic inhibitory receptors while stimulating the slow excitatory receptors on the cell bodies of the AH/ Type 2 neurons in the network. A presynaptic braking action appears to control excitability in the circuitry and thereby, the strength of outflow to the effector system. The positive feedback configuration of driver circuits, as described above, would be expected to produce runaway amplification if left unchecked after activation by histamine H2 receptors on the cell bodies. Simultaneous activation of histamine H3 receptors leading to suppression of the strength of transmission at synapses in the network applies a brake on excitability in the whole circuit and in this way maintains a check on the strength of outflow from the circuit to the effector system. The presence of two selective receptor subtypes for histamine at two key locations in the circuit seems to be basic for neuromodulatory


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reconfiguration of the circuit into a different output mode when the circuit is exposed to a paracrine overlay of histamine.

IMMUNO-NEUROPHYSIOLOGY IN THE SENSITISED INTESTINE NEURAL BEHAVIOUR IN THE ANTIGEN-SENSITISED INTESTINE The effects of histamine and other mast cell mediators on neurophysiological functions predict similar effects when mast cells are degranulated by sensitising antigens. Guinea-pig models have proved useful for investigation of the neurophysiology of antigen-sensitised bowel. The general protocol is to induce gastrointestinal immunity and consequently hyper-sensitivity to milk protein or the intestinal parasite T. spiralis (Frieling, Cooke and Wood, 1994; Frieling et al., 1994a). Milk hypersensitivity is produced in guinea-pigs by ingestion of cow’s milk in place of drinking water for a 3-week period. Hypersensitivity to T. spiralis is produced by oral inoculation of guinea-pigs with live infective-stage larvae. After 3 weeks of milk ingestion, or 6–8 weeks after T. spiralis infection, submucous or myenteric plexus preparations are set up for intracellular recording from the neurons. Electrophysiological and synaptic behaviour of the sensitised tissue and non-sensitised controls is then compared during application of β-lactoglobulin for the milk sensitised animals or an extract of T. spiralis larvae for the nematode infection. Submucous neurons in guinea-pig colon sensitised to either milk protein or T. spiralis antigens are found to have a higher incidence of spontaneously occurring action potentials and fast nicotinic excitatory post-synaptic potentials than neurons from non-sensitised animals (Frieling, Cooke and Wood, 1994; Frieling et al., 1994a). This activity is reminiscent of the spontaneous spike discharge evoked by application of histamine in submucous plexus preparations. Application of antigens from T. spiralis larvae or β-lactoglobulin produce no changes in neuronal behaviour in preparations from nonsensitised guinea-pigs. In neurons from sensitised animals, the same application of antigen evokes electrical changes similar to slow excitatory synaptic transmission mentioned earlier in the chapter. The changes consist of slowly activating membrane depolarisation, increased input resistance, suppression of postspike hyperpolarising potentials and enhanced excitability reflected by increased frequency of repetitive spike discharge during injection of depolarising current (Figure 9.8). Application of a selective histamine H2 receptor antagonist prevents or reverses the excitatory action of antigen exposure in the sensitised tissues (Figure 9.8). This is reminiscent of the action of histamine and reversal of its action by histamine H2 antagonists. The effects of antigen exposure on fast excitatory neurotransmission at nicotinic synapses in the sensitised preparations are also reminiscent of the effects of histamine on fast synaptic behaviour. Introduction of T. spiralis antigen to the parasite-sensitised submucous plexus preparations, or β-lactoglobulin to the milk-sensitised preparation, results in suppression of fast nicotinic transmission in both (Figure 9.8). Like the presynaptic inhibitory action of histamine on fast nicotinic transmission, the inhibitory action

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Figure 9.8 Effects of exposure to β-lactoglobulin or Trichinella spiralis antigen in submucous neurons from the colon of guinea-pigs previously infected with T. spiralis or cow’s milk. (A) Excitation of an AH/Type 2 submucous neuron in a milk-sensitised preparation. Each action potential evoked by injection of depolarising current pulses was followed by after-hyperpolarisation (AH) characteristic of AH/Type 2 neurons. Application of β-lactoglobulin resulted in suppression of the AH and enhanced excitability reflected by spike discharge evoked by each depolarising current pulse. (B) Enhanced neuronal excitability evoked by application of T. spiralis was reversed by application of histamine antagonists. Intrasomal injection of depolarising current pulse (lower traces) evoked 2 spikes (upper traces) at the onset of the pulse, in the control, prior to application of the antigen in the superfusion solution. In the presence of T. spiralis somatic antigen, enhanced excitability in the same neuron is seen as repetitive spike discharge at increased frequency throughout the depolarising current pulse. The excitatory action of the antigen was suppressed by cimetidine with the antigen still present.

of antigen exposure can be reversed by treatment with the histamine H3 antagonist burimamide (Figure 9.9).

EFFECTOR BEHAVIOUR IN THE ANTIGEN-SENSITISED INTESTINE Effector behaviour in the antigen-sensitised intestine generally fits with predictions derived from the actions of histamine on neural elements of the enteric nervous system. Mastocytosis occurs in the T. spiralis sensitised guinea-pig model and levels of histamine in the intestinal wall are elevated (Russell and Castro, 1989). Basal secretion from the mucosa is enhanced and secretory responses to substance P, carbachol and electrical field stimulation are facilitated in Ussing chamber studies (Wang, Palmer and Cooke, 1991).


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Figure 9.9 Suppression of fast excitatory postsynaptic potentials (EPSPs) occurs during exposure to β-lactoglobulin in a neuron from a milk-sensitised guinea-pig. The histamine H3 receptor antagonist, burimamide, reversed suppression of the EPSP. In the control prior exposure to β-lactoglobulin, focal electrical stimulation of an interganglionic connective evoked a fast EPSP. Exposure to β-lactoglobulin suppressed the fast EPSP. Application of burimamide with the antigen present reversed the antigen effect. The amplitude of the EPSP returned to near pre-challenge level after washout of the antigen and burimamide.

Introduction of the sensitising antigen evokes recurrent cyclical secretory behaviour. Each of these forms of behaviour in the sensitised intestine is suppressed by histamine H2 antagonists. Exposure of isolated segments of intestine from sensitised animals to T. spiralis antigen evokes powerful propulsive motility (Alizadeh, Castro and Weems, 1987; Alizadeh, Weems and Castro, 1989). Histamine application mimics the propulsive motor responses and neural blockade by tetrodotoxin eliminates both the response to antigen and histamine. The highly propulsive motility evoked by antigen in the sensitised intestine in vitro is reproduced in vivo in a dog model. Instillation of T. spiralis into the large intestine of conscious dogs, after an infection with the parasite, triggered motor behaviour characteristic of power propulsion (“giant migrating contractions”) associated with watery diarrhoea (Cowles and Sarna, 1990, 1991; Sethi and Sarna, 1991).

CONCLUSIONS The immunoneurophysiological evidence leads to the conclusion that the minute-tominute behaviour of the bowel, whether it be normal or pathologic, is determined primarily

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by integrative functions of the enteric nervous system. Information input processed by the brain-in-the-gut is derived from local sensory receptors, the central nervous system and a compliment of immunological cells including mast cells. Intestinal mucosal mast cells utilise the capacity of the immune system for detection of new antigens and long-term memory, for recognition of the antigen if it ever reappears in the gut lumen. Should the antigen reappear, the mast cells signal its presence to the enteric nervous system. The enteric nervous system interprets the mast cell signal as a threat and calls up from its program library secretory and propulsive motor behaviour organised for rapid and effective eradication of the threat. Operation of the “alarm” program protects the integrity of the bowel, but at the expense of side effects that include abdominal distress and diarrhoea. The signalling function of intestinal mast cells is analogous to that of sensory neurons. Sensory neurons detect changes in stimulus energy and transfer the information to integrated neural circuits for processing. Mast cells, like sensory neurons are detectors. However, unlike sensory neurons that are endowed with mechanisms for detection and transduction of a single form of stimulus, mast cells utilise immunological memory functions to detect foreign antigens as they appear throughout the life of the individual. Unlike sensory neurons, mast cells use paracrine signalling mechanisms for direct transfer of chemical information to the integrative neural circuits of the brain-in-the-gut. The integrative circuits can receive and interpret the chemical signals from the mast cells. The enteric nervous system responds to the signals as if they were a labelled code for the presence of a threat in the intestinal lumen. The histoanatomical proximity of intestinal mast cells and enteric neural elements and an expanding collection of immunophysiological observations described in this chapter indicate that mast cells in the mucosa and elsewhere in the intestine are strategically positioned to establish a first line of defence against foreign intrusion at a vulnerable interface between the body and the outside world. Evidence in this chapter implicates histamine as one of the significant paracrine signals from mast cells to the enteric nervous system. When the enteric microcircuits detect histamine, the message is processed as a labelled signal indicating mast cell degranulation, which in turn is a specific label for the presence of a sensitising antigen. Microelectrode recording from neurons in the sensitised bowel has clarified the neurophysiological events associated with antigen detection at the interface for information transfer between mucosal mast cells and enteric neurons. This approach confirmed specificity for detection and signalling of the sensitising antigen and added to the evidence for significant involvement of histamine in immuno-neural signalling. Work at the whole organ level reviewed in this chapter has established that an overlay of histamine in the enteric nervous system results in an organised pattern of coordinated behaviour of mucosal secretion and motility. The pattern of effector behaviour generated by the neural circuitry emerges from two actions of histamine at the level of the individual neural elements that make-up the circuits. One action is dramatically enhanced excitability of the membrane of the neuronal cell bodies mediated by histamine H2 receptors. The second is presynaptic inhibition mediated by histamine H3 at nicotinic synapses in the circuitry. How the actions of histamine at the cell bodies and nicotinic synapses are transformed by the integrated circuitry into the neurally programmed behaviour seen at the level of the whole organ is not yet fully understood.


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ACKNOWLEDGEMENTS The concepts of enteric neuro-immunophysiology reviewed in this chapter emerged, in part, from the collaborative work of my former colleague, Prof. Helen J. Cooke and several postdoctoral visitors to our laboratories. These include: Fedias Christofi, Thomas Frieling, Jeffrey M. Palmer, Kenji Tamura, Yu-Z. Wang, Yun Xia and Dimiter Zafirov. The work in my laboratory on neuro-immune signalling in the enteric nervous system was supported by National Institutes of Health Grants RO1 NS17363, RO1 AM26742, and RO1 DK37238.

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Thomas, E.A., Bertrand, P.P. and Bornstein, J.C. (1999). Genesis and role of coordinated firing in a feed forward network: A model study of the enteric nervous system. Neuroscience, 93, 1525–1537. Vanner, S. and Surprenant, A. (1996). Neural reflexes controlling the intestinal microcirculation. American Journal of Physiology, 271, G223–G230. Wade, P.R. and Wood, J.D. (1988). Actions of serotonin and substance P on myenteric neurons of guinea-pig distal colon. European Journal of Pharmacology, 148, 1–8. Wang, Y.Z. and Cooke, H.J. (1990). H2 receptors mediate cyclical chloride secretion in guinea pig distal colon. American Journal of Physiology, 258, G887–G893. Wang, Y., Palmer, J. and Cooke, H. (1991). Neuro-immune regulation of colonic secretion in guinea pigs. American Journal of Physiology, 260, G307–G314. Wang, L., Stanisz, A.M., Wershil, B.K., Galli, S.J. and Perdue, M. (1995). Substance P induces ion secretion in mouse small intestine through effects on enteric nerves and mast cells. American Journal of Physiology, 269, G85–G92. Williams, R.M., Berthoud, H.R. and Stead, R. (1995). Association between vagal afferent nerve fibers and mast cells in rat jejunal mucosa. Gastroenterology, 108, A941. Wood, J.D. (1989). Electrical and synaptic behavior of enteric neurons. In Handbook of Physiology, The Gastrointestinal System, Motility and Circulation, edited by J.D. Wood, pp. 465–517. Bethesda, MD: American Physiological Society. Wood, J. D. (1991). Communication between minibrain in gut and enteric immune system. News in the Physiological Sciences, 6, 64–69. Wood, J.D. (1994). Physiology of the enteric nervous system. In Physiology of the Gastrointestinal Track, 3rd edition, edited by L.R. Johnson, J. Christensen, M.J. Jackson, E.D. Jacobson and J.H. Walsh, pp. 423–482. New York: Raven Press. Wood, J.D. (1995a). Gastrointestinal neurophysiology. In Medical Physiology, edited by R.A. Rhoades and G.A. Tanner, pp. 487–504. Boston: Little, Brown Co. Wood, J. D. (1995b). Gastrointestinal motility. In Medical Physiology, edited by R.A. Rhoades and G.A. Tanner, pp. 505–529. Boston: Little, Brown Co. Wood, J.D. (1995c). Physiological and pathophysiological paracrine functions of intestinal mast cells in enteric neuro-immune signalling. In Gastrointestinal Tract and Endocrine System, edited by M.F. Singer, R. Ziegler and G. Rohr, pp. 254–263. Dordrecht: Kluwer Academic Publishers. Wood, J.D. and Mayer, C.J. (1979). Serotonergic activation of tonic-type enteric neurons in guinea-pig small bowel. Journal of Neurophysiology, 42, 582–593. Xia, Y., Fertel, R.H. and Wood, J.D. (1994). Stimulation of formation of cAMP by 5-hydroxytryptamine in myenteric ganglia isolated from guinea pig small intestine. Life Sciences, 55, 685–692. Xia, Y., Zafirov, D.H., Cooke, H.J. and Wood, J.D. (1995). Actions of tumor necrosis factor (TNF-α) on electrical and synaptic behavior in the submucous plexus of guinea-pig small intestine. Gastroenterology, 108, A945. Xia, Y., Fertel, R. and Wood, J. (1996). Stimulation of formation of adenosine 3’,5’-phosphate by histamine in myenteric ganglia isolated from guinea-pig small intestine. European Journal of Pharmacology, 316, 81–85. Xia, Y., Lu, G., Zafirov, D.H., Sarna, S.K. and Wood, J.D. (1996). Effects of platelet-activating factor on electrical and synaptic behavior of neurons in the submucous plexus of guinea-pig small intestine. Gastroenterology, 110, A782. Xia, Y., Fertel, R.H. and Wood, J.D. (1997). Suppression of cAMP formation by adenosine in myenteric ganglia of guinea-pig small intestine. European Journal of Pharmacology, 320, 95–101. Xia, Y., Hu, H.-Z., Liu, S., Ren, J., Zafirov, D. and Wood, J.D. (1999). IL-1β and IL-6 excite neurons and suppress nicotinic and noradrenergic neurotransmission in guinea-pig enteric nervous system. Journal of Clinical Investigation, 103, 1309–1316. Zafirov, D.H., Palmer, J.M. and Wood, J.D. (1985). Adenosine inhibits forskolin-induced excitation in myenteric neurons. European Journal of Pharmacology, 113, 143–144.

10 Cellular Organisation of the Mammalian Enteric Nervous System Simon J.H. Brookes and Marcello Costa Department of Human Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide, South Australia 5001 The enteric nervous system plays a central role in the control of gastrointestinal function. To understand how its circuits operate, it is necessary to identify and characterise the different functional classes of enteric neurones and determine their cellular properties and connectivity. The most powerful way to distinguish the different classes of neurones has been to study the combinations of neurochemicals (“chemical coding”) that they contain in their cell bodies and axons. Some of the neurochemicals in the chemical codes, such as neurotransmitters, may be functionally important in their own right. The role of other molecules, such as calcium binding proteins, are currently unclear, however they are still useful as markers to distinguish various classes of neurones. In combination with axonal tracing techniques, chemical coding has provided a quantitative account of all of the major classes of neurones in one preparation, the guinea-pig ileum. Extensive electrophysiological and morphological studies have been readily incorporated into this account. This has revealed an exquisite degree of organisation, comparable to many parts of the central nervous system. Each class of neurone has characteristic combinations of neurotransmitters, neuromodulators, synaptic inputs, projections and soma-dendritic morphology. The methodologies developed in the guinea-pig small intestine are increasingly being applied to other regions of gastrointestinal tract and to preparations from other species. While many characteristics appear to be shared by different preparations, substantial differences are beginning to emerge, even between neurones with apparently identical functions. It is clear that the guinea-pig ileum is not representative of the enteric nervous system in general, nor of the human enteric nervous system in particular. However, neither is any other animal model. Fortunately, with the techniques now available, it is possible to study directly specimens of human gut, reducing the reliance on animal models to answer fundamental questions of the cellular organisation of the human enteric nervous system. Changes in the chemical coding during development and in diseases will cast light onto both the mechanisms underlying plasticity of the enteric nervous system and the processes underlying physiopathological changes. KEY WORDS: enteric nervous system; afferent neurones; motor neurones; interneurones; neurotransmission; chemical coding.

INTRODUCTION REASONS FOR STUDYING THE ENTERIC NERVOUS SYSTEM The enteric nervous system plays an important role in the day to day survival of the organism. The control of motility is essential for the digestion and absorption of nutrients. 393

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The coordinated movements of the gut allow mixing and sufficient exposure of the contents to the absorptive surface of the wall while avoiding stasis, which would allow the gut flora to overwhelm the body’s defences. In addition, by optimising the rate of propulsion, together with mucosal secretory activity and blood flow, the enteric nervous system plays a profoundly important role in setting the Na+, K+ and water balance of the whole body. When these control mechanisms are compromised, for example by infection, severe diarrhoea can result. Many of the readers of this volume live in relatively wealthy countries, with reliable water supplies, good infrastructure and adequate provision of medical services. In this case, such diarrhoea is unpleasant and inconvenient, as are many of the other disorders involving the enteric nervous system. In less wealthy parts of the world, where water supplies are limited and sometimes contaminated and where medical services are unavailable or unaffordable, dehydration caused by diarrhoea is a major cause of mortality. Children and the elderly people are especially vulnerable. Even in western countries, chronic dysfunction of the enteric nervous system has an enormous impact on quality of life. In the most severe cases, such as pseudo-obstruction or Hirschsprung’s disease, radical surgical treatment is urgently required. The vital role played by the enteric nervous system in the functioning of the entire body, provides a powerful justification for basic research aimed at understanding how this circuitry operates in health and disease. For many of us working in this field, there is another rationale for our study. Understanding how spindly little nerve cells can interact to give rise to adaptive, coordinated behaviour holds a deep intellectual fascination. For neurobiologists of a faint-hearted disposition, who are intimidated by the complexity of the central nervous system, the enteric nervous system is a wonderful alternative. The gut is the only organ in mammals (with the debatable exception of the heart) where entire neuronal circuits including sensory neurones, interneurones and motor neurones can be isolated from the central nervous system and still show reflex responses of physiological significance. Thus it is possible to study the cellular basis of entire behaviours which merely involve tens of thousands of neurones, rather than the uncountable millions potentially influencing central pathways. Understanding enteric neuronal circuits still represents a considerable intellectual challenge, which hopefully will identify some of the principles of organisation which may be shared by other neuronal circuits including those in the brain. AIMS OF THIS REVIEW The enteric nervous system and its control of gastrointestinal function have been extensively studied using morphological, electrophysiological, biochemical and pharmacological approaches. Each approach provides different types of information about the cells of the ENS. It is clear that to understand the cellular basis of neuronal control, it is necessary to have an accurate characterisation of the types of nerve cells that make up the circuitry. An adequate description of a nerve cell must incorporate morphological, electrophysiological, biochemical and pharmacological aspects. Fortunately, a substantial number of “multi-disciplinary” studies of the ENS have been carried out, in which electrophysiological and pharmacological approaches have been combined. This combinatorial approach

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has been most extensively pursued in the guinea-pig small intestine, by a number of groups around the world. The aim of this review is to attempt to synthesise some of the more significant findings into an account of the different functional classes of nerve cells in this preparation. We will then discuss some of the similarities and differences between functional classes of cells in other regions of the guinea-pig gut and in the gastrointestinal tract of other species.

A BRIEF DESCRIPTION OF THE ENTERIC NERVOUS SYSTEM The enteric nervous system, classified as the third division of the autonomic nervous system (Langley, 1921), is made up of the enormous number of nerve cells which lie within the wall of the gut. These nerve cells have cell bodies located in two ganglionated plexuses. The myenteric plexus (Auerbach’s plexus) lies between the outer longitudinal smooth muscle layer and the circular smooth muscle (see Figure 10.1). The submucous plexus is closely associated with the thick layer of connective tissue lying between the

Figure 10.1 Dimensions of the enteric nervous system of the guinea-pig ileum. A tube of intestine, maximally distended has a diameter of approximately 6 mm. From this a 1 mm ring is shown at higher magnification, with the myenteric plexus schematically shown. At the right is a higher magnification of the myenteric plexus, with ganglia arranged in rows. At maximal stretch there are approximately 2–2.5 rows of ganglia per millimetre.

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circular muscle and the innermost mucosal tissue. In small animals such as the guinea-pig, the submucous plexus forms basically a single network, however in larger animals, including humans; it can be anatomically subdivided. There is an inner submucous plexus (Meissner’s plexus) and an outer submucous plexus (Schabadasch’s or Henle’s plexus) with an intermediate plexus, at least in humans (Hoyle and Burnstock, 1989). It has been estimated that the guinea-pig small intestine contains 2,750,000 myenteric neurones and 950,000 submucous neurones (Gabella, 1987). The mouse small intestine has a higher density of neurones, but lower overall numbers (about 400,000 myenteric and 330,000 submucous neurones) due to its shorter length, whereas the sheep, with its extended small intestine, was estimated to have over 30,000,000 myenteric and over 50,000,000 submucous neurones. It appears that, on average, the individual nerve cells have a greater size in the larger species (Gabella and Trigg, 1984). These estimates of numbers of enteric neurones may in fact be underestimates, since they are based on a histochemical method that does not stain every nerve cell body (Young et al., 1993a; Karaosmanoglu et al., 1996).

CLASSIFICATION OF ENTERIC NEURONES As in the rest of the nervous system, the millions of enteric nerve cells are not entirely different from one another; they appear to be organised into a relatively small number of different classes. Neurones of each class share a combination of characteristics, which distinguishes them from neurones in all other classes. These characteristics include where they project to (i.e. their targets), their soma-dendritic morphology, the combinations of transmitters and modulators that they contain (“chemical coding”), their electrophysiological properties, their synaptic inputs and their synaptic outputs. The expression of these features is under genetic control, played out as a cascade of interacting mechanisms during the development of the enteric nervous system (see chapter by Michael Gershon in this volume). It is likely that a relatively small number of key developmental events determine the phenotype of each and every enteric neurone. Thus, the ultimate way to distinguish classes of enteric nerve cells would be to identify the crucial decision points in their ontogeny. While enormous advances have been made in the last decade in understanding the colonisation of the gut by enteric nerve cell precursors (Gershon, 1997; Gershon, 1998; Taraviras and Pachnis, 1999), the factors which determine most of the differences between classes of cells have yet to be identified. For this reason, to identify the classes of enteric nerve cells, it is necessary to examine their characteristics in the mature ENS, at the single cell level, and determine how these vary systematically.

CHEMICAL CODING Since the development of indirect immunohistochemical staining methods (Coons, 1958), the localisation of neurochemicals has been one of the most successful means to distinguish different classes of nerve cells from one another. An enormous number of studies have now been carried out analysing the distribution of one substance at a time in nerve


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cells and other cells of the gastrointestinal tract. Immunohistochemical localisation is now a standard tool for determining where a particular protein or peptide is expressed. A recent search has shown that over 1000 studies have used immunohistochemical methods to localise substances in enteric nerve cells. Many of these studies have been carried out with the aim of characterising a substance and its distribution. They give invaluable information about the range of neurochemicals present in enteric ganglia. The ability of such data to distinguish between different functional classes of nerve cells however, is usually rather limited, since this has not been the primary aim of most of the studies. We will not attempt to summarise all of the data in these many studies. Rather we will attempt to review those studies in which systematic attempts have been made to distinguish between different types of enteric neurones. The finding that cells can contain more than one releasable, bioactive compound was an important discovery. First suggested in endocrine cells (Pearse, 1969), co-existence of neurochemical markers was demonstrated for enteric neurones in the early 1980s (Schultzberg, 1980). Subsequent use of immunohistochemical double-labelling, and later multiple-labelling, led to the concept of “chemical coding” which suggests that each class of neurone, with its characteristic projections, morphology and other features, can be distinguished by its particular combination of neurochemicals (Costa, Furness and Gibbins, 1986). This has turned out to be a very powerful way to distinguish different classes of neurones, which has been widely applied to enteric neurones and to other parts of the nervous system. In combination with selective lesions (“myectomies” and “myotomies”, as well as transmural crushes), chemical coding has been used to identify the gross projections of neurochemically defined classes of enteric neurones. It should be mentioned that this approach, however, has consistently under-estimated the lengths of projections of enteric neurones which can be measured more accurately and quantitatively using retrograde tracing techniques (Brookes, 2001). It has become clear that to distinguish different classes of enteric neurones from one another requires systematic and quantitative analysis of patterns of co-existence of multiple markers (see Figure 10.2). This has been most thoroughly achieved in the guinea-pig small intestine (Costa et al., 1996). For this reason, we will describe in detail the classes of nerve cells in the guinea-pig small intestine, integrating data on the chemical coding, projections, morphology, electrophysiological characteristics and connectivity of these neurones. We will then compare this data with the rather more sparse information about classes of enteric nerve cells in other parts of the guinea-pig gastrointestinal tract. Lastly, we will briefly discuss some of the major similarities and differences that have been identified in the gastrointestinal tract of other species that have been systematically studied with multiple labelling immunohistochemistry, lesions or retrograde tracing techniques. TERMINOLOGY Throughout this review the word “type” will be used to describe a subset of neurones which can be distinguished by the presence of a particular characteristic. For example, neurones which are immunoreactive for vasoactive intestinal polypeptide (VIP) are defined as a VIP-immunoreactive “type”. It is likely that types can be further subdivided

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A

B

C

D

Figure 10.2 Distinguishing nerve cell bodies in myenteric ganglia by chemical coding. Double labelling for the calcium binding proteins calbindin and calretinin (A) is shown combined in a single micrograph. Although colour information is lost, calretinin cells appear paler. The outlines of the cells are shown in (B). Note the smooth outlines of calbindin-immunoreactive Dogiel type II cells (oblique crosshatching) and the more irregular, Dogiel type I outlines of the calretinin immunoreactive cells (vertical hatching). In (C) a preparation was triple labelled for NOS, calretinin and NFP triplet. In (D) the chemical coding of the labelled cell bodies is shown as horizontal hatching (Neurofilament protein triplet), vertical hatching (calretinin) and as grey tint (NOS). Note that NOS and calretinin do not coexist, but NOS and NFP are found in many nerve cell bodies.

on the basis of other characteristics. The term “class” will be applied to groups of neurones which share a number of characteristics and which cannot be subdivided on the basis of current knowledge. A “functional class” is one in which a role can be deduced from the projections of the cells, their pattern of activation and their transmitter content. It should be noted that within any class, there may be variability in some characteristics. It is important to avoid subdividing classes unnecessarily, since this undermines the aim of classification, which is to reduce number of cells to manageable limits. A useful criterion for identifying classes is that the cells in each class differ from those of other classes by at least two co-varying characteristics. This largely overcomes the problem of mistaking variability within a single characteristic (e.g. intensity of immunoreactivity or size of the cell soma) for differences between functional classes of cells. For example, some Dogiel type II cells (which are characterised by multiple long processes arising from a smooth cell body) are immunoreactive for calbindin, while others are not (Iyer et al., 1988; Brookes et al., 1995). In this review they will be grouped in the same class because, according to current knowledge, they do not differ consistently on any other characteristic such as projection, morphology or neurochemical coding. In contrast, some Dogiel type II cells have a “dendritic” appearance with numerous short filamentous dendrites (Stach, 1989; Bornstein et al., 1991b; Brookes et al., 1995). Many of these “dendritic” cells have long descending projections within the myenteric plexus which “non-dendritic” Dogiel type II cells lack (Brookes et al., 1995). They also make rather fewer varicose endings in their ganglion of origin, compared to other Dogiel type II cells (Bornstein et al., 1991b). This suggests that “dendritic” Dogiel type II cells may comprise a separate class, although their function has not yet been unequivocally identified.


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PRIMARY AFFERENT NEURONES IDENTIFICATION OF ENTERIC PRIMARY AFFERENT NEURONES Early studies on extrinsically denervated intestine by Bayliss and Starling indicated that the enteric nervous system was capable of mediating entire reflexes, including peristalsis, in the absence of connections with the central nervous system (Bayliss and Starling, 1899). In their landmark paper, they wrote: “Local stimulation of the gut produces excitation above and inhibition below the excited spot. These effects are dependent on the activity of the local nervous mechanism.” Their observations were supported by later studies on isolated preparations of intestine, which confirmed that the enteric nervous system was entirely capable of producing integrated, adaptive responses to physiological stimuli (Langley and Magnus, 1905; Trendelenburg, 1917). An inescapable conclusion of those studies was that there must be primary afferent neurones within the enteric nervous system, which are capable of transducing physiological stimuli into neuronal activity, in order to activate enteric neuronal circuitry. In the last decade, this view has been challenged (Wood, 1994a). It has been reported that motor reflexes evoked by distension in the isolated rat colon were abolished by prior extrinsic denervation, suggesting that intramural collaterals of extrinsic afferent nerve fibres (either spinal or vagal in origin) were required for the reflex activity (Grider and Jin, 1994). It is clear that spinal afferent neurones have extensive collateral branching within enteric ganglia (Gibbins et al., 1985) and the same applies to vagal afferent nerve fibres (Berthoud and Powley, 1992). However, extrinsic denervation of the guinea-pig ileum has been demonstrated to have little effect on distension-activated motor reflexes (Furness et al., 1995). In this study, the effectiveness of extrinsic denervation was tested by confirming the disappearance of immunoreactivity for tyrosine hydroxylase, a marker of extrinsic sympathetic nerve fibres. In our laboratory, we have demonstrated that slow circumferential stretch of isolated segments of guinea-pig ileum evokes, at a sharp threshold, an abrupt contraction of the circular muscle corresponding to the initiation of peristalsis (Brookes et al., 1999). This motor activity, which is somewhat more complex in nature than the simple ascending reflex, is preserved after 4 days in organ culture, when extrinsic nerve fibres have degenerated (unpublished observations). These studies strongly suggest that intrinsic sensory neurones are present, and trigger motor reflexes, in the absence of extrinsic afferent collaterals within the gut wall. For a considerable period, the identity of the primary afferent neurones in the enteric nervous system remained uncertain. It was speculated, on the basis of morphology, that the multipolar enteric neurones, known as Dogiel type II neurones, were likely to be sensory in function (Dogiel, 1899). Early intracellular recordings from enteric neurones revealed a type of cell with a long after-hyperpolarisation following their action potentials, which have come to be known as AH cells (Hirst, 1974) or AH type II cells (Wood, 1994a). These cells were reported to lack fast synaptic inputs to drive their firing, unlike the majority of cells recorded. It was speculated that they might be primary afferent neurones, with no need for a synaptic drive (Hirst, 1974). Later, it was shown that AH cells have Dogiel type II morphology (Bornstein et al., 1984a; Erde, Sherman and Gershon, 1985; Katayama, Lees and Pearson, 1986; Iyer et al., 1988; Hendriks, Bornstein

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and Furness, 1990; Brookes et al., 1995). Interestingly, a few years after their initial characterisation, AH cells were shown to receive excitatory synaptic inputs, although these had a much slower time course than the fast, nicotinic synaptic inputs recorded in other types of cells (Wood and Mayer, 1978).

FUNCTIONAL IDENTIFICATION OF ENTERIC PRIMARY AFFERENT NEURONES Mucosal stimuli Identification of the enteric primary afferent neurones required functional studies. The first of these used expression of the immediate early gene product, Fos, to identify nerve cell bodies in the submucous ganglia of the guinea-pig small intestine which had been activated by cholera toxin or mechanical stimulation (using bubbles of nitrogen, blown against the villi) (Kirchgessner, Tamir and Gershon, 1992). Hexamethonium, a blocker of nicotinic neurotransmission, was used to prevent indirect activation of pathways, which might otherwise have confounded the results. One particular class of cells was identified in the submucous ganglia that had developed Fos expression in responses to the stimuli. These cells were immunoreactive for the calcium binding protein, calbindin, and/or the neuropeptide, substance P. Later studies have shown that these neurones comprise approximately 13% of all submucous neurones in the guinea-pig small intestine (Song et al., 1992) and that they can be retrogradely labelled from both the mucosa and from the myenteric plexus. Intracellular dye fills have shown that these cells have morphology similar to the Dogiel type II cells in the myenteric plexus, with several long axonal processes which appear to contact neurones in nearby ganglia (Bornstein et al., 1989; Evans, Jiang and Surprenant, 1994). In addition they have electrical properties similar to the AH cells of the myenteric plexus, with rare fast synaptic inputs, broad action potentials with a characteristic inflection on the falling phase and a long after-hyperpolarisation (Bornstein et al., 1989). Later it was shown that similar submucous primary afferent neurones were involved in entero-pancreatic reflexes evoked by stimulating the mucosa with either nitrogen bubbles or glucose (Kirchgessner, Liu and Gershon, 1996). Interestingly, these submucous primary afferent neurones appear to be activated indirectly by mechanical stimuli applied to the mucosa. Superfusion with the 5-HT1P receptor antagonist N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, blocked the activation of these neurones, suggesting that non-neuronal enterochromaffin cells in the mucosa may have activated the nerve terminals of the primary afferent neurones via the release of 5-hydroxytryptamine (5-HT) (Kirchgessner, Tamir and Gershon, 1992). A recent study has suggested that an additional class of submucous neurones may also function as primary afferent neurones. Using both expression of Fos and uptake of the styryl dye FM2–10, it has been suggested that a subset of calcitonin gene-related peptide (CGRP)immunoreactive neurones in the submucous plexus may also function as primary afferents, probably being activated by 5-HT (Kirchgessner, Tamir and Gershon, 1992) released from enterochromaffin cells in the mucosa (Pan and Gershon, 2000). This is interesting because CGRP has been reported in a separate class of unipolar submucous neurones which are also immunoreactive for neuropeptide Y (NPY), choline acetyltransferase


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(ChAT) and a number of other markers (see section “Submucosal neurones and other mucosally-projecting neurones”). Numerous myenteric neurones expressed Fos when the mucosa was stimulated by nitrogen bubbles or by choleragenoid, however this was largely blocked when hexamethonium was included in the bathing medium. It was suggested that myenteric neurones were indirectly activated by the stimuli and that there were no primary afferent neurones in the myenteric plexus which responded to these stimuli (Kirchgessner, Tamir and Gershon, 1992). Later it was shown that myenteric Dogiel type II neurones in the small intestine are quite resistant to the expression of Fos and only expressed low levels, even in response to prolonged electrical stimulation (Ritter, Costa and Brookes, 1997). This leaves open the possibility that myenteric Dogiel type II neurones might function as primary afferents to the mucosa. Earlier studies, using retrograde labelling in vitro with the carbocyanine dye, DiI, had shown that probably all Dogiel type II cells in the myenteric plexus have a projection to the mucosa, in the guinea-pig ileum (Song, Brookes and Costa, 1994a). Direct evidence supporting a role for Dogiel type II cells in transducing mucosal stimuli came from the elegant studies of Kunze and colleagues (Kunze, Bornstein and Furness, 1995; Bertrand et al., 1997) who made intracellular recordings from AH cells while applying substances to the mucosa. AH cells fired action potentials in response to solutions of high and low pH and to neutral solutions of short chain fatty acids. Many of the responses were not blocked in AH cells by bathing solutions containing a low concentration of calcium ions, indicating that the responses were not indirectly mediated by other cells synapsing onto the afferent terminal. In contrast, many S cells responded to mucosally applied chemicals with bursts of fast excitatory post synaptic potentials: these were blocked in low calcium solution, indicating that they reflected indirect activation, presumably via primary afferent neurones. Thus it can be concluded that some myenteric Dogiel type II neurones respond to chemical stimulation of the mucosa and then activate other enteric neurones.

Stretch stimuli It has been known since the nineteenth century that stretch is also a powerful stimulus for activating enteric neuronal circuitry. Bayliss and Starling demonstrated that distension of the dog small intestine triggers characteristic polarised reflexes in the smooth muscle layers (Bayliss and Starling, 1899). Later it was shown that distension of isolated intestine by fluid could trigger more complex propagating, peristaltic activity (Trendelenburg, 1917). It is likely that the transduction sites of the primary afferent neurones involved in these responses lie outside the mucosa. Removal of the mucosa and submucosa does not prevent the expression of stretch-induced polarised reflexes in the guinea-pig small intestine (Smith, Bornstein and Furness, 1990), although it does block reflex responses to mucosal distortion (Smith and Furness, 1988), nor does it prevent peristalsis in this preparation (Yokoyama et al., 1990; Tsuji et al., 1992). Identification of the stretch-sensitive myenteric neurones was hampered for a long time by movements of the preparation dislodging the microelectrode. This was solved by a modification of the floating microelectrode technique (Woodbury and Brady, 1956) which allowed impalements to be maintained while the level of stretch of the preparation was changed (Kunze, 1998).

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Using this approach, it was shown that a subset of Dogiel type II neurones fired spontaneously when the preparation was stretched to 140% of its resting length. The preparations consisted of longitudinal muscle and myenteric plexus, with most of the circular muscle removed. This is important because it indicates that there is a transduction site for mechanical stimulation located in the primary or secondary branches of the myenteric plexus. Hyperpolarising the nerve cell body during stretch affected the amplitude but not the frequency of action potentials, indicating that transduction did not occur in the cell body. This makes it highly likely that there are specific mechanically-sensitive sites located in the axons of these Dogiel type II cells. In a later study, it was shown that these neurones appear to be primarily sensitive to intramural tension, rather than length. Drugs which decreased smooth muscle activity also reduced stretch-induced firing in these primary afferents (Kunze et al., 1999) whereas Bay K8644, which opens L-type calcium channels, and hence activates smooth muscle contractility, increased firing. Circular muscle was largely absent from the preparations, but this did not alter their responses to circular stretch. This suggests that the neurones are probably responsive to any distortion of the ganglia in the region of their transduction sites. Further support for this comes from the observation that applying fine probes to the top of the ganglion activated stretch-sensitive Dogiel type II neurones even though force was applied vertically to the ganglion surface (Kunze et al., 2000). It is interesting to note that similar mechanisms seem to operate in the vagal mechanoreceptors to the upper gastrointestinal tract which also have transduction sites in myenteric ganglia (Zagorodnyuk and Brookes, 2000). Vagal mechanoreceptors have also been established to function as intramural tension receptors (Iggo, 1955). In a recent study, Kunze et al. (2000) succeeded in recording whole cell currents in Dogiel type II neurones in situ, using the methods developed by Gola and colleagues to enzymatically remove the surface of sympathetic ganglia (Gola et al., 1992). This confirmed that the transduction sites were located on the axons, since small depolarising generator potentials were evoked by mechanical stimulation of axons, whereas cell bodies responded with a hyperpolarisation, apparently mediated by stretch-sensitive BK channels (Kunze et al., 2000). ELECTROPHYSIOLOGICAL CHARACTERISTICS OF ENTERIC PRIMARY AFFERENT NEURONES There has been a lot of discussion over the relationship between electrophysiological and morphological properties of enteric neurones. It has become clear that many of the characteristics used to classify enteric nerve cells are actually shared by neurones belonging to several different functional classes. In addition, many of the characteristics show variability, both between cells, and within the same cell under different physiological conditions. For example, the long after-hyperpolarisation which follows action potentials in some neurones, was originally used to define AH cells (Hirst, 1974). In another influential paper, published a year earlier, enteric nerve cells were classified as type I and type II, on the basis of the number of action potentials evoked by depolarising current pulses (Nishi and North, 1973). Since these classifications largely overlap, the two schemes were quite reasonably combined and an electrophysiological type of AH/type II cells was identified (Wood, 1994). However, both the long after hyperpolarisation and the relative inexcitability


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of these cells are substantially modified by slow excitatory synaptic inputs (Wood and Mayer, 1978). In addition, both are affected by the level of activation of calcium dependent potassium currents (Hirst, Johnson and van Helden, 1985a,b). Since these currents may be activated by a leak conductance of calcium ions, caused by the intracellular microelectrode, the criteria of excitability, and amplitude of the long after-hyperpolarisation cannot be used by itself to identify the functional class to which an impaled neurone belongs. This uncertainty should be borne in mind in the following sections where the electrophysiological characteristics of enteric primary afferent neurones, identified as AH cells, are reviewed. However, it should also be noted that enteric primary afferent neurones are amongst the largest nerve cell bodies in enteric ganglia, making them easy to impale with microelectrodes. It is likely therefore that the great majority of studies on AH cells have probably been on enteric primary afferent neurones.

Currents in AH cells AH cells, have been demonstrated to have a substantial calcium component to their action potentials, leading to a tetrodotoxin-resistance of spikes evoked by depolarising current pulses (North, 1973). This calcium influx gives rise to a pronounced inflection on the falling phase of the action potential, and is associated with a longer duration, measured at half peak amplitude, of soma action potentials. The inflection has been proposed to be a useful criterion for identifying AH cells, since it is largely resistant to the effects of membrane potential, synaptic inputs etc. (Schutte, Kroese and Akkermans, 1995). However, a similar inflection is seen on the action potentials of some somatostatin (SOM)-immunoreactive interneurones in the guinea-pig ileum (Song et al., 1997a), thus it is not unique to AH cells. The long after-hyperpolarisation following action potentials is largely due to the activation of a calcium-dependent potassium current (Hirst, Johnson and van Helden, 1985b) which has subsequently been shown to be due to opening of BK channels, which are sensitive to iberiotoxin and charybdotoxin (Kunze et al., 1994). A persistent calcium-dependent potassium conductance appears to contribute to the resting membrane potential of AH cells (North and Tokimasa, 1987), explaining the observation that calcium-free bathing solution causes depolarisation of AH cells (Wood et al., 1979; Grafe, Wood and Mayer, 1980). This is likely to be substantially due to BK channels too, since charybdotoxin depolarised AH cells (Kunze et al., 1994). A number of studies have characterised other ion channels in AH cells in the guinea-pig small intestine. It has been shown that in addition to inward calcium currents and outward potassium currents mediated via calcium-activated potassium channels, there are also inward sodium currents, which are likely to contribute to the depolarisation during action potentials. There is also a delayed rectifier (potassium current) and a transient outward current (Hirst, Johnson and Helden, 1985a) and a background potassium conductance (Galligan, North and Tokimasa, 1989). Another current present in AH cells is the inwardly rectifying cation current IH, which may contribute to stabilising the membrane potential (Galligan et al., 1990b) of these neurones during the after-hyperpolarisation. This current causes a characteristic “sag” in the response of AH cells to hyperpolarising current pulses, but is also seen in other classes of enteric neurones.

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MORPHOLOGY OF ENTERIC PRIMARY AFFERENT NEURONES The intrinsic primary afferent neurones of the gut have AH cell electrophysiological characteristics and Dogiel type II soma-dendritic morphology. The details of their projections have been characterised using immunohistochemical labelling combined with lesions, intracellular dye filling, and retrograde tracing techniques. The first firm indication of the projections and morphology of these cells came with the recognition that a large proportion of them, at least in the guinea-pig ileum, contain immunoreactivity for the calcium binding protein, calbindin (Furness et al., 1988). The distinctive soma-dendritic morphology indicated that a substantial proportion of Dogiel type II cells contained this marker. This was confirmed when immunoreactivity for calbindin was detected in over 80% of electrophysiologically identified myenteric AH cells (Iyer et al., 1988). Importantly, calbindin appeared to be confined to this group of cell bodies, in the myenteric plexus, and was not present in S cells, which do not have Dogiel type II morphology. Large number of calbindin-immunoreactive varicosities were present in myenteric and submucous ganglia and in the mucosa, but not in the circular or longitudinal muscle layers. This is consistent with a role for these neurones as primary afferent neurones or as interneurones, but not as motor neurones. It was suggested at the time of these studies that the presence of calbindin might be related to the calcium-dependence of action potentials and the long after-hyperpolarisation in these neurones. However, many Dogiel type II neurones with apparently identical electrophysiological characteristics lack detectable calbindin immunoreactivity (Iyer et al., 1988; Brookes et al., 1995). In addition, many calbindin-immunoreactive neurones in the guinea-pig proximal colon have Dogiel type I morphology and have S cell electrophysiological characteristics (Messenger, Bornstein and Furness, 1994), thus the functional significance of this calcium binding protein remains to be determined. Studies of the effects of lesions on calbindin-immunoreactive nerve fibres in the ileum suggested that Dogiel type II neurones projected for short distances circumferentially as well as to the submucous ganglia and mucosa (Furness et al., 1990b). Early studies on the soma-dendritic morphology of enteric neurones used dyes or tracers such as Procion Yellow (Hodgkiss and Lees, 1983) or Lucifer Yellow (Bornstein et al., 1984a), which appear to have limited ability to diffuse through the cytoplasm of nerve cells. The use of more rapidly diffusing tracers such as biocytin (Horikawa and Armstrong, 1988) allowed for considerably better filling of enteric nerve cells, revealing their finer processes. It became apparent that Dogiel type II neurones formed two major morphological classes. Most of the cells had several long processes which ran circumferentially, giving rise to many varicose endings in the neuropile of their ganglion of origin and other ganglia further circumferentially (Bornstein et al., 1991b). In fact, retrograde labelling studies in our laboratory have shown that when tracers are applied selectively to myenteric ganglia (after careful removal of all overlying circular muscle) Dogiel type II neurones make up all of the circumferentially projecting neurones more than 3 mm from the application site. About 10% of the cells had numerous short dendritic processes and were classified as “dendritic Dogiel type II” neurones according to the morphological classification scheme of Stach (Stach, 1989). These neurones also projected circumferentially, but appeared to give rise to relatively few endings within their ganglion of origin (Bornstein et al., 1991b).


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No polarised projections up or down the gut were apparent from this study. Using retrograde tracing techniques, it was later demonstrated that the “dendritic Dogiel type II neurones” had long aboral projections within the myenteric plexus, up to 100 mm in length (Brookes et al., 1995). In fact, this class of cells actually comprised the major single source of aborally directed axons in the guinea-pig small intestine (Brookes et al., 1995; Song, Brookes and Costa, 1996). During all of the intracellular dye filling experiments it was reported that the axons of Dogiel type II neurones often ended abruptly as a retraction bulb, in the circular muscle. This was due to the removal of the bulk of the circular muscle prior to impalement of myenteric nerve cell bodies. This observation suggested that many Dogiel type II cells projected to overlying layers of the gut wall. Using retrograde tracing it was later shown that all calbindin immunoreactive myenteric neurones in the guinea-pig small intestine, and in fact probably all Dogiel type II neurones, project to the overlying mucosal layers (Song, Brookes and Costa, 1994a). The projections of enteric primary afferent neurones in the submucous plexus have also been largely established. Early studies on their immunoreactivity gave differing estimates of the proportion of submucous neurones which contained calbindin immunoreactivity (Furness et al., 1990b; Kirchgessner, Tamir and Gershon, 1992). It has recently become clear that some antisera to calbindin reveal many more neurones than other antisera (Reiche et al., 1999) and this may explain some of the discrepancies between studies. Nevertheless, a population of tachykinin-immunoreactive neurones in the submucous ganglia, with and without calbindin immunoreactivity, were identified in a number of studies (Bornstein and Furness, 1988). Intracellular recording and dye filling revealed that, like their myenteric counterparts, these cells have AH-cell-like properties with multipolar morphology (Bornstein et al., 1989; Evans, Jiang and Surprenant, 1994). They make extensive projections within the submucous ganglia, often appearing to contact other nerve cell bodies (Bornstein et al., 1989; Evans, Jiang and Surprenant, 1994). They can be retrogradely labelled by tracers applied to both the mucosa and to the myenteric plexus (Kirchgessner, Tamir and Gershon, 1992; Song et al., 1992) and thus project to both targets.

CHEMICAL CODING OF ENTERIC PRIMARY AFFERENT NEURONES The submucous primary afferent neurones were reported to be immunoreactive for the calcium binding protein calbindin and to contain immunoreactivity for tachykinins (Kirchgessner, Tamir and Gershon, 1992). In addition to these neurochemicals, this class of submucous neurones is known to be immunoreactive for ChAT (Furness, Costa and Keast, 1984) and was later shown to contain neuromedin U (NMU) (Furness et al., 1989a). The myenteric primary afferent neurones have very similar chemical coding. The majority of them contain immunoreactivity for calbindin (Iyer et al., 1988) and for tachykinins (Song, Brookes and Costa, 1991) and for ChAT (Steele, Brookes and Costa, 1991) and many also contain NMU (Furness et al., 1989a). It is very clear from this that primary afferent neurones in the submucous plexus and in the myenteric plexus are strikingly similar to one another, showing nearly identical patterns of projection, electrophysiological properties, chemical coding and morphology.

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SYNAPTIC INPUTS TO ENTERIC PRIMARY AFFERENT NEURONES In early studies, myenteric neurones were classified as AH cells because of the presence of the long after-hyperpolarisation. In contrast, other cells which usually lacked the after-hyperpolarisation, had prominent fast excitatory synaptic inputs following focal electrical stimulation of the preparation. This led to their being named “S cells” (Hirst, 1974). Subsequently, it was suggested that AH cells do in fact receive fast synaptic inputs, but that these are of small amplitude and require signal averageing to be visualised clearly (Grafe et al., 1979). This is consistent with recent reports that calbindin-immunoreactive myenteric neurones (which include many of the enteric primary afferent neurones of the guinea-pig ileum), maintained in culture, generate smaller currents to nicotine than do non-calbindin neurones (Zhou and Galligan, 2000). Nevertheless, there have been reports of some AH cells receiving prominent fast excitatory synaptic inputs, in the small intestine (Bornstein, Furness and Kunze, 1994; Wood, 1994a), large intestine (Wade and Wood, 1988) and rectum (Tamura and Wood, 1989). It should also be remembered that in many of these studies, recorded cells were not routinely dye filled, so it cannot be assumed that they had Dogiel type II morphology. In the small intestine, very few Dogiel type II neurones receive fast synaptic inputs (Brookes et al., 1995; Kunze et al., 1999). It seems likely that much of the confusion over this matter may result from the fact that AH cells and Dogiel type II cells are not identical. It is likely that some AH cells, classified on the basis of the long afterhyperpolarisation or inflection on their action potentials, do not have Dogiel type II morphology. Some of these AH/non-Dogiel type II cells almost certainly receive powerful fast synaptic inputs. Prominent among these are the SOM-immunoreactive descending interneurones which have been electrophysiologically characterised (Song et al., 1997a). Many of these cells had a long after-hyperpolarisation and they often had an inflection on their action potentials, which had a longer duration than most S cells. These interneurones also appear to have the IH current which contributes greatly to the “look and feel” of AH cells. Intracellular dye fills of these neurones reveal a smooth ovoid cell body with numerous long processes, similar to AH cells. However, when biocytin or biotinamide are used as labels, it becomes clear that these neurones have a single long axon, which consistently runs aborally (Song et al., 1997a; Meedeniya et al., 1998), and numerous shorter filamentous dendrites (Portbury et al., 1995b; Song et al., 1997a; Meedeniya et al., 1998). It seems very likely that these neurones may have been classified as AH cells by some investigators but not by others. This matter is important because the presence of fast synaptic inputs would profoundly alter the understanding of how enteric primary afferent neurones are likely to function in enteric neuronal circuitry. On the current balance of evidence, it seems reasonable to assume, in the guinea-pig ileum, that Dogiel type II neurones function as enteric primary afferent neurones, transducing both mucosal and/or mechanical stimuli, without being synaptically driven via fast synaptic inputs from other classes of neurones. Other types of AH cells probably play very different roles and it is likely that most of these lack Dogiel type II morphology. However, it is not possible to exclude the possibility that a small subset of AH cells, with Dogiel type II morphology do receive fast synaptic inputs.


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Slow synaptic inputs to enteric primary afferent neurones Although AH cells show few fast synaptic inputs, it became apparent that following trains of electrical stimulation, many AH cells showed long-lasting depolarisation which was due to slow synaptic excitation (Wood and Mayer, 1978). These slow excitatory postsynaptic potentials (slow EPSPs) were mimicked by 5-HT and it was proposed that this might be the neurotransmitter that is responsible for them (Wood and Mayer, 1978). This was supported, a few years later, by the observation that large number of enteric nerve fibres in both myenteric and submucous ganglia, contained immunoreactivity for 5-HT (Costa et al., 1982; Furness and Costa, 1982). Slow EPSPs were associated with a depolarisation, which lasted from 30 s up to several minutes. Typically there was a decrease in input impedance of the cells at the peak of the potential, suggesting that there was a net closure of ion channels. Under current clamp conditions it was clear that slow EPSPs had a reversal potential near –90 mV, the equilibrium potential for potassium ions. During the slow EPSP, the long after-hyperpolarisation which follows action potentials in AH cells was suppressed and there was a profound increase in excitability, with cells firing repeatedly during depolarising current pulses and often showing spontaneous firing. All of these effects were mimicked by 5-HT and, furthermore, desensitisation with 5-HT reduced electrically stimulated slow EPSPs (Wood and Mayer, 1979). A number of studies were then carried out to determine whether or not 5-HT was the only transmitter that might mediate slow EPSPs. An early study demonstrated that substance P (SP) also mimicked the slow EPSP in AH cells (Katayama and North, 1978) and that desensitisation with SP, or chymotrypsin treatment, reduced or blocked the synaptic potential (Morita, North and Katayama, 1980). This led to some debate as to whether SP or 5-HT was “the transmitter”. An elegant study used lesions to the myenteric plexus to distinguish between these possibilities. It was known that 5-HT immunoreactive neurones had descending projections within the myenteric plexus (Furness and Costa, 1982) whereas SP immunoreactive neurones gave rise to many varicosities close to their cell bodies (Costa et al., 1980a). Lesions were used to remove long descending inputs from 5-HT containing neurones and intracellular recording were made in these partially denervated islands. It was found that both fast and slow EPSPs persisted in these areas, indicating that local pathways were sufficient, although there was a reduction in amplitude of synaptic events, which was attributed to the loss of long descending pathways (Bornstein et al., 1984b). Later studies have shown that much of the SP (or tachykininlike) immunoreactivity in the myenteric plexus is likely to arise locally from Dogiel type II neurones (Song, Brookes and Costa, 1991) which have profuse circumferential projections (Hendriks, Bornstein and Furness, 1990; Bornstein et al., 1991b; Song, Brookes and Costa, 1991, 1996; Brookes et al., 1995). This evidence does not rule out a role for 5-HT in slow synaptic inputs to AH cells, but suggests that SP, or a related tachykinin is likely to be involved, under some circumstances. An early ultrastructural study suggested that 5-HT immunoreactive nerve varicosities are associated with the cell bodies of dye filled AH cells (Erde, Sherman and Gershon, 1985) although a subsequent study failed to find any special association (Young and Furness, 1995). The story is also complicated by the fact that a number of other endogenous substances have been demonstrated to mimic slow EPSPs on AH cells. For example pituitary adenyl cyclase activating peptide (Christofi and

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Wood, 1993a), VIP, bombesin and gastrin-releasing peptide (GRP) (Zafirov et al., 1985a), cholecystokinin (CCK) and the gastrin analogue, pentagastrin (Nemeth, Zafirov and Wood, 1985) and CGRP (Palmer et al., 1986) all mimic slow EPSPs. Using selective antagonists, evidence has been provided that endogenous CCK may contribute to slow EPSPs via both CCKA and CCKB receptors (Schutte et al., 1997). In addition, histamine also mimics slow EPSPs, although it is unlikely to be an endogenous mediator of these synaptic events (Nemeth, Ort and Wood, 1984). The intracellular mechanisms underlying slow EPSPs are of considerable interest, given the long time course of these events. All of the candidate receptors belong to the superfamily of receptors with 7 transmembrane regions and are likely to be coupled via G-proteins to second messenger pathways within cells. The observation that slow EPSPs were mimicked by intracellular injection of cyclic AMP (Zafirov et al., 1985b) and by membrane permeant cAMP analogues (Palmer, Wood and Zafirov, 1986) suggested a role for adenylcyclase in generating slow EPSPs. This effect was also mimicked by forskolin which raises intracellular cAMP levels (Nemeth et al., 1986). Adenosine, hyperpolarises AH cells, increase the amplitude of long after-hyperpolarisations, reduces their input impedance and reduces excitability (Palmer, Wood and Zafirov, 1987). It has been proposed that this is due to reduction in intracellular cAMP concentrations. Direct measurement suggests that this is one of the effects of adenosine (Xia, Fertel and Wood, 1997). Adenosine also blocked the effects of forskolin (Palmer, Wood and Zafirov, 1987). Using adenosine as a tool, it was demonstrated that the effects of several of the potential transmitters of the slow EPSP were also blocked by adenosine, however the effects of exogenous SP, CGRP and 5-HT were not reduced. Unfortunately, it was not possible to use adenosine as a tool to investigate the nature of second messenger pathways activated during the slow EPSP because adenosine also causes potent presynaptic inhibition of both fast and slow synapses onto enteric neurones (Christofi and Wood, 1993b). Early studies had suggested that during the slow EPSP, a calcium-dependent potassium current was inhibited (Grafe, Wood and Mayer, 1980). Later it was shown that SP reduced such a current which contributed to both resting membrane potential and the long afterhyperpolarisation (Morita and Katayama, 1992). Such a reduction in a resting potassium conductance would explain the observation that there was a marked increase in input impedance in most cells during the slow EPSP. However, in many recordings it had been noticed, particularly during the early phase of the slow EPSP that there was sometimes an initial decrease in input impedance. The currents underlying this event were systematically studied using single electrode voltage clamp (Bertrand and Galligan, 1994). It was shown that slow EPSPs are frequently associated with an increase in chloride currents which sum with the decrease in potassium conductance. While forskolin and cAMP analogues mimic the decrease in potassium conductances, the second messenger pathways associated with the increase in chloride conductance have not been identified (Bertrand and Galligan, 1995). Sources and significance of slow EPSPs Experimentally, slow EPSPs are typically evoked by repetitive focal electrical stimulation relatively close to the recorded nerve cell body. Sometimes the stimulating electrode is


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placed on an internodal strand or on a nearby part of the ganglion. From our experience with retrograde labelling techniques, we estimate that such stimuli probably activate 200–2000 axons synchronously, which makes them rather “un-physiological”. It is important therefore to determine under what conditions slow EPSPs are likely to occur, in vivo, which cells are likely to give rise to them and what their role is likely to be. Several studies have examined the presence of slow EPSPs without using electrical stimuli. Using paired intracellular recording, it was shown that single AH cells, with Dogiel type II morphology, could give rise to measurable slow EPSPs in other myenteric neurones (Kunze et al., 1993) including AH cells. This was significant because it demonstrated that it was not necessary to stimulate large number of axons simultaneously. However, repetitive stimulation was required, indicating that multiple action potentials were required to evoke measurable potentials. When chemical stimuli were applied to the mucosa, some AH cells were activated directly by the stimulus, whereas others were activated indirectly, via slow excitatory synaptic inputs. In contrast, all S cells were activated indirectly, via synaptic inputs (Bertrand et al., 1997). This suggests that firing in chemosensitive primary afferent neurones, presumably myenteric AH cells, can evoke slow EPSPs in other primary afferent neurones. The same mechanism appears to operate when primary afferent neurones are activated by stretch (Furness et al., 1998). Thus it appears that primary afferent neurones in the guinea-pig small intestine are organised into self-exciting assemblies, which are then capable of giving synchronised responses to stimuli. This idea was proposed by Jackie Wood, long before type II/AH neurones had been identified as enteric primary afferents. He suggested that the slow EPSP may modulate the spread of excitability across the soma of Dogiel type II neurones (Wood, 1989) (see also Chapter 9 of this volume). ENTERIC PRIMARY AFFERENT NEURONES IN OTHER REGIONS AND SPECIES It is clear that a great deal of data has been gathered about the projections, chemical coding, electrophysiological characteristics and morphology of the intrinsic primary afferent neurones of the guinea-pig small intestine. The great bulk of this data was obtained before the function of these cells had been identified. This particular type of enteric neurone has been the focus of a great deal of study, for several rather arbitrary reasons. First of all, they are large and easy to impale with intracellular microelectrodes and are frequently encountered, because they make up a substantial proportion of all enteric neurones. Their electrophysiological characteristics are intriguingly unusual and they have a distinctive morphology that allows them to be unequivocally distinguished from other classes of cells in this preparation. This raises the question then, as to whether primary afferent neurones are similar in other regions of the gut and in other species. At present, this question cannot be answered directly, since it is only in the guinea-pig ileum that functional studies of sensory transduction by enteric neurones have been carried out. However, it is possible to determine whether or not there are electrophysiological AH cells, and cells with Dogiel type II morphology present in other preparations. It should be stated, at the outset, that the only absolute requirement for an enteric neurone to function as a primary afferent is the ability to produce a generator potential in response to a mechanical or chemical stimulus. This must reflect the presence of appropriate stretch-activated channels or ligand-gated mechanisms in the processes of these cells. Since none of the

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molecules responsible for transduction have yet been identified in enteric primary afferent neurones, they cannot be used to identify them. It should be pointed out that all of the other electrical characteristics found in AH/type II cells, such as their action potential shape, after-hyperpolarisation, channels and currents, probably function only to modulate the responses of these neurones to physiological stimuli, or to influence transmitter release from their terminals. There is no reason, per se, to believe that any of these characteristics is absolutely essential for a neurone to function as a primary afferent. It seems intuitively possible that a number of different combinations of electrical characteristics could meet the integrative requirements of enteric primary afferent neurones. Thus the presence or absence of after-hyperpolarisations, spike inflections etc. may not be a good indicator of whether a particular neurone functions as a primary afferent in any gut preparation other than the guinea-pig ileum. For example, the absence of a calcium component to the soma action potential, or the lack of an after-hyperpolarisation could not be taken to rule out the possibility that a particular neurone functions as a primary afferent in the mouse, rat, pig or human gut, for example. Perhaps the best correlation with a role as a primary afferent is the soma-dendritic morphology of the neurone. Throughout the body, primary afferent neurones are generally bipolar, multipolar or pseudo-unipolar – this applies to those with cell bodies in dorsal root ganglia, nodose or petrosal ganglia, trigeminal and other cranial ganglia. It also applies to myenteric and submucous Dogiel type II neurones in the guinea-pig small intestine, which have been shown to function as primary afferent neurones. The multipolar morphology may be functionally important since it ensures spatial separation between transduction sites and synaptic release sites. It would be reasonable to expect that enteric primary afferent neurones in other species and regions of gut would also be multipolar. In his original work, Dogiel (1899) described multipolar (type II cells) in several species and regions of gut, including small and large intestine of guinea-pigs, in the gall bladder and in human intestine. In the guinea-pig, Dogiel type II cells have been described in the ileum, duodenum (Clerc et al., 1998), and proximal colon (Messenger, Bornstein and Furness, 1994; Neunlist and Schemann, 1997; Neunlist, Dobreva and Schemann, 1999), distal colon (Lomax et al., 1999) and rectum (Tamura, 1992; Tamura, 1997). In each of these regions some correlation has been noted between Dogiel type II morphology and AH cell electrophysiological characteristics. However, Dogiel type II neurones do not appear to be present in the proximal guinea-pig stomach, based on retrograde labelling of large number of neurones (Brookes et al., 1998). Neither is the AH cell electrophysiological type present in this area (Schemann and Wood, 1989). Dogiel type II cells also appear to be absent from the oesophageal myenteric plexus (Brookes et al., 1996) although intracellular recordings have not been made from these neurones. Interestingly, neurones with AH cell electrophysiological characteristics have been recorded from the distal stomach of the guinea-pig (Tack and Wood, 1992) but were not morphologically identified. Immunohistochemical and retrograde labelling studies have shown a small population of neurones in the myenteric plexus of the guinea-pig antrum which have large smooth cell bodies and several long processes. This suggests that a small population of Dogiel type II neurones may be present in this region (SJH Brookes and GW Hennig, unpublished observations). Thus it appears that multipolar neurones, with AH cell electrophysiological characteristics (a calcium-dependent action potential, with an inflection on


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the falling phase, usually followed by a long after-hyperpolarisation) are present throughout the enteric nervous system of the guinea-pig, at least distal to the mid stomach. It should be noted, however that the long after-hyperpolarisation, in particular, shows considerable variability in amplitude, being powerfully suppressed when slow excitatory synaptic inputs have been activated (Wood, 1994b). Dogiel type II neurones, with AH cell electrophysiological characteristics have also been described in the myenteric plexus of other species, including the rat small intestine (Brookes, Ewart and Wingate, 1988), rat large intestine (Browning and Lees, 1996) and in the pig small intestine (Cornelissen et al., 2000). It should be noted however that the correlation in the pig small intestine was not as straightforward as reported elsewhere. In a systematic study of the long after hyperpolarisation, in porcine myenteric cells with Dogiel type II morphology, it was reported that only a small proportion (17%) showed a slow after hyperpolarisation comparable to that seen in the guinea-pig (Cornelissen et al., 2000). Cells classified as AH cells have been recorded with intracellular microelectrodes in the mouse colon (Furukawa, Taylor and Bywater, 1986) and occasionally in the human colon (Brookes, Ewart and Wingate, 1987), although their morphology was not determined, so it is not clear whether or not they were multipolar neurones. Thus it is clear that neurones with Dogiel type II morphology and neurones with AH cell electrophysiology are found in the enteric nervous system of most of the preparations of small or large intestine in a range of species. Where tested, there has usually been some correlation between the characteristics, although they do not always match one-for-one. The functional significance of this variability remains to be determined. It may reflect minor differences in the way that stimuli are frequency coded by afferents or it may indicate that some Dogiel type II cells play roles other than as primary afferents. Concerning the chemical coding of Dogiel type II neurones, differences between the guinea-pig and other species have been reported. In the guinea-pig, Dogiel type II enteric primary afferent neurones are immunoreactive for ChAT (Steele, Brookes and Costa, 1991; Costa et al., 1996) with or without immunoreactivity for the calcium binding protein calbindin (Iyer et al., 1988; Brookes et al., 1995). Similar coding is seen in Dogiel type II/AH neurones in the proximal colon of the guinea-pig (Neunlist and Schemann, 1997; Neunlist, Dobreva and Schemann, 1999). In the small intestine, the majority of these primary afferent neurones are also immunoreactive for tachykinins (Song, Brookes and Costa, 1991) and for NMU (Furness et al., 1989a). In other species, Dogiel type II neurones have been reported to contain immunoreactivity for CGRP, notably in the small intestine of the pig (Scheuermann et al., 1991) and the human (Timmermans et al., 1992). While care has to be taken to identify soma-dendritic morphology purely on the basis of immunoreactivity, a recent study has confirmed that porcine Dogiel type II neurones, retrogradely labelled from the mucosa, frequently contain immunoreactivity for CGRP, in addition to ChAT and tachykinins (Hens et al., 2000). Myenteric Dogiel type II cells in the rat ileum have also been shown to contain ChAT (Mann, Furness and Southwell, 1999), at least those that were immunoreactive for calbindin. These differences in chemical coding may have profound importance for interpreting functional studies. CGRP immunoreactive nerve cell bodies are found throughout the rat enteric ganglia (Su et al., 1987; Sternini and Anderson, 1992). If some of these are the Dogiel type II cells, this could explain the observations of Grider and colleagues which suggest that enteric

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primary afferent neurones may release CGRP to activate reflex circuits in the rat colon (Grider, 1994). ENTERIC PRIMARY AFFERENT NEURONES: SUMMARY From functional studies it is clear that enteric neuronal circuits can be specifically activated by physiological stimuli such as stretch, distortion of the villi and chemical stimulation, indicating the existence of enteric primary afferent neurones. To date, neurones which respond to these stimuli have been directly demonstrated only in the myenteric and submucous ganglia of the guinea-pig small intestine. These neurones all appear to have a large smooth cell soma, sometimes with short filamentous dendrites, but always characteristically have several long axonal processes (and hence have “Dogiel type II” morphological features). In the guinea-pig ileum these neurones also have characteristic electrophysiological characteristics, with a significant inward calcium current during their action potentials which are followed by a long after-hyperpolarisation. These cells receive powerful slow excitatory synaptic inputs, substantially arising from other primary afferent neurones, but receive small and sparse fast synaptic inputs. Intuitively, this is compatible with the neurones being activated by physiological stimuli (rather than being synaptically driven by powerful fast synaptic inputs) but with their excitability being modulated by slow synaptic contacts from other neurones of the same class, which are thus organised into coordinated assemblies. Exactly how many classes of intrinsic primary afferent neurones exist in the guinea-pig ileum is not currently clear. At least three classes can be distinguished on morphological grounds. These include the cells located in submucous ganglia, myenteric “dendritic” Dogiel type II neurones, with long descending projections (Brookes et al., 1995) and “non-dendritic” Dogiel type II neurones. Three physiological types have also been identified, including the submucous neurones responding to mechanical stimulation of the mucosa, myenteric neurones responding to chemical stimuli and myenteric neurones that are stretch sensitive. Whether or not any of the cells respond to two or more modes of stimulation remains to be determined, but it seems likely given estimates of the proportions of cells that are activated under different circumstances (Furness et al., 1998; Kunze and Furness, 1999). The calcium influx during the action potential, and the related long after-hyperpolarisation may not be directly involved in sensory transduction. It seems likely that these characteristics may have little to do with the ability of these neurones to detect physiological stimuli, but may rather be involved in modulating the output from enteric primary afferent neurones onto other neurones in the enteric ganglia. As such, the long after-hyperpolarisation and calcium spike may well be epi-phenomena, associated with a sensory role, but not directly, causally involved in it. If this is the case, primary afferent neurones in other regions of gut and in other species may show different combinations of such electrophysiological characteristics. It would be premature to expect all enteric primary afferent neurones to appear morphologically, neurochemically and electrophysiologically identical to those of the guinea-pig small intestine. Lastly, it is worth pointing out that although recent evidence has demonstrated convincingly that many Dogiel type II cells function as primary afferents, this does not exclude other roles for them. Slow synaptic input to Dogiel type II neurones may, under some


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circumstances, drive them to produce action potentials in the absence of physical or chemical stimuli (Wood, 1994b). This suggests that Dogiel type II neurones may, under some circumstances, function as interneurones in the enteric nervous system. All of the classes of Dogiel type II neurones discussed above have axonal projections to the mucosa (Song, Brookes and Costa, 1994a). It is possible that the axons within the mucosa may not be exclusively afferent in function. It is possible that they might influence epithelial cells in the mucosa, since they give rise to abundant calbindin-immunoreactive endings in close apposition to the mucosal epithelium. In addition, these cells are likely to be cholinergic (from the presence of ChAT immunoreactivity) and cholinergic mechanisms play such an important role in regulating epithelial secretion (Cooke and Reddix, 1994) (see also Chapter 7 of this volume).

ENTERIC MOTOR NEURONES Since the earliest studies on isolated or denervated preparations of intestine (Bayliss and Starling, 1899; Langley and Magnus, 1905; Cannon, 1912; Trendelenburg, 1917) it has been clear that there are intrinsic motor neurones within the enteric nervous system. It is technically very straightforward to record mechanical activity of the smooth muscle layers of preparations of gastrointestinal tract. Since the first studies using electrical field stimulation (Paton, 1955), the guinea-pig ileum, in particular, has become a standard pharmacological preparation, in which to test an enormous range of pharmacological agents on neuromuscular transmission and smooth muscle contractility (see review by Marcello Tonini and colleagues in this volume). Despite the widespread use of this preparation, the motor neurones responsible for this transmission have only been identified in the last decade. For many years it was unclear which major morphological type enteric motor neurones belonged to, with Dogiel suggesting that type I cells were probably motor (Dogiel, 1899), whereas some later authors considered that type II cells were likely to function as motor neurones (Hill, 1927). The first real insights came with the use of immunohistochemical methods to analyse the axons of enteric motor neurones within the muscle layers. In particular, extensive branching by VIP-immunoreactive nerve axons was seen in the circular muscle of the guinea-pig ileum (Costa et al., 1980b; Costa and Furness, 1983). A later study demonstrated that the nerve cell bodies that gave rise to VIP-containing axons were largely of the Dogiel type I morphological class with S cell electrophysiological characteristics (Katayama, Lees and Pearson, 1986). A similar finding was made for enkephalin (ENK)-immunoreactive axons (Furness, Costa and Miller, 1983) and cell bodies (Bornstein et al., 1984a). Since either ENK or VIP is present in the majority of motor axons in the circular muscle layer of the guinea-pig ileum (Llewellyn Smith et al., 1988), it could be concluded that a substantial proportion of motor neurones must have Dogiel type I morphology. This was later supported by observations that immunoreactivity for the calcium binding protein, calbindin, was present in a substantial proportion of Dogiel type II neurones (Iyer et al., 1988) but was not present in many varicose motor axons within the circular muscle layer (Furness et al., 1988, 1990b).

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LONGITUDINAL MUSCLE MOTOR NEURONES The longitudinal muscle layer of the guinea-pig ileum is a thin layer of tissue lying just below the serosa, from 15–40 µm thick, depending on the degree of stretch of the tissue. It is innervated by the ramifying network of varicose nerve fibres making up the tertiary plexus, which lies on its inner face, but which does not penetrate deeply in the muscle layer (Llewellyn-Smith et al., 1993; Gabella, 1994). Retrograde labelling, with the carbocyanine dye 1,1′-didodecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) applied in solid form to the tertiary plexus, labelled longitudinal muscle motor neurones in the myenteric plexus (Brookes et al., 1992). All of the nerve cell bodies were located within 3 mm of the DiI application site, indicating that the motor neurones have relatively short projections to the region of muscle that they innervate. The cell bodies were typically very small (12–15 µm in the short axis) with a single axon. Some had clear Dogiel type I morphology, with short lamellar dendrites, but in others, the dendrites were so restricted that they were better classified as “simple cells" (Furness, Bornstein and Trussell, 1988). Longitudinal muscle motor neurones were often located in small clusters, located near the points at which internodal strands entered the myenteric ganglia. Intracellular dye fills of these neurones have revealed that they branch extensively within the tertiary plexus, close to their cell bodies (Bornstein et al., 1991a; Furness et al., 2000). Intracellular recordings have shown that they are highly excitable S cells which fire tonically to depolarising current pulses (Smith, Burke and Shuttleworth, 1999). Analysis of the chemical coding of retrogradely labelled longitudinal muscle motor neurones revealed that the great majority in the guinea-pig ileum (>97%) were immunoreactive for ChAT and hence likely to be cholinergic (Brookes et al., 1992). These motor neurones are likely to release the acetylcholine that mediates the excitatory junction potential evoked by electrical stimulation (Cousins et al., 1993) and which underlies the widely-studied cholinergic twitch of the guinea-pig ileum (Paton, 1955). It is well established that inhibitory junction potentials are relatively small in the longitudinal muscle of the guinea-pig ileum (Bywater and Taylor, 1986), at least in comparison to those recorded in the circular muscle layer. This suggests that the longitudinal muscle layer probably receives relatively little direct inhibitory innervation. Consistent with this is the relatively sparseness of VIP-immunoreactive (Costa et al., 1980b; Costa and Furness, 1983) and nitric oxide synthase (NOS)-immunoreactive (Costa et al., 1992) varicose nerve fibres in the tertiary plexus of this region. This was matched by the observation that only 3% of retrogradely labelled motor neurones to the longitudinal muscle were immunoreactive for VIP (Brookes et al., 1992). However it is still possible to record relaxations of the longitudinal muscle in responses to electrical stimulation (Osthaus and Galligan, 1992; Yunker and Galligan, 1998). This suggests either that the 3% of motorneurones can still be functionally important, or that overflow of inhibitory transmitters from the circular muscle can occur following repetitive, synchronous activation of large number of axons. Of the retrogradely labelled cell bodies of motor neurones to the longitudinal muscle of the guinea-pig ileum, approximately 87% were immunoreactive for the calcium binding protein, calretinin (Brookes et al., 1992). Calretinin immunoreactivity is present in many varicose nerve fibres in the tertiary plexus (Brookes, Steele and Costa, 1991a), where it coexists with ChAT, but not with neurofilament protein (NFP) triplet. Calretinin is found


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in two classes of neurones within the myenteric plexus of the guinea-pig ileum, which together comprise 26% of all cells. Approximately 21% of all myenteric neurones are calretinin-immunoreactive longitudinal muscle motor neurones, indicating that about 24% of all myenteric neurones innervate the longitudinal muscle in this preparation. About half of all longitudinal muscle motor neurones were immunoreactive for tachykinins (Brookes et al., 1992), all of which were also immunoreactive for ChAT. It is well established that the longitudinal muscle of the guinea-pig ileum receives a non-cholinergic excitatory innervation (Ambache and Freeman, 1968) which is substantially mediated by SP (Franco, Costa and Furness, 1979; Bjorkroth, 1983). The cholinergic twitch can be effectively stimulated by electrical stimuli delivered at low frequencies (typically 0.1 Hz) whereas non-cholinergic excitation, recorded in the presence of muscarinic antagonists, requires trains of stimuli. This led to speculation that tachykinins, or other mediators of non-cholinergic excitation, may be released from different classes of nerves from those releasing acetylcholine. It appears from the anatomical data that tachykinins are probably released by a subset of cholinergic nerve fibres and that the apparent frequency-dependence may reflect differential release of transmitters stored in different vesicles in the same nerve endings in response to repetitive action potentials (Maggi, Holzer and Giuliani, 1994). Many other neurochemicals found in enteric neurones are strikingly absent from the tertiary plexus. There are very few axons immunoreactive for SOM (Costa et al., 1980c), 5-HT (Costa et al., 1982), calbindin (Furness et al., 1988; Furness et al., 1990b) which each mark other substantial populations of enteric neurones. However, immunoreactivity for γ-amino butyric acid (GABA) is present, particularly after pre-loading the preparation with exogenous GABA (Jessen, Hills and Saffrey, 1986; Furness et al., 1989b). It appears that GABA is present within a subset of motor neurones to both the circular and longitudinal muscle layers, largely (but not exclusively) in those immunoreactive for NOS (Williamson, Pompolo and Furness, 1996). Longitudinal muscle innervation: other regions The relative paucity of direct, inhibitory innervation of the longitudinal muscle of the guinea-pig ileum is not the case for most other regions of gut. The longitudinal muscle layer of the stomach, proximal colon and distal colon of the guinea-pig all show more pronounced nerve mediated relaxations (Costa, Furness and Humphreys, 1986) in response to electrical stimulation. Indeed, the taenia coli (or more properly taenia caeci) was one of the first preparations at which non-cholinergic, non-adrenergic inhibitory innervation of the gastrointestinal tract was described (Burnstock et al., 1963; Burnstock, Campbell and Rand, 1966). It is tempting to speculate why the ileum may be rather differently organised. It is clear from functional studies that during peristaltic emptying, there is a marked lengthening of the longitudinal muscle, while the circular muscle is contracting to expel the fluid contents (Trendelenburg, 1917). This led to the suggestion that the two muscle layers may be reciprocally innervated, such that one muscle layer contracts while the other relaxes (Kottegoda, 1970). A good deal of evidence now suggests that this is not the case. In particular, in the guinea-pig ileum, the longitudinal muscle remains contracted during circular muscle contraction, if the preparation is first opened up into a flat sheet, preventing passive interactions due to the incompressibility of fluid contents (Brookes

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et al., 1999). If contractions of circular and longitudinal muscle are carefully monitored during peristaltic emptying in tubular preparations, the longitudinal muscle starts to lengthen only after circular muscle contractions have occluded the lumen of the intestine (Hennig et al., 1999). When contractions of the two layers are monitored independently, using force transducers attached to regions where the layers have been separated, the two are seen to contract simultaneously (Spencer, Walsh and Smith, 1999). Thus it appears that during peristaltic emptying, contraction of the circular muscle is sufficiently powerful to overcome the active contraction of the longitudinal muscle. It is possible, then, that active relaxation of the longitudinal muscle may not be functionally important in this region. Under similar conditions, in the colon, there is evidence for a direct, functional inhibitory innervation of the longitudinal muscle causing relaxation in response to physiological stimuli (Smith and Robertson, 1998). Longitudinal muscle motor neurones: other regions, other species As described above, the longitudinal muscle of the guinea-pig ileum is innervated mostly by excitatory motor neurones with short, local projections. The excitatory motor neurones did not have a preferential direction of projection and the inhibitory motor neurones were too few in number to determine whether they showed any polarity. The only other preparation in which longitudinal muscle motor neurones have been studied in such detail is in the upper stomach of the guinea-pig (Michel, Reiche and Schemann, 2000). Here it was found, not surprisingly, that there was a substantial inhibitory innervation of the muscle layer, with approximately 44% of all motor neurones being immunoreactive for NOS and hence probably inhibitory in function. In addition, the neurones had a marked polarity, with inhibitory neurones being located orally and excitatory motor neurones being located anal to the muscle that they innervated. There were also significant differences in the chemical coding of excitatory motor neurones to the longitudinal muscle of the gastric corpus compared to those of the ileum. For example, about half of the gastric excitatory motor neurones contained ENK immunoreactivity, whereas this was very sparsely distributed in the tertiary plexus of the ileum (Furness, Costa and Miller, 1983). Fewer than 4% of gastric motor neurones to the longitudinal muscle contained calretinin immunoreactivity. This points to substantial functional differences between regions within the same species, presumably reflecting the different physiological requirements related to specialised motor activity. It seems likely that the polarised distributions of inhibitory and excitatory motor neurones seen in the stomach may be more typical than the specialised innervation of the ileum. Since inhibitory reflexes have been evoked in the longitudinal muscle of the guinea-pig colon (Smith and Robertson, 1998), it seems likely that polarised inhibitory innervation of the longitudinal muscle layer may exist here too. Intracellular dye fills of longitudinal muscle motor neurones in the distal colon reveal them to be small unipolar cells with short dendrites, often with a filamentous appearance (Lomax et al., 1999). Their chemical coding has not been investigated in detail and it is not currently clear whether inhibitory motor neurones have a different polarity to excitatory motor neurones. In the human intestine, longitudinal muscle motor neurones have been retrogradely labelled and seen to be numerous, small cells, with relatively short projections to the muscle that they


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innervate (Wattchow et al., 1995). Their chemical coding has not, as yet, been investigated so it is not clear whether excitatory and inhibitory motor neurones show a polarised distribution. CIRCULAR MUSCLE MOTOR NEURONES In most regions of the gastrointestinal tract, the circular muscle layer is the major component of the muscularis externa. It is densely innervated by enteric excitatory and inhibitory motor neurones. In the guinea-pig ileum, it had been shown from myectomy operations that the circular muscle is exclusively innervated by motor neurones with cell bodies in the myenteric plexus (Wilson et al., 1987). Four types of junction potential have been recorded in the circular muscle of the guinea-pig ileum. These include cholinergic excitatory junction potentials (Hirst, Holman and McKirdy, 1975), non-cholinergic excitatory junction potentials (Bywater, Holman and Taylor, 1981; Bauer and Kuriyama, 1982a,b) and non-cholinergic, non-adrenergic (NANC) fast inhibitory junction potentials (Hidaka and Kuriyama, 1969; Hirst, Holman and McKirdy, 1975; Holman and Weinrich, 1975). The addition of apamin, a toxin which blocks channels that mediate the fast inhibitory junction potential (Shuba and Vladimirova, 1980), reveals an additional, slow hyperpolarising junction potential (Niel, Bywater and Taylor, 1983). To identify and characterise the motor neurones that give rise to junction potentials, retrograde tracing studies were carried out, in vitro. DiI was applied in solid form to the microdissected surface of the circular muscle where it was taken up by the axons of motor neurones and transported back to their cell bodies of origin over the following 3–4 days in organotypic culture. Using this methodology, circular muscle motor neurones could be distinguished from all intermingled classes of nerve cell bodies in the myenteric ganglia (Brookes and Costa, 1990). It was confirmed that the great majority of nerve cell bodies labelled from the circular muscle had Dogiel type I morphological characteristics and that they were located both oral and anal to the region of the muscle that they innervated. Combining immunohistochemical labelling with retrograde tracing allowed results from this approach to be integrated with the enormous volume of immunohistochemical data already available. It quickly became apparent that excitatory motor neurones, identified by their immunoreactivity for ChAT, accounted for nearly three quarters of all circular muscle motor neurones. They were located either very close to the circular muscle that they innervated (local projections ET-2 >> ET-3 (Yanagisawa, 1994). The discovery of ET-1 resulted from its properties as a strong vasoconstrictor produced by vascular endothelial cells (Yanagisawa et al., 1988). The endothelins and the endothelin receptors, however, have now been found to be widely distributed (Rubanyi and Polokoff, 1994). The endothelins are synthesised with a signal sequence (as a preproendothelin) that enables the cell that secretes them to translocate the molecules across the membranes of the rough endoplasmic reticulum (RER) to the cisternal space. From the cisternal space of the RER, the proteins can be transported to the Golgi apparatus, and packaged into vesicles for secretion by exocytosis. The signal sequence is cleaved co-translationally, to convert the preproendothelins to the inactive big endothelins which are secreted. The big endothelins are split by a specific membrane-bound metalloprotease, the endothelinconverting enzyme-1 (ECE-1), to produce the active ET-1, ET-2, or ET-3 (Xu et al., 1994). Transgenic knockout mice that fail to produce ET-1 exhibit craniofacial defects arising from abnormal development of the first branchial arch (Kurihara et al., 1994). Missense mutations in the genes encoding ETB occur in sl/sl mice and so are associated with megacolon and coat spotting (Hosoda et al., 1994). Similar mutations can be found in the analogous human locus in a subset of human patients with Hirschsprung’s disease (Puffenberger et al., 1994). When the gene encoding ETB is knocked out in transgenic mice by homologous recombination, the colon becomes aganglionic, mimicking the megacolon seen in sl/sl mice, confirming the causal relationship between ETB and the aganglionosis of the terminal colon (Hosoda et al., 1994). Lethal spotting, the aganglionosis of the colon in rats, also arises as a result of a mutation that prevents expression of the rat

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ETB; in this case the mutation is an interstitial deletion in an exon of the gene encoding ETB (Ceccherini et al., 1995; Karaki et al., 1996). An arginine is replaced by a tryptophan residue in the C-terminus of big ET-3 as a result of the mutation of the ET-3 gene in ls/ls mice (Baynash et al., 1994). The consequence of this mutation is that ECE-1 is unable to convert big ET-3 to ET-3. Since big endothelins are inactive, this mutation essentially ablates ET-3, suggesting that loss of the ligand, ET-3, in ls/ls mice has the same effect as the loss of the receptor, ETB is sl/sl mice. The suggestion is confirmed by the occurrence of an identical aganglionosis of the terminal colon in mice in which the gene encoding ET-3 has been knocked out by homologous recombination. While it is clear that ET-3 and ETB play essential roles in the development of the ENS of the terminal colon, it is not clear what these roles are. Since ETB can be stimulated by ET-1 and ET-2, as well as by ET-3 (Yanagisawa, 1994), the acquisition of aganglionosis in animals that lack only ET-3 indicates that the endothelins do not circulate in the fetus at times that are critical in the formation of the ENS (Baynash et al., 1994). On the contrary, the endothelins are likely to be factors that act only locally, so that the loss of the ligand, ET-3, will be manifest in the area where it is expressed. Conceivably, aganglionosis might result, when ET-3 is absent, because crest-derived neuronal precursors express ETB and require ET-3 stimulation. This possibility does not explain why ET-3 should only be required by those crest-derived precursors that develop in the terminal colon. A second possibility is that ET-3 might be required by a non-neuronal resident cell of the terminal colon, which provides a component of the local microenvironment that crest-derived precursors need to colonise the colon or develop there. Obviously, these two possibilities are not mutually exclusive. Both crest-derived and non-neuronal cells may express ETB and be influenced by ET-3. In any case, the issue of why enteric neurons, other than those in the colon, develop perfectly well in the absence of ET-3 or ETB is an issue that must be resolved.

AGANGLIONOSIS IS NOT EXPLAINED BY THE FAILURE OF ET-3 TO STIMULATE CREST-DERIVED NEURAL PRECURSORS IN THE COLON One hypothesis that has been advanced to account for the critical role of ET-3 in the formation of the ENS of the terminal colon is that ET-3 may be an autocrine growth factor produced and required by enteric crest-derived neural and glial precursors and by melanocytes (Baynash et al., 1994). This hypothesis nicely explains the universal correlation of aganglionosis of the colon with spotting of the coats in animals lacking either ET-3 or ETB. The autocrine hypothesis supposes that the migrating crest-derived cells that colonise the intestine express big ET-3, ECE-1, and ETB. The cells can thus convert big ET-3 to ET-3 and respond to the ET-3 they produce. If ET-3, were to be an autocrine growth factor required by cells migrating from the vagal crest, however, the congenital absence of either ET-3 or ETB would be expected to cause an aganglionosis of the entire bowel (like the defects induced by the knockouts of GDNF or Ret; Figure 11.3), not just the terminal colon, as is actually seen in ls/ls (Baynash et al., 1994), sl/sl mice (Hosoda et al., 1994; Kapur et al., 1995) and spotting lethal rats (Ceccherini et al., 1995; Gariepy, Cass and Yanagisawa, 1996; Karaki et al., 1996).

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The vagal crest after all, colonises the entire gut below the oesophagus and rostral stomach (Figure 11.1). If the autocrine hypothesis applied only to sacral crest-derived cells, no part of the bowel would be expected to be completely aganglionic, because both vagal and sacral cells participate in colonising the post-umbilical gut and there is no region of the ENS that is entirely sacral in origin. Conceivably, the target of ET-3’s action might not be the colon. The target could instead be the pre-migratory crest (Lahav et al., 1996). For example, a critical mass of vagal crest cells might be needed in order for them to extend their colonising range to the terminal colon. The pre-migratory vagal crest cells may require stimulation by ET-3 in order to proliferate sufficiently to reach this critical mass. In the absence of the stimulation of their putative ETB receptors by ET-3, therefore, the population of vagal crest-derived cells might be too small for any of them to migrate as far as the terminal colon, which lies at the end of their migratory pathway. If this hypothesis were to be correct, then ET-3 could still be an autocrine growth factor that produces only localised effects. Applied to the skin, an analogous argument would account for coat spotting. The starting population of melanogenic precursors might have to be built up by ET-3-stimulated proliferation in order to reach a size that is big enough to spread throughout the skin. If the population is too small, patches devoid of melanocytes may occur, thereby accounting for the spotted coats of the affected animals. This hypothesis that ET-3 is a mitogen that is required by pre-migratory (or earlymigrating) crest cells has been supported by the demonstration that ET-3 actually does stimulate the proliferation of avian cells cultured from the pre-migratory neural crest (Lahav et al., 1996). These cells are induced by the addition of ET-3 in vitro to proliferate massively and, after having done so, to go on to develop primarily as melanocytes. These data thus only partially support the hypothesis that the production of a critical mass of crest-derived precursors depends on stimulation by ET-3. The hypothesis works well when applied to the skin, but the apparent action of ET-3 to skew development toward the generation of melanocytes is not compatible with the production of large number of enteric neuronal precursors; therefore, to explain why a loss of stimulation by ET-3, should deprive the gut of neuronal precursors, one would have to postulate that the action of ET-3 on pre-migratory crest cells in vivo is different from the effect that has been observed in vitro. That presumption could be valid, but there is, as yet no evidence to support it. In any case, if one assumes that the action of ET-3 on crest cells in vivo is different from that which it exerts in vitro, then the in vitro data cannot be used to support any hypotheses about the development of crest cells in the gut. The crest-derived cell population that colonises the bowel does not contain any cells with a melanogenic potential (Rothman et al., 1990, 1993) and is itself a proliferating population (Teitelman et al., 1981; Baetge and Gershon, 1989; Baetge, Pintar and Gershon, 1990). The ability of crestderived cells to proliferate as they migrate would argue that the putative effect of ET-3 as a mitogen is more likely to be manifest within the gut than in the pre-migratory crest. In this location, moreover, the loss of the option to develop as melanocytes might permit ET-3 to activate mitosis without pushing the crest cells counterproductively to develop along a melanocytic lineage. Crest-derived cells from the bowel are not identical to their predecessors in the pre-migratory crest. Relative to the pre-migratory crest, the developmental potential of


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intra-enteric crest-derived cells has been reduced (Rothman et al., 1990; Lo and Anderson, 1995), enteric crest-derived cells respond differently from pre-migratory crest cells to NT-3 (Kalcheim, Carmeli and Rosenthal, 1992; Pinco et al., 1993; Chalazonitis et al., 1994), and the two cells express different cell surface proteins (Pomeranz et al., 1991). Crestderived cells immunoselected from the gut have recently also been found to respond differently than pre-migratory crest cells to ET-3. Whereas ET-3 is a mitogen for premigratory crest cells that increases the number of melanocytes differentiating in vitro (Lahav et al., 1996), the in vitro action of ET-3, when it is applied to enteric crest-derived cells, immunoselected from the developing murine bowel with antibodies to p75NTR, is actually to inhibit the generation of neurons (Wu et al., 1999). Exposure of these cultured crest-derived cells to ET-3 or to other ETB agonists decreases the number of neurons that arise in vitro. The addition of an ETB antagonist to the medium, moreover, does not have any visible effect at all on the differentiation of neurons, suggesting that neuronal differentiation in vitro cannot be influenced significantly by an autocrine effect of ET-3 on ETB receptors on crest-derived cells. Such an effect should be inhibited by an ETB antagonist. Since ET-3 inhibits the formation of enteric neurons from crest-derived precursors, it seems paradoxical that the absence of ET-3 or ETB is associated with aganglionosis of the terminal colon. The loss of an inhibitor of neuronal differentiation would seem at first glance to be an unlikely cause of the failure of neurons to develop in a given region of the bowel. The crest-derived precursors of neurons, however, are migratory, while neurons are sedentary cells. As a result, the differentiation of crest-derived precursors should also mean that neurogenic cells stop migrating. The implication of this consideration is that timing of neuronal differentiation is probably very important in the colonisation of the bowel by crest-derived cells. If neurons differentiate too soon, then the pool of migrating neurogenic cells will become depleted. Should this occur, then the bowel distal to the point where neurons differentiate prematurely will not become colonised by crest-derived precursors. ET-3 might thus be required to inhibit the premature development of crestderived cells as neurons and glia and to sustain them in a migratory (possibly still proliferating) state. This is a speculative hypothesis; however, the observation that ET-3 affects the development of enteric crest-derived cells in vitro, implies that crest-derived cells must express ETB receptors and be ET-3 responsive, even if the effect of ET-3 on them is not what it was first thought likely to be. The fact that ET-3 affects crest-derived cells, however, does not mean that these are the only cells in the bowel upon which ET-3 acts. THE AGANGLIONOSIS THAT ARISES IN ET-3 OR ETB-DEFICIENT ANIMALS IS NOT NEURAL CREST AUTONOMOUS Aganglionosis of the terminal colon could be induced if the absence of ET-3 or the loss of the ETB receptor were to make the enteric microenvironment of this region of the bowel inhospitable for colonisation by crest-derived cells (Kapur, Yost and Palmiter, 1993; Rothman, Goldowitz and Gershon, 1993; Coventry et al., 1994; Kapur et al., 1995). Expression of the ETB receptor by non-neuronal cells of the bowel wall might thus, as noted previously, be an indirect contributor to aganglionosis. Alternatively, crest-derived cells may secrete a factor in response to ET-3 that causes non-neuronal cells of the terminal colon to make the enteric microenvironment amenable for colonisation by émigrés

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from the neural crest. The advancing wave of crest-derived pioneers migrating to and within the developing gut cannot be recognised morphologically, but the wave front can be detected indirectly by explanting the bowel and determining whether neurons develop in vitro (Rothman and Gershon, 1982, 1984; Rothman, Tennyson and Gershon, 1986). Neurons will arise in explants of the normal murine terminal colon removed from fetuses after stage 33 (Jacobs-Cohen et al., 1987). In contrast, cultures prepared from the terminal 2 mm of an ET-3 deficient (ls/ls) gut never contain neurons, no matter when they are explanted (Rothman and Gershon, 1984; Rothman, Tennyson and Gershon, 1986). The final segment of the ls/ls bowel, therefore, is presumptively aganglionic, suggesting that viable crest-derived cells may never enter this region. Crest-derived cells from a variety of co-cultured sources, including the ganglion-containing proximal gut of ls/ls mice, can enter explants of terminal bowel from control mice and give rise to neurons in vitro; however, in similar co-culture experiments, no source of crest-derived cells can colonise an ls/ls terminal colon (Jacobs-Cohen et al., 1987); the ls/ls colon also lacks the normal ability of the bowel, when co-cultured with crest cells, to promote their expression of gut-appropriate phenotypes (Coulter, Gershon and Rothman, 1988). These observations suggest that the wall of the terminal colon, not just crest-derived cells, is abnormal in mice that lack ET-3. The observations that normal crest-derived cells will not enter the presumptive aganglionic region of the ls/ls colon are inconsistent with the hypothesis that a deficiency of ET-3 causes aganglionosis only because ET-3 is an autocrine factor that crest-derived cells require. On the one hand, ls/ls crest-derived cells are unable to produce active ET-3, but they can give rise to neurons if they are allowed to colonise a normal colon. Moreover, wild-type crest-derived cells should be capable of synthesising active ET-3, but they cannot colonise the ls/ls colon. There may be additional sources of ET-3 in the colon and the crest-derived cells may not be the only cells in the wall of the colon that express ETB. Both neurogenic cells and non-neuronal cells could express ETB and each could play roles in enabling crest-derived cells to complete the colonisation of the bowel. The idea that ET-3 affects non-neuronal cells of the gut wall has been supported by data derived from studies of chimeric animals. For example, aganglionosis of the terminal colon does not develop in ls/ls-C3H aggregation chimeric mice, as long as some of the cells in the bowel wall are C3H; moreover, ls/ls neurons, identified with an endogenous marker (3-glucuronidase activity), are found throughout the colon of the chimeric animals (Rothman, Goldowitz and Gershon, 1993). Similarly, mutant neurons, marked by the expression of the DBH-lacZ transgene, develop in the terminal colon of aggregation chimeras constructed between wild-type and either ls/ls (Kapur, Yost and Palmiter, 1993; Coventry et al., 1994) or sl/sl embryos (Kapur et al., 1995). While it is conceivable, in chimeric embryos, that the autocrine secretion of ET-3 by their normal crest-derived neighbours, might rescue ET-3-deficient cells, it is inconceivable that such an effect could rescue crest-derived cells that do not express ETB. Since the cells of ls/ls mice lack ET-3, but not ETB, they might thus respond to ET-3 supplied by nearby wild-type cells in chimeric embryos. In contrast sl/sl cells lack ETB (Hosoda et al., 1994); nevertheless, they too colonise the terminal colon in chimeric mice. Cells that lack ETB should not be able to respond to ET-3 and thus are not amenable to rescue by nearby normal cells. The simple autocrine model, therefore, in which crest-derived cells are both the source and


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target of ET-3, does not explain the development of aganglionosis in animals that lack ET-3 or ETB. If ET-3 is not an essential autocrine growth factor, why are enteric neurons missing from the terminal colon when either ET-3 or ETB are absent? It has been proposed that there are critical cells that are “downstream” from the cells that express ETB and that these “downstream” cells are required for the terminal gut to become colonised by crest-derived cells (Kapur et al., 1995). This hypothesis thus invokes the participation by another cell in the process by which crest-derived cells colonise the colon, thereby accounting for why the defect in ETB is not crest autonomous. In a chimeric embryo, the normal crest-derived cells presumably respond to ET-3 and signal the “downstream” cells, which then allow both normal crest-derived cells and their ET-3- or ETB-deficient neighbors to develop in the terminal colon. The hypothesis supposes, therefore, that an intercellular conversation is initiated by ET-3. An alternative hypothesis, which also invokes the participation of another cell, is less complex. This alternative possibility considers that the additional, nonneuronal cell, itself expresses ETB and is ET-3 responsive. Both hypotheses thus postulate a role for a non-neuronal cell in making it possible for the colon to be colonised by crestderived neuronal precursors; however, the first idea sees ET-3 as stimulating only the crest-derived cells, while the second idea dispenses with the intercellular conversation and assumes that the non-neuronal cell responds directly to ET-3. These ideas can each be tested. The location of ETB is important in these evaluations. This idea that the colon itself is abnormal in animals lacking ET-3 is supported by observations on the progression down the gut of vagal crest-derived cells, visualised by their expression of the DBH-LacZ transgene. The progression of these cells in ls/ls mice is entirely normal until the cells reach the colon. As soon as the cells cross the ileo-cecal junction, however, the migration of vagal crest-derived cells becomes abnormal and remains so until migration ceases short of the end of the colon (Kapur, Yost and Palmiter, 1992; Coventry et al., 1994). The ability of crest-derived cells to migrate in ET-3 deficient ls/ls mice is thus normal until the cells become exposed to the microenvironment of the colon, which by implication must itself be abnormal. The suggestion that the colonic microenvironment is intrinsically abnormal has been confirmed by experiments in which segments of wild-type or ls/ls mouse colon were back-transplanted into neural crest migration pathways of quail embryos (Rothman, Goldowitz and Gershon, 1993). The grafts each survive well; however, the behaviour of the quail crest-derived cells is different depending upon which of the two grafts they encounter (Figure 11.5). When quail crest-derived cells encounter a wild-type colon, they enter the grafts and/or pass on through (Figure 11.5A). The normal mouse colon thus does not obstruct the migration of cells from the quail crest. In marked contrast, quail crest-derived cells do not enteric backgrafts of ls/ls colon (Figure 11.5B). Instead, they form large ganglia just proximal to, but outside, the mouse gut. The quail crest-derived cells in these experiments are normal and thus should not be deficient either in ET-3 or in ETB. Their failure to enter the ls/ls colon, therefore cannot be explained by their loss of an essential autocrine growth factor. Rather, the absence of ET-3 in the mouse gut would appear to have prevented its colonisation by the presumably normal crest-derived cells of the quail. The simplest explanation of these data is that the absence of ET-3, which is confined in a transplantation experiment to the grafted tissue, affects non-neuronal elements of the bowel wall, causing them to render the

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Figure 11.5 Quail crest-derived cells can migrate into backtransplanted segments of wild-type, but not ls/ls fetal mouse gut. (A) Quail crest-derived cells, identified by demonstrating HNK-1 immunoreactivity, migrate through the control mouse gut. Mouse cells can be distinguished from those of the quail, by their more scattered and abundant nuclear heterochromatin (Feulgen stain). (B) Quail cells fail to enter the ls/ls colon. Instead they form an immense ganglion at the border of the murine tissue.

bowel resistant to an influx of cells from the neural crest. The absence of active ET-3 in the ls/ls colon evidently makes the environment of the colon one that crest-derived cells do not enter. In summary, crest-derived cells appear to be capable of colonising the gut and giving rise to enteric neurons, whether or not they produce ET-3 or respond to ET B; however, the colon becomes abnormal in the absence of ET-3/ETB stimulation and resistant to colonisation by crest-derived cells, whether or not these crest-derived cells produce ET-3 or express ETB. In fact, abnormalities of smooth muscle-produced basal laminae have been described, both in the colon of ls/ls mice, and in human patients with Hirschsprung’s disease (see below).

BASAL LAMINAE ARE ABNORMAL IN THE COLON OF ls/ls MICE Extracellular matrix abnormalities occur in the aganglionic colon of ls/ls mice (Payette et al., 1988; Tennyson et al., 1990; Rothman et al., 1996). Similar abnormalities have


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also been observed in human patients with Hirschsprung’s disease (Parikh et al., 1992; Parikh et al., 1995). All of these matrix defects include an overabundance and/or maldistribution of molecules that are normally found in basal laminae. Among the molecules that accumulate in the aganglionic colon are laminin, type IV collagen, nidogen, nonsulfated glycosaminoglycans, and proteoglycans. Although the mucosal basal lamina is thicker than normal in the developing colon of ls/ls mice, the most striking abnormality is in the diffuse distribution of these molecules in the mesenchyme of the colon and the surrounding pelvis (Payette et al., 1988; Tennyson et al., 1990; Rothman et al., 1996). This distribution places the accumulated matrix molecules directly in the migratory pathways followed by vagal crest-derived cells within the bowel (Tucker, Ciment and Thiery, 1986) and by sacral crest-derived cells within the pelvis (Pomeranz and Gershon, 1990). Contacts of HNK-1 immunoreactive crest-derived cells migrating in the enteric mesenchyme with laminin-immunoreactive tufts of electron opaque material have been demonstrated by double label electron microscopic immunocytochemistry (Pomeranz et al., 1991). The excessive accumulation of laminin and type IV collagen appears to arise in the ls/ls mouse hindgut before (Payette et al., 1988) crest-derived cells would be expected to colonise the terminal bowel (Jacobs-Cohen et al., 1987). This timing and the location of the matrix abnormalities make it possible that the abnormal extracellular matrix is causally related to the development of aganglionosis. If so, then the accumulation of basal lamina molecules would have to occur independently of the failure of crest-derived cells to develop in the colon. This independence is suggested by the timing of the appearance of the matrix abnormalities and the arrival of crest-derived cells, but the timing alone does not rule out the possibility that the matrix becomes abnormal because crest-derived cells are absent from the terminal bowel of ls/ls mice. Crestderived cells, for example, might secrete a factor that regulates the secretion of matrix components in advance of their migratory front. Direct evidence of whether the matrix abnormalities are a direct or indirect consequence of the ET-3 deficiency in ls/ls mice is thus needed. The accumulation of laminin and type IV collagen in the ls/ls fetal colon has recently been shown to be associated with an increase in the abundance of transcripts encoding the molecules (Rothman et al., 1996). Quantitative Northern analysis has revealed that mRNA encoding the β1 and γ1 chains of laminin, as well as that encoding the αl and α2 chains of type IV collagen, is increased in the ls/ls colon. Messenger RNA encoding laminin α1 is also increased, but because of its relatively low abundance, the increase in laminin αl mRNA had to be demonstrated by reverse transcription in combination with the competitive polymerase chain reaction (RT-cPCR), rather than by Northern analysis. The relative abundance of mRNA encoding laminin α1 is greatest at E11 and then declines as a function of age, until it stabilises at a very low level that is maintained in adult life. The production of laminin αl, therefore, is developmentally regulated. The laminin-1 isoform (α1-β1-γ1) thus is associated with development and is present in the fetal bowel while enteric ganglia are in the process of formation. The age-related decline in the abundance of laminin α1 transcripts occurs in the fetal colon of both wildtype and ls/ls mice; nevertheless, laminin α1 mRNA is significantly more abundant in the ls/ls fetal colon than in that of wild-type animals throughout development. Prior to day E15, cells that synthesise laminin α1 and β1 and those that produce the α2 chain of

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collagen type IV have each been found, by in situ hybridisation, to be most concentrated in the endodermal epithelium. At later ages, however, cells that contain mRNA encoding these molecules are more abundant in the outer gut mesenchyme than in the epithelium, and more of this mRNA is found in the ls/ls than in the wild-type colonic and pelvic mesenchyme. The expression of laminin α1 in ls/ls and wild-type animals has been compared to that in age-matched E15 and newborn c-ret knockout mice (Rothman et al., 1996). As noted above, the entire gut is aganglionic below the oesophagus and proximal stomach of c-ret knockout mice (Figure 11.3); however, this pan-intestinal aganglionosis occurs because enteric neural and glial precursor cells are GDNF/Ret-dependent (Schuchardt et al., 1994; Durbec et al., 1996b; Jing et al., 1996; Sanchez et al., 1996; Trupp et al., 1996), not because of an abnormality in ET-3, the mutation that gives rise to the colonic aganglionosis in ls/ls mice (Baynash et al., 1994). If the increase in laminin αl mRNA that characterises the aganglionic colon of ls/ls is due to the absence of crest-derived cells, then the same defect should occur in the aganglionic gut of c-ret knockout mice. In contrast, this increase in mRNA should not be seen in the intestine of c-ret knockout mice if the defect in ls/ls is caused by the loss of an effect of ET-3 on non-neuronal cells of the colonic and pelvic mesenchyme. The abundance of mRNA encoding laminin αl was found to be the same in the intestines of wild-type and c-ret knockout mice both at E15 or in newborn animals (Rothman et al., 1996); moreover, the amount of laminin immunoreactivity in the colon of E15 and newborn c-ret knockout mice cannot be distinguished from that in agematched controls. These results suggest that the abnormal extracellular matrix of the ls/ls mouse colon is a primary effect of the ET-3 deficiency in these animals, and is not a secondary consequence of the absence of crest-derived cells from the ls/ls colon. The ls/ls defect probably includes an overabundance of the laminin-1 isoform, because the αl, β1, and γ1 subunit are all present in excess in the ls/ls mouse colon. The excess of laminin-1 occurs in the pelvic mesenchyme as well as in the bowel. An overabundance of this molecule, therefore, is present in the paths of each of the populations of crest-derived cell, vagal and sacral, that colonise the colon. The location of the extracellular matrix defect, in ls/ls mice is thus compatible with the idea that the matrix abnormalities are causally related to the pathogenesis of aganglionosis. The extracellular matrix in some regions of the embryo normally inhibits the migration of crest cells, which thus tend to avoid these locations. Matrix molecules that inhibit crest cell migration have been found between the ectoderm and the somites (Erickson, Duong and Tosney, 1992; Oakley et al., 1994), which coincides with the dorsolateral pathway of neural crest migration, the posterior sclerotome (Rickmann, Fawcett and Keynes, 1985; Norris, Stern and Keynes, 1989; Keynes et al., 1990), and the perinotochordal mesenchyme (Pettway, Guillory and Bronner-Fraser, 1990). Basal lamina components, however, have not been observed to accumulate in any of these regions (Norris, Stern and Keynes, 1989; Keynes et al., 1990; Oakley and Tosney, 1991; Oakley et al., 1994). The extracellular matrix in the zones that are normally avoided by migrating crest cells moreover, differs from that found in the aganglionic ls/ls (Payette et al., 1987) or Hirschsprung’s bowel (Holschneider et al., 1994; Miura et al., 1996) in that the regions that normally exclude crest-derived cells also inhibit the outgrowth of axons (Oakley and Tosney, 1991). In contrast, the aganglionic bowel is heavily innervated, both by axons of neurons from the more rostral


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hypoganglionic gut and from extrinsic ganglia (Payette et al., 1987). The enteric aganglionosis, therefore, extends only to crest-derived cells, and does not include their axons. Crest cells normally adhere well to laminin (Lallier and Bronner-Fraser, 1991; Lallier et al., 1994); moreover, the migration of cells away from the neural crest is promoted by laminin-1 (Bilozur and Hay, 1988; Perris, Paulsson and Bronner-Fraser, 1989). The migration of cranial crest cells, furthermore, can be inhibited by the in vivo administration of antibodies to integrins, which antagonise the attachment of crest-derived cells to laminin (Bronner-Fraser, 1985, 1986), as well as by antibodies that recognise the complex formed in situ between laminin and proteoglycans (Bronner-Fraser and Lallier 1988). An overabundance of laminin, therefore, such as that which occurs in the ls/ls colon, should provide an excellent adhesive substrate for the crest-derived cells that colonise the bowel. Certainly, since laminin promotes neurite extension and axonal growth (Manthorpe et al., 1983; Calof and Reichardt, 1985; Lander, Fujii and Reichardt, 1985; Engvall et al., 1986; Kleinman et al., 1988; Liesi et al., 1989) the abundance of laminin-1 in the aganglionic colon of ls/ls mice and human patients with Hirschsprung’s disease probably accounts for the observations that the aganglionic tissue is very well supplied by the axons of neurons the cell bodies of which lie outside the aganglionic zone. The association of a laminin-rich substrate with aganglionosis, however, seems, at least superficially, counter-intuitive. If laminin interferes with the colonisation of the gut, then the response to laminin of crest-derived cells in the bowel would have to be different from those of pre-migratory crest cells, migrating crest cells that have not yet reached the target organ, or axons.

LAMININ-1 PROMOTES THE DEVELOPMENT OF ENTERIC NEURONS Extracellular matrix molecules are biologically active and can affect the terminally differentiated phenotypes expressed by the progeny of neural crest stem cells in vitro (Stemple and Anderson, 1992). The extracellular matrix, therefore, is not just the framework to which migrating crest-derived cells adhere; it is also a source of signalling information and is probably capable of influencing the fate of the crest-derived cells that differentiate in contact with it. Specifically, with regard to the matrix, one of the molecules that accumulates in the aganglionic colon, laminin-1, has been found to promote the in vitro development of cells that express neuronal markers, such as peripherin, neurofilament proteins, neuron specific enolase, or PGP9.5 (Gershon et al., 1993; Pomeranz et al., 1993; Tennyson et al., 1995; Chalazonitis et al., 1997). The promotion of enteric neuronal development by laminin-1 was first demonstrated by using crestderived cells immunoselected from the embryonic avian or fetal rat gut with antibodies to HNK-1. A similar effect is seen when cells are immunoselected from the mouse gut (Chalazonitis et al., 1992, 1997; Gershon et al., 1993; Tennyson et al., 1995) with antibodies to a cell-surface laminin-binding protein, known a LBP110 (Douville, Harvey and Carbonetto, 1988; Kleinman et al., 1988; Kleinman and Weeks, 1989; Jucker et al., 1991).

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AN IKVAV SEQUENCE OF THE α1 SUBUNIT OF LAMININ-1 IS RESPONSIBLE FOR THE PROMOTION OF ENTERIC NEURONAL DEVELOPMENT LBP110 is a non-integrin laminin-binding protein that is similar to a β-amyloid precursor protein (Kibbey et al., 1993). The amino acid sequence, isoleucine-lysine-valine-alaninevaline (IKVAV), of the domain of laminin that binds to LBP110 is located on the laminin α1 chain, near its globular C-terminal end (Sephel et al., 1989a,b; Kleinman et al., 1991). PC12 cells express LBP110 and this expression can be down regulated by transfecting the cells with an antisense amyloid precursor protein cDNA (Kleinman et al., 1991; Kibbey et al., 1993). This antisense treatment inhibits NGF-induced neurite extension on a laminin-1 substrate. These observations have led Hynda Kleinman and her colleagues to suggest that LBP110 is a receptor for laminin-1, which mediates the effects of laminin-1 on neurite outgrowth (Kleinman et al., 1991; Kibbey et al., 1993, 1994). Kleinman and others have also implicated LBP110 as a laminin receptor that mediates many of the responses of non-neuronal cells to laminin (Haralabopoulos et al., 1994; Weeks et al., 1994; Bresalier et al., 1995; Corcoran et al., 1995; Nomizu et al., 1995). Crest-derived cells are the only ones in the developing bowel that express LBP110, which is thus co-localised in the fetal gut with crest markers (Pomeranz et al., 1991); moreover, neurons or glia preferentially differentiate in cultures of cells immunoselected from the fetal mouse gut with antibodies to LBP110 (Figure 11.6A) (Pomeranz et al., 1993). A synthetic peptide that contains the IKVAV sequence (IKVAV-peptide) of the domain of laminin α1 that binds to LBP110, competitively blocks the promotion of enteric neuronal and glial development in vitro by laminin-1 (Figure 11.6B) (Chalazonitis et al., 1992, 1997; Gershon et al., 1993). A nonsense peptide, a peptide with the same amino acids in a different sequence, or a peptide with a sequence found elsewhere in the laminin-1 molecule all fail to influence the response of enteric crest-derived cells to laminin-1. The development of enteric neurons and glia is not affected by the IKVAV-peptide when the crest-derived cells are cultured on poly-D-lysine or fibronectin. The action of the IKVAV-peptide, therefore, is both sequence-specific and laminin-specific. The IKVAVpeptide, furthermore, affects only the differentiation of crest-derived cells and does not reduce the total number of cells in culture. This observation suggests that the IKVAVpeptide does not interfere with the adhesion of cells to laminin-1. It seems likely that adhesion depends more on the binding of laminin by plasmalemmal integrins (BronnerFraser, 1985, 1986), than LBP110 (Kleinman et al., 1991). Antibodies to laminin αl, like the IKVAV-peptide, inhibit the ability of laminin-1 to promote the development of neurons (Chalazonitis et al., 1997). This effect of antibodies to laminin α1 is not shared either by pre-immune sera, nor is it shared by antibodies to the β1 chain of laminin. Neither the antibodies to laminin α1, nor those to laminin β1, cause cells to detach from a laminin-1 containing substrate. The observation that the IKVAV-peptide does not reduce the total number of cells in culture suggests that it does not block the adhesion of most of the cells in culture. The possibility remains, however, that the adherence of a neurogenic subset of crest-derived cells, the size of which is negligible in comparison to the total number of cells in cultures, is inhibited by the IKVAV-peptide. To investigate this possibility, laminin-1 was added in


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Figure 11.6 The ability of laminin-1 to promote the development of enteric neurons in vitro is blocked by an IKVAV-peptide. Crest-derived cells were immunoselected from the fetal murine bowel with antibodies to p75NTR and cultured on a substrate of poly-D-lysine. The presence of neurons in the cultures is demonstrated by immunostaining with antibodies to PGP9.5. (A) The development of neurons is stimulated by the addition of soluble laminin. (B) When laminin is added, together with an IKVAV-peptide, the development of neurons is inhibited.

soluble form to cells that had previously been allowed to adhere for 24 h to poly-D-lysine (Chalazonitis et al., 1992, 1997; Gershon et al., 1993; Tennyson et al., 1995). When this was done, laminin-1 was equally effective in promoting the differentiation of neurons and glia as when it was used as a component of the substrate upon which cells were plated. This experimental paradigm rules out the explanation that laminin-1 promotes neuronal development because it selectively permits the adherence of cells able to differentiate as neurons. The observation, however, that because soluble laminin-1 is efficacious, does not establish that soluble laminin-1 is necessarily able to stimulate the receptors that promote enteric neuronal development. Soluble laminin-1might have to bind to the substrate before it can activate receptors on cell surfaces. Such a requirement could explain the observation that the IKVAV-peptide is a pure antagonist, and not an agonist at the LBP110 site. Still, whether laminin-1 activates LBP110 as a bound or soluble molecule, the fact that it is effective in promoting neuronal differentiation many hours after cells have become adherent indicates that promotion of neuronal development by laminin-1

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is due to an effect on differentiation and not adherence. In this sense, therefore, laminin-1 is a growth factor for enteric neurons. As is also the case when crest-derived cells are exposed to other growth factors, a rapid, but transient induction of the expression of the c-fos proto-oncogene accompanies the response of immunoselected crest-derived cells to laminin-1 (Chalazonitis et al., 1992, 1997; Gershon et al., 1993; Tennyson et al., 1995). Expression of c-fos becomes apparent within one hour of the addition of laminin-1, but after 24 h c-fos expression can no longer be detected. The laminin-1-induced expression of c-fos, like laminin-1-promoted neuronal and glial development, is antagonised by the IKVAV-peptide, but not by control peptides. These observations suggest that both of these responses are LBP110-mediated. Since the IKVAV-peptide is an antagonist and not an agonist, it seems likely that the binding of the IKVAV domain to laminin-1 is necessary but not sufficient to activate the LBP110 receptor. LAMININ-1 EXERTS DIFFERENT EFFECTS ON PRIMARY CREST CELLS AND CREST-DERIVED CELLS IMMUNOSELECTED FROM THE FETAL BOWEL Pre-migratory and early-migrating crest cells express integrins and thus attach to laminin (Bronner-Fraser, 1986; Bilozur and Hay, 1988; Bronner-Fraser and Lallier, 1988; Lallier and Bronner-Fraser, 1991; Lallier et al., 1994), which these cells encounter in the embryonic mesenchyme and when they come into contact with basal laminae (Martins-Green and Erickson, 1987; Erickson, Loring and Lester, 1989; Pomeranz and Gershon, 1990; Pomeranz et al., 1991). In contrast to crest-derived cells immunoselected from the fetal gut, pre-migratory and early-migrating crest cells do not express LBP110 (Pomeranz et al., 1991). This receptor is not expressed by the crest-derived cells that colonise the bowel until after these cells enter the gut. Neural crest stem cells do not express LBP110 and exposure of these cells to laminin-1 does not induce them to differentiate as neurons (Stemple and Anderson, 1992, 1993). In fact, laminin stimulates the migration of crest cells, not their differentiation (Bilozur and Hay, 1988). The ability of crest-derived neuronal precursors to respond to laminin-1 must thus be a characteristic the cells acquire, either while migrating to the bowel, or after they enter it. It is likely that the difference between the responses to laminin-1 of neural crest stem cells on the one hand, and their enteric crest-derived successors on the other, can be accounted for by the failure of the stem cells to express LBP110 and the acquisition of LBP110 by the crest-derived neuronal precursors in the gut. The absence of LBP110 from crest-derived cells migrating to the bowel is probably important in enabling these cells to reach their destinations. Clearly, if laminin were to induce crest cells to differentiate as neurons as soon as such cells encountered it, differentiation would occur too soon and the bowel would never become colonised. Neurons do not migrate, their precursors do. Differentiation of crest-derived cells as neurons must thus be postponed until after the bowel has been colonised. The delay in LBP110 expression, therefore, means that enteric neuronal precursors can adhere to laminin-1 while they are migrating to the gut, without being induced by laminin α1 to turn into neurons before they reach the bowel. Within the gut, moreover, crest-derived cells


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acquire LBP110 asynchronously. Presumably, the vagal crest-derived émigrés that express LBP110 as soon as they enter the proximal bowel are the precursors of neurons in foregut ganglia. Those that acquire LBP110 later are likely to be able to move more distally and differentiate into neurons in the midgut and eventually the hindgut (Pomeranz et al., 1991). The asynchrony in the timing of LBP110 expression within the bowel may thus make it possible for a subset of vagal crest-derived cells to delay differentiating and continue to migrate until they reach the terminal bowel. AN EXCESS OF LAMININ-1 IN THE COLON MAY CAUSE NEURONS TO DIFFERENTIATE PREMATURELY The seemingly paradoxical association of an overabundance of laminin-1 with aganglionosis in the terminal colon of mice that lack ET-3 and human patients with Hirschsprung’s disease may be causally related to the stimulation of the LBP110 on crest-derived cells by the excess laminin-1. Since the IKVAV domain of the laminin α1 subunit of laminin-1, acting on crest-derived cells that have acquired LBP110, would be expected to promote their differentiation as neurons, the laminin-1 rich environment of the ET-3-deficient colon and pelvis may provoke premature neuronal differentiation. ET-3, moreover, is itself an inhibitor of neuronal differentiation. The crest-derived cells migrating to and within the ET-3-deficient gut, therefore, are subjected simultaneously to positive and negative influences that are likely to combine to provide these cells with a very strong stimulus to differentiate along a neuronal pathway. Because ET-3 is absent, the crestderived cells are deprived of a factor that inhibits them from differentiating as neurons, while at the same time they encounter an environment rich in laminin-1 that stimulates them to do so. If crest-derived cells respond to these strong influences to differentiate as neurons before they have completed their task of colonising the bowel, the terminal colon, which is the last part of the gut to be colonised, will become aganglionic. A congenital deficiency of ET-3 or its receptor, ETB, may thus prevent the formation of ganglia in the terminal bowel by exerting direct and indirect effects, which synergise to promote neuronal differentiation at the expense of migration and proliferation. Since the overabundance of laminin α1 is present throughout the ls/ls colon (Rothman et al., 1996), this hypothesis predicts that the progression of the vagal crest-derived cells down the bowel of ls/ls mice would become abnormal for the first time when these cells enter the proximal colon. This prediction has been confirmed. The descent of crest-derived cells has been mapped in ls/ls mice that express the DBH-lacZ transgene (Coventry et al., 1994). The migration of these cells appears to be comparable in ls/ls and wild-type animals until the crest-derived cells cross the ileo-cecal junction. As soon as the cells enter the ls/ls colon, the rate at which they descend becomes slower and their progress become erratic. Laminin-1 accumulates not only in the colon of ls/ls mice, but also in the pelvic mesenchyme that surrounds it. The hypothesis that the excess of laminin-1 and the ET-3 deficiency of ls/ls mice combine to induce premature neuronal differentiation thus also predicts that sacral crest-derived precursors would stop short of the gut (Payette et al., 1988). Ectopic ganglia have been found outside the terminal bowel, in the pelvis of ls/ls mice (Rothman and Gershon, 1984; Payette et al., 1987). These ganglia actually fuse with myenteric ganglia in the hypoganglionic zone of the ls/ls colon. To do so, the ectopic ganglia have to penetrate through the

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longitudinal muscle layer to form odd structures that are half in the bowel and half out. The ectopic ganglia, which are not present in wild-type mice, are likely to be sacral crestderived cells that have differentiated before reaching the colon. A new hypothesis to explain the aganglionosis of ET-3 or ETB deficiency is that the genetic defect causes the developmental regulation of the secretion of components of basal laminae to become delayed in the colon. This delay leads to an excess of laminin-1 in the matrix through which the crest-derived cells that colonise the colon must migrate. This excess drives inadequately resistant crest-derived progenitors to differentiate before reaching the terminal bowel. The hypothesis accounts for the fact that the aganglionosis of ET-3 deficiency is not neural crest autonomous.

ET-3 AFFECTS BOTH CREST-DERIVED AND NON-NEURONAL CELLS OF THE COLON Both crest-derived and non-crest-derived cells appear to be targets for ET-3 in the colon. Laminin is known, in the gut of normal mice, to be produced by epithelial cells and by one or more cells of the enteric mesenchyme (Simo et al., 1991). In situ hybridisation has revealed that mRNA transcripts encoding subunits of laminin are predominantly found in the epithelium early in development (Rothman et al., 1996). Later, however, about the time that ganglia begin to form, synthesis of these subunits switches to the mesenchyme. The locations of transcripts encoding subunits of lamini-1 and those encoding collagen IV are qualitatively similar in control and ls/ls mice, but quantitatively different. Messenger RNA encoding each of these components of basal lamina is more concentrated in the enteric mesenchyme of fetal ls/ls than in that of wild-type mice. Crest-derived cells are thus not the source of laminin-1 and are not responsible for its overproduction in the ls/ls bowel. The overproduction of laminin-1 that occurs in the aganglionic bowel of ls/ls mice does not occur in the aganglionic gut of c-ret knockout mice. The overproduction of laminin-1 in the ls/ls animals is thus explained most straightforwardly by the hypothesis that ET-3 down-regulates the secretion of laminin-1 by pelvic mesenchymal cells, including those of the fetal bowel. This hypothesis that ET-3 downregulates laminin-1 secretion implies that fetal mesenchymal cells and/or epithelial cells express ETB. Precursors of smooth muscle, fibroblasts, and ICCs are all found in the fetal enteric mesenchyme. ETB has been demonstrated on smooth muscle cells in the mature large (Okabe et al., 1995) and small intestines (Yoshinaga et al., 1992). Both of which respond to ET-3. When these receptors are first acquired by developing smooth muscle cells is unknown. The totally aganglionic bowel of c-ret knockout mice contains transcripts of mRNA encoding ET-3 and those encoding ETB (J. Chen, T. Rothman and M. Gershon, unpublished data). This observation confirms that the biosynthesis of these molecules in the bowel is not confined to crest-derived cells. In situ hybridisation carried out in mice in which the crest-derived cells are marked by their expression of the DBH-lacZ transgene (R. Kapur and M. Yanagisawa, reported at the 1996 meeting of the American Motility Society) has shown that ETB mRNA is present both in lacZ-expressing and non-lacZ-expressing cells. The location of the lacZ-negative cells that contain ETB transcripts is close to presumptive ganglia. In this location, the cells


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could be ICCs or a subset of smooth muscle. This location is interesting, because ICCs have been found to be abnormal in patients with Hirschsprung’s disease (Yamataka et al., 1995; Vanderwinden et al., 1996).

INTERSTITIAL CELLS OF CAJAL ARE ABNORMAL IN THE AGANGLIONIC BOWEL OF PATIENTS WITH HIRSCHSPRUNG’S DISEASE The identity and origin of ICCs has been a contentious issue for a long time (Kobayashi et al., 1989; Sanders, 1996; Torihashi, Ward and Sanders, 1997). At one time, ICCs were thought to be fibroblasts (Cook and Burnstock, 1976). More recently, they have been postulated to be modified or primitive smooth muscle cells (Faussone-Pellegrini, 1985; Torihashi et al., 1993). This idea remains a current concept, but ICCs can be distinguished from banal smooth muscle, by their expression of, and dependence on, the c-kit protooncogene (Ward et al., 1994; Huizinga et al., 1995; Torihashi et al., 1995; Ward et al., 1995). This gene encodes a receptor tyrosine kinase, Kit, and is allelic with white spotting (W) (Besmer, 1991). The ligand for Kit has been given various names including Kit ligand (KL), stem cell factor and Steel factor. The gene encoding KL is allelic with steel (sl). A great deal of evidence indicates that Kit and KL are necessary for the development and/ or maintenance of ICCs. For example, both W (Ward et al., 1994; Huizinga et al., 1995) and sl (Ward et al., 1995) mutations inhibit the development of ICCs. Administration of antibodies that neutralise Kit is associated with the disappearance of ICCs from the mouse gut (Maeda et al., 1992; Torihashi et al., 1995). Finally, the in vitro development of Kit-expressing ICCs (Figures 11.1C, 11.7) is dependent on the presence of KL in the culture medium (Wu, Rothman and Gershon, 1996). Myogenic intestinal slow waves are impaired when the network of ICCs is lost or fails to develop (Maeda et al., 1992; Ward et al., 1994, 1995; Huizinga et al., 1995; Torihashi et al., 1995); therefore, ICCs are probably the pacemakers for these waves (Sanders, 1996). Intestinal motility becomes abnormal after the disruption of the ICC network causes slow waves to be lost and the bowel dilates in a manner that resembles that seen in aganglionosis. In the mature longitudinal muscle and elsewhere in the bowel wall during fetal life, ICCs and smooth muscle cells express markers in common (Torihashi, Ward and Sanders, 1997). Markers expressed by both ICCs and smooth muscle include the intermediate filament protein, desmin, and smooth muscle isoforms of actin and myosin. The crest-derived cell marker, Ret (Pachnis, Mankoo and Costantini, 1993; Schuchardt et al., 1994; Tsuzuki et al., 1995) is not expressed by ICCs (Torihashi, Ward and Sanders, 1997). These observations suggest that ICCs may be derived from a common smooth muscleICC progenitor. Studies of stably marked crest-derived cells in avian interspecies chimeras have also indicated that ICCs are mesenchymal derivatives and not cells of neural crest origin (Lecoin, Gabella and Le Douarin, 1996). The morphology of Kit-immunoreactive ICCs differs in various sites within the wall of the bowel (Figure 11.7) and may represent subtypes of ICC that diverge at different times from the putative common smooth muscleICC progenitor (Torihashi, Ward and Sanders, 1997). The evidently different subtypes of ICCs that surround myenteric ganglia and those that reside in the deep muscle plexus,

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Figure 11.7 ICCs develop in vitro and can be demonstrated in the wall of the gut with antibodies to Kit. Note that some of the cells are thin and orientated parallel to muscle fibers, while others are thicker and more random in their orientation.

circular, and longitudinal muscle layers have been proposed to constitute functionally distinct cell classes. When crest-derived and non-crest-derived cells are separated by immunoselection with antibodies to p75NTR, ICCs develop in the cultures of non-crestderived cells (Figure 11.1C) (Wu, Rothman and Gershon, 1996). The abnormality of ICCs in the aganglionic region of the bowel of patients with Hirschsprung’s disease (Yamataka et al., 1995; Vanderwinden et al., 1996) demonstrates that the genetic defect in these patients is not restricted to cells of neural crest origin. Despite the fact that the normal pattern of ICCs is disrupted and their number reduced in the aganglionic colon in Hirschsprung’s disease, at least some ICCs are still present. ICCs can also be found in the terminal colon of ls/ls mice and in the aganglionic intestine of c-ret knockout mice. Again, in both cases the abundance of ICCs is less than that in wildtype mice; moreover, the network of ICCs is abnormal in the mutant bowel (Wu, Rothman and Gershon, 1996). ICCs thus can develop in the absence of ET-3 (in the ls/ls gut) and also in the absence of neurons (in the c-ret knockout mouse). The reduced numbers and the disruption of the network of ICCs in the aganglionic bowel of patients with Hirschsprung’s disease and ls/ls mice, however, may be the secondary result of the absence of neurons. Neurons may thus influence the development of at least some ICCs. This possibility is supported by the observation, made by in situ hybridisation, that enteric neurons


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contain mRNA encoding KL (Torihashi, Ward and Sanders, 1997). Enteric neurons are probably, therefore, a source of KL. Since the isoform of KL that is physiologically active is membrane-bound (Miyazawa et al., 1995; Wehrle-Haller and Weston, 1995), neurons probably must make contact with the plasma membranes of ICCs to stimulate the Kit they express. A requirement for such cell-to-cell interaction might explain the close spatial relationship of one of the ICC subtypes to myenteric ganglia (Sanders, 1996). Stimulation of Kit on ICCs by KL expressed on the plasma membranes of neurons, therefore, could explain the ICC abnormalities associated with aganglionosis in ls/ls mice and patients with Hirschsprung’s disease. Since ICCs are not totally absent when the bowel is aganglionic, however, neurons must not be the only source of KL in the gut. In fact, mRNA encoding KL, as well as that encoding Kit, can be detected in the aganglionic bowel of c-ret knockout mice (J. Chen, T. Rothman and M. Gershon, unpublished data). Since ICCs develop in ls/ls mice, they clearly do not require ET-3 for their development; nevertheless, even if they do not need ET-3 to develop, they might still express ET B and be ET-3 responsive. The abnormal number and aberrant network of ICCs in the aganglionic Hirschsprung’s and ls/ls colon are consistent with this idea. The location of the non-neuronal cells in the DBH-lacZ mouse colon that contain ETB mRNA coincides with that of one subset of Kit-immunoreactive ICCs. ET-3 stimulates the development of smooth muscle in vitro (Wu et al., 1999). In the presence of ET-3 or an ETB agonist, the number of desmin-immunoreactive cells increases. ET-3 could thus promote the development of smooth muscle and/or ICCs from their common progenitor. Conceivably, this progenitor cell secretes more laminin-1 than does either of its successors. Laminin-1 is developmentally regulated (Rothman et al., 1996). The α1 chain decreases as a function of developmental age, while the β1 and γ1 subunits do not. Presumably, therefore, as laminin α1 declines it is replaced by another α subunit, such as α2. This putative shift in secretion from laminin-1 to laminin-2 would make the enteric mesenchyme far less likely to promote neurogenesis. If laminin-2 is secreted by mature smooth muscle and/or ICCs, then the shift from laminin-1 to laminin-2 secretion may reflect the development of smooth muscle and ICCs from their common progenitor, events that are likely to be promoted by ET-3. A delay in the development of smooth muscle and ICCs, resulting from the absence of ICCs, may thus prolong the secretion of laminin-1, causing excessive amounts of it to be present in the bowel at the time the colon is colonised by crest-derived cells.

DIFFERENT GENETIC ABNORMALITIES CAN GIVE RISE TO HIRSCHSPRUNG’S DISEASE Neuromuscular defects of the human gut are common perinatal problems. In addition to Hirschsprung’s disease, these disorders include a variety of related conditions such as hypoganglionosis, neuronal intestinal dysplasias (hyperganglionosis), ganglion cell immaturity, and dysganglionoses. Other neuromuscular defects that may involve ENS abnormalities include hypertrophic pyloric stenosis, volvulus, and intussusception. Hirschsprung’s disease itself is not uncommon and occurs in about 1/5000 live births (Angrist et al., 1995). A subset of patients with Hirschsprung’s disease have loss-of-function mutations in

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the RET proto-oncogene (Edery et al., 1994; Romeo et al., 1994; Angrist et al., 1995; Borrello et al., 1995; Pasini et al., 1995). These patients account for only a relatively small fraction of the total set of Hirschsprung’s disease (Edery et al., 1994; Romeo et al., 1994; Angrist et al., 1995). Identical RET abnormalities can be associated with either long or short segment diseases; moreover, some patients not only have aganglionosis of the colon, but may also exhibit multiple endocrine neoplasia type A (which is more commonly associated with gain-of-function mutations in RET), maternal deafness, talipes, and malrotation of the gut. Distinctly different phenotypes can thus arise from identical mutations in RET. The relationship between the RET genotype and the Hirschsprung’s phenotype is, therefore, not entirely obvious; furthermore, the relatively low frequency of RET mutations in Hirschsprung’s disease indicates that additional genetic and/or environmental conditions must explain the majority of cases. Mutations in ETB have also been associated with Hirschsprung’s disease (Puffenberger et al., 1994). Again, however, Hirschsprung’s disease can occur in patients who exhibit mutations in neither ETB nor RET and mutations can occur in these genes without necessarily giving rise to the Hirschsprung’s disease phenotype (Puffenberger et al., 1994). Mutations of genes encoding ET-3, have also recently been linked to some cases of Hirschsprung’s disease. In the ET-3-deficient patients, the phenotype resembles that of ls/ls mice. Hirschsprung’s disease occurs in a setting of cutaneous pigmentary abnormalities and is combined with a Waardenburg type 2 phenotype (Shah-Waardenburg syndrome) (Edery et al., 1996; Hofstra et al., 1996). Since a wide variety of mutations (many of which are still to be identified) predispose toward Hirschsprung’s disease, the condition is a multigene abnormality (Puffenberger et al., 1994; Angrist et al., 1995). The genetic abnormalities found in patients with Hirschsprung’s disease are not completely comparable to the related mutations in animals. For example, c-ret knockout causes aganglionosis in mice only when the mutated gene is homozygous (Schuchardt et al., 1994; Durbec et al., 1996b). Even mice that are doubly heterozygous for both c-ret and ls do not become aganglionic (T. Rothman, M. Gershon and F. Costantini, unpublished observations). In contrast, patients with Hirschsprung’s disease who have RET mutations have only been heterozygous. The background and environment thus affect the penetrance of the aganglionic phenotype when Ret is mutated. The ET-3/ETB-deficient mice would seem to be useful models for studying the pathogenesis of Hirschsprung’s disease. From both a genetic and an anatomical point of view, these models strikingly resemble Hirschsprung’s disease. The molecular abnormalities that have been found in the extracellular matrix of the ET-3 deficient ls/ls mice (Rothman et al., 1996) also occur in patients with Hirschsprung’s disease (Parikh et al., 1992, 1995). These include a massive increase in the thickness of the muscularis mucosa and the excessive secretion of laminin and type IV collagen. Fujimoto and colleagues have concluded that laminin and type IV collagen accumulate at the sites where ganglia will form before neurogenesis begins, suggesting that the promotion of the development of neurons by laminin may determine where ganglia arise (Fujimoto et al., 1989). In any case, the sum of observations made from studies of ls/ls (Rothman and Gershon, 1984; Jacobs-Cohen et al., 1987; Payette et al., 1988; Kapur, Yost and Palmiter, 1993; Rothman, Goldowitz and Gershon, 1993; Rothman et al.,1993; Coventry et al., 1994), sl/sl (Kapur et al., 1995), and Dom (Kapur et al., 1996) mice have all indicated that the murine aganglionosis involves an intrinsic


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abnormality of the colon in addition to, or instead of, defects of crest-derived neuronal precursors. It is thus highly likely that the pathogenesis of Hirschsprung’s disease will also be the result of abnormalities of both crest and non-crest-derived cells.

SUMMARY The ENS is a unique region of the PNS. It is larger, more complex, and more independent than any other. Its development must thus involve factors and/or mechanisms that are different from those which operate in the formation of extra-enteric ganglia. The ENS is formed by the progeny of immigrants that migrate to the bowel from vagal, rostral-most truncal, and sacral regions of the neural crest. Crest-derived progenitors are multipotent when they arrive in the gut, although their developmental potential is less than it was in the pre-migratory crest. Enteric crest-derived émigrés, for example, do not give rise to ectomesenchyme or melanocytes. Enteric crest-derived cells, however, can develop as sympathetic neurons or Schwann cells if they are experimentally induced to re-migrate and develop outside the bowel. The fate of crest-derived émigrés in the gut is thus powerfully influenced by the enteric microenvironment. The microenvironment does not, however, work alone. The effects exerted by the growth factors and matrix constituents of the bowel wall vary on different crest-derived cells because the receptors expressed by crest-derived cells also vary. The receptors expressed by crest-derived cells reflect the lineages and sublineages into which these cells have been sorted. Two such lineages have been identified. One, which gives rise to serotonergic neurons and other types of cell, is transiently catecholaminergic, born early, and mash-1-dependent. The other, which gives rise to CGRP-containing and other types of neuron, is never catecholaminergic born late, and mash-1-independent. Many signals that influence the differentiation and/or survival of enteric neurons have been identified, One is GDNF, which is the functional ligand that activates the Ret receptor. This factor acts early and is evidently required by the multipotent crest-derived cell that colonises the bowel distal to the oesophagus and most proximal stomach. A second factor is the neurotrophin, NT-3, which is additive with a cytokine that activates the CNTFRα, but which is not CNTF or LIF. The knockout of genes, such as those encoding GDNF or Ret that encode growth or transcription factors required by crestderived precursors while these cells are still multipotent produce large defects in the ENS. The knockout of genes, such as those encoding CNTFRα, which are required later in ontogeny, produce more limited neuronal abnormalities, although the defects may still be lethal if the cells that are lost, such as a motor neurons, are functionally vital. A different kind of abnormality, which involves the entire ENS, but in a restricted region, occurs in mice lacking ET-3 or ETB. The terminal colon of these animals is aganglionic. The mutation probably affects both the crest-derived precursors of enteric neurons and the nonneuronal cells that produce the matrix through which the crest-derived cells that colonise the gut must migrate. The precursors of smooth muscle and/or ICCs oversecrete the α1 subunit of laminin-1 which promotes neuronal differentiation. ET-3 inhibits the in vitro differentiation of crest-derived cells as neurons. The crest-derived cells that attempt to colonise the ET-3-deficient colon may thus encounter an overly strong drive to differentiate at the same time that they are deprived of a differentiation inhibitor. The result appears to

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be premature differentiation at the expense of migration, leaving the terminal portion of the bowel uncolonised by crest-derived neuronal or glial precursors. Hirschsprung’s disease of human has been associated with mutations of RET, ETB, and ET-3, but is a multigene abnormality that has not yet been adequately explained. All of the factors that play roles in the development of the ENS are potential targets of mutations that causes birth defects in humans. The genetic bases of hypoganglionosis, neuronal intestinal dysplasias, and intestinal dysganglionoses, as well as additional genes that contribute to Hirschsprung’s disease are likely soon to be discovered. Understanding the pathogenesis of Hirschsprung’s disease and birth defects of the ENS can be expected to provide improved means of treating these conditions and, eventually, some hope of preventing them.

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Index accommodatory reflex (intestine/colon), 8 acetylcholine (ACh), 1, 215–17, 300–1, pharmacology, 215–17 secretion, 315 acid-evoked gastric hyperaemia mediators, 146–7 model and species differences, 147–8 neural pathways, 144–6 physiological relevance, 148–9 somatic vasoconstriction associated with, 148 adenosine triphosphate (ATP), 232–3 inhibitory neuromuscular transmission, 304–7 secretion, 319–20 sympathetic co-transmitter in colon, 180–4 vasomotor effects, 352 adrenoreceptor subtypes postjunctional, 225–7 prejunctional, 224–5 afferent neurons acid-induced secretion of bicarbonate, 149 “autonomic” impact of, 122–7 control mediators, 127–8 motor effects and mediators, 128–9 pathological inhibition of gastric motor activity, 149–50 physiological implications in the gastric motor control, 129–32 afferent vasodilator nerve fibres in the stomach, 116–21 role in circulation, 116–21 vagal efferent activation, 121 after-hyperpolarizing potential (AHP), 11

aganglionosis of colon, 492–513 AH/type II cells, 10–12, 60 γ-aminobutyric acid (GABA), 22, 240–3 effects on neuromuscular preparations, 241 electrophysiological effects on neurons, 241–2 role of endogenous GABA, 242–3 α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA), 244 δ-aminovaleric acid (DAVA), 241 angiotensin, 263–4 anti-secretory transmitters, 320–1 neuropeptide Y and related peptides, 320–1 noradrenaline, 320 arylazidoaminopropionyl (ANAPP3), 305 ascending interneurons, 422–5 regions of gut, 424 species, not guinea-pig, 424–5 Auerbach’s (myenteric) plexus, 395 autonomic nervous system, plasticity in the, 321–3 chronic extrinsic denervation, 322 chronic sympathectomy, 322–3 autoregulatory escape, 120, 352, 353, 354 basal lamina in colon of ls/ls mice, 500–3 biliary tract, 189, 204, 206 blood vessels to gut, 341–58 anatomy, 342–3 autoregulatory escape, 120, 352–4 CGRP and related peptides, 113–14 enteric nervous system, 348–53 innervation, 344–55 nitric oxide, 114–15 pathways and mediators of neurogenic vasodilatation, 113–16

527

528

INDEX

blood vessels to gut (Continued) post-prandial vasodilation, 354 prostaglandins and mast cell mediators, 116 sympathetic control, 344–6 spinal afferent nerves, 346–8 tachykinins, 115–16 vasoactive intestinal polypeptide and PACAP, 116 vasodilation by afferent nerves, 108–13 vasodilator reflex, 354–5 bradykinin, 263 brain-derived neurotrophic factor (BDNF), 485 brain-in-the-gut concept, 365–6 brainstem reflexes, 84–8 convergent vagal inputs, 87–8 modulation of the vagal outflow, 86 spontaneous activity, 85–6 calcitonin gene-related peptide (CGRP), 39, 138–9, 256–8 in extrinsic afferent nerves, 105–8 gastric vasodilation, 113–14 gastric mucosal protection, 138 gastric hyperaemia, 146–7 capsaicin-sensitive afferents, 150–1 central reflex pathways, 83–91 chemical coding, 11, 32 acetylcholine and nitric oxide as motor neurotransmitters, 443–4 enteric nerves in the stomach, 63–9 functional significance of chemical coding gallbladder neurons, 196–9 limitations of chemical coding, 449–52 opioid peptides as enteric transmitters, 445–6 related to motor activity in the isolated stomach, 66–9 substance P as neurotransmitters, 445 vagal fibres, 66 variability hypothesis, 448 VIP as neurotransmitter, 444–5 cholecystokinin (CCK), 27 enteric nervous system, 254–5 gallbladder function, 195–6 sphincter or Oddi neurons, 202–3 choline acetyltransferase (ChAT), 22 ciliary neurotrophic factor (CNTF), 490 ciliary neurotrophic factor receptor (CNTFR), 490

circular muscle (CM) motor neurons, 417–21 regions of gut, 420 species, not guinea-pig, 420–1 command neurons, 88–90 constipation (idiopathic, chronic), 324 co-transmission, 295, 296, 345 c-ret proto-oncogene, 483–4 crest-derived cells in the gut, 475–7 Crohn’s disease, 357, 367 cyclic adenosine monophosphate (cAMP), 343 cytokine, 377–8 development of enteric motor neurons, 490–2 descending interneurons, 425–32 5-HT immunoreactive interneurons, 427–8 classes, 428–30 regions of gut, 430–1 somatostatin-immunoreactive interneurons, 426–7 species not guinea-pig, 431–2 development of enteric nervous system, 469–514 diabetes mellitus, 323–4, 356–7 DiI, 69, 205–6, 401, 414, 417, 419, 425 dimethylphenylpiperazinium (DMPP), 68 4-diphenylacetoxy-methylpiperidine (4-DAMP), 301 dopamine β-hydroxylase (DBH), 65 dorsal motor nucleus of the vagus (DMNV), 85 dynorphin (DYN), 214 electrical field stimulation (EFS), 68 endothelins, 132, 303–4, 494–500, 508 endothelin-B (ETB) receptor, 494–500 endothelin-converting enzyme-1 (ECE-1), 494 endothelin-like peptide, 264 endothelium, 347, 349–50 endothelium-derived hyperpolarizing factor (EDHF), 343 endothelium-derived relaxing factor (EDRF), 343 endotoxicosis, 68 enkephalin (ENK), 32 enteric immunology, 367 enteric mast cells, 367–70 enteric motor neurons, 413–21

INDEX

enteric nervous system (ENS), 58–77, 298 animal models for the human, 449 cellular organisation in mammalian, 393–452 chemical coding, 396–7 classification of enteric neurons, 10–12, 396, 441–3 description, 395–8 development, 469–514 plasticity, 321–7, 450–2 stomach, 59–62 enteric primary afferent neurons, see intrinsic primary afferent neurons enterochromaffin cells, 38, 127, 128, 217, 233, 238, 239, 240, 321, 355, 376, 400 EPSPs, see synaptic ET-3 affects both crest-derived and non-neuronal cells of the colon, 508–9 excitatory amino acids, 243–5 effects on neuromuscular preparations, 244–5 effects on neurons, 245 excitatory junction potential (EJP), 12 excitatory neuromuscular transmission, 300–4 acetylcholine, 300–1 endothelin, 303–4 noradrenaline, 303 substance P, 301–3 excitatory synaptic potentials (EPSPs), 1 extravasation caused by extrinsic afferent neurons, 121–1 extrinsic afferent nerves gastric mechanorecptors, 80–2 inputs to the CNS, 80 intestinal afferents and feedback mechanism, 82–3 mucosal afferents, 82 nerve fibres in the stomach, 105–8 extrinsic innervation, 77–83, 298 denervation, 322 fast excitatory post-synaptic potentials (fEPSP), 60 opioids, 248 Fos, 20, 30, 39, 74, 76, 84, 89, 154, 239, 400, 401, 489, 506

529

GABA, see γ-aminobutyric acid galanin (GAL), 62, 261–2 gallbladder ganglion and neurons chemical coding, 196–9 extrinsic sensory fibres, 194–5 hormonal cholecystokinin, 195–6 interactions with intestine, 204–5 interactions with sphincter of Oddi, 206 morphological properties, 190–1 regulatory inputs, 193–6 electrical properties, 191–3 sympathetic postganglionic fibres, 194 synaptic inputs, 192–3 vagal efferent fibres, 193–4 gastric enteric neurons chemical coding, 63–9 circuits, 69–74 electrophysiological properties, 60–1 neuropharmacology, 60–3 projections and circuits within gastric myenteric plexus, 69–74 synaptic properties, 61–2 gastric mucosal blood flow (GMBF), 108 gastric mucosal hyperaemia, 141 gastric motor reflexes (types), 57–8 permissive control of, 90–1 gastrin releasing peptide (GRP), 22 gastrin, cholecystokinin and caerulein, 253–5 effects of CCK-like peptides on neuromuscular preparations, 253–4 electrophysiological effects of CCK-like peptides on neurons, 254–5 role of endogenous CCK-like peptides, 255 gastrin-releasing peptide and neuromedin B, 260–1 giant migrating contraction (GMC), 4 glial and enteric neuronal progenitors, source of, 471–5 glial cell line-derived neurotrophic factor (GDNF), 482–4 glial fibrillary acidic protein (GFAP), 488 glutamic acid decarboxylase (GAD), 240 glyceryl trinitrate (GTN), 311 hexahydrosiladifenidol (HHSiD), 215 Hirschsprung’s disease, 295, 324–6, 357, 394, 493, 494, 500, 501, 503, 507, 509, 510, 511, 512, 513, 514

530

INDEX

Hirschsprung’s disease (Continued) abnormal intestitial cells of Cajal in aganglionic bowel, 509–13 histamine, 371 mimics slow synaptic excitation, 371–2 pattern generation evoked, 373–4 presynaptic inhibition, 375–6 receptors for slow excitatory responses, 373 signal transduction for slow EPSPs, 372 histidine decarboxylase (HDC), 107 5-hydroxytryptamine, see serotonin hyoscine, 31, 34, 215, 219, 228, 241, 247, 254, 258, 262–3 hyperaemia (gastric mucosal), 140–9 mediators, 146–7 mechanisms, 147–9 hyperaemia-independent mechanisms, 141–2 hypoganglionosis, 511, 514 ileus, 58, 68, 88, 103, 150, 363 immuno-physiology of gut, 363–89 effector behaviour, 386–7 neural behaviour, 385–6 sensitised intestine, 385–7 inflammatory bowel disease, 68, 357 inhibitory neuromuscular transmission, 304–14 ATP, nitric oxide and vasoactive intestinal peptide, 304–7 inhibitory junction potential (IJP), 12 nitric oxide and intestinal cells of Cajal, 311–12 nitric oxide, 307–11 noradrenaline, 313–14 pituitary adenylate cyclase-activating peptide, 313–12 inhibitory synaptic potentials (IPSPs), see synaptic innervation of intestinal blood vessels, 344–53 cholinergic transmission, 349–51 extrinsic sensory nerves, 346–8 extrinsic vasoconstrictor nerves, 344–6 intrinsic nerves, 348–9 non-cholinergic transmitters, 351–3 interleukin-1β (IL-1β), 150, 377–8 interleukin-6 (IL-6), 371, 377–8, 490 interleukin-II (IL-II), 490 interneurons, identities of, 22, 421–36 ascending (orally-projecting), 422–5 descending (aborally-projecting), 425–32

interstitial cells of Cajal (ICCs), 218, 509–11 nitric oxide, 311–12 intestinal transmission neuroepithelial, 295 neuromuscular, 295 intestinofugal neurons, see viscerofugal neurons intraganglionic laminar endings (IGLE), 78, 402 intrinsic primary afferent neurons (IPANs), 16–21, 399–413 chemical coding, 405 electrical properties, 402–3 identities of, 1, 16–21, 400–2 morphology, 404–5 motor activity, 40–4 muscle mechanoreceptors, 18–20, 401–2 mucosal mechanoreceptors, 20–1 neurotransmission between, 36–8, 382–4, 406–9 species differences, 409–12 stomach, 75–7 synaptic inputs, 406–9 ischaemia, 82, 151, 354, 355–7 Kit ligand, 488, 509 β-lactoglobulin, 385–7 laminin-1 promotes the development of enteric nerves, 503–8 excess in the colon, 507–8 IKVAV peptide effects, 504–6 laminin-binding protein, 110 (LBP-110), 504–10 primary crest cells and crest-derived cells, 506–7 Law of the Intestine, 3, 470 leucocyte recruitment afferent nerves, 122–3 mast cells, 370 leukaemia inhibitory factor (LIF), 490–2 leukotrienes B4, E4, D4 and C4 (LTB4, LTE4, LTD4 and LTC4), 378–9 Lissauer’s tract, 83 longitudinal muscle (LM) motor neurons, 1, 414–17 innervation in other regions, 415–16 other regions and other species, 416–17 longitudinal muscle/myenteric plexus (LM-MP) preparation, 213

INDEX

mash-1, 479–81, 487 mast cells (enteric), 367–79 adenosine, 377 cytokines, 377–8 histamine, 371–6 serotonin, 376 Meissner’s (submucous) plexus, 396, 440 Met-enkephalin, 174, 248, 249, 253 N-methyl-D-aspartate (NMDA), 244 α,β-methylene-ATP (α,β-MeATP), 182 β,γ-methylene-ATP (β,γ-MeATP), 182 2-methylthio-ATP (2-MeSATP), 182 migrating myoelectric complex (MMC), 4 monoaxonal neurons, 10, 11 Nw-monomethyl-L-arginine (L-NMMA), 174 monosynaptic reflex, 3 motilin, 259–60 motility, see motor patterns motor neurons, identities of, 22–3, 413–21 longitudinal muscle, 414–17 circular muscle, 417–21 motor patterns, programs and enteric reflexes, 2–3, 40–2, 363–5 ascending excitatory reflex, 1, 12–14, 32–4, 40, 61, 76, 172, 216, 239, 247, 256, 299, 423 descending inhibitory reflex, 12–14, 35, 75, 76, 216, 227, 232, 253, 255, 299, 313, 429 gastric motor reflexes (types), 57–8, 74–5, 90–1 identities of neurons in the reflex pathways, 16–23 in vivo, 3–5 intestinal reflexes, 1, 3, 6, 7, 16 isolated systems, 5–9 peptidergic afferent nerves, 128–32 peristalsis, 3, 6, 7, 40–4, 58, 68, 74, 91, 172, 204, 215–21, 233, 236, 242, 244, 248, 253, 255, 257, 302, 323–4, 399, 401, 415, 423, 445, 470 permissive control of, 90–1 physiological studies, 3–9 mucosa histamine, 379–80 mediators of protection, 138–40 peptidergic afferent nerves protect, 136–49 restitution, healing and afferent nerves, 143–4 muscarinic receptors, 215–16

531

myectomies, 397 myogenic tone, 57 myotomies, 397, 424 NANC transmission, 171, 213 natural IL-1 receptor antagonist (nIL-1ra), 377 nerve growth factor (NGF), 322 neural basis of reflex control, 58–9 neural circuits mediating reflexes evoked by stationary stimuli in vitro, 9–16 interactions evoked by different stimuli, 14–15 local distensions, 12–13 mechanical or chemical stimulation of the mucosa, 13–14 responses recorded from neurons, 15–16 neural crest, 471–5 source of enteric neuronal progenitors, 471–5 pluripotent cells, 475 neural interactions between gallbladder and the sphincter of Oddi, 206 gut and the gall bladder, 204–7 gut and the sphincter of Oddi, 205–6 neural networks and effector systems behaviour, 379–85 coordinated recruitment, 382–3 gating functions, 383–4 neural pattern generation, 380–1 presynaptic inhibition, 384–5 responses to histamine and leukotrienes, 379–80 slow synaptic excitation, 381–2 neurodegenerative disease, 298 neuroeffector dysfunction in pathological conditions, 323–7 diabetes mellitus, 323–4 Hirschsprung’s disease, 324–6 idiopathic chronic constipation, 324 ulcerative colitis, 326–7 neuroeffector transmission in the intestine, 295–327 autonomy of the enteric nervous system, 297–8 co-transmission and neuromodulation, 296–7 extrinsic innervation, 298 neuroepithelial transmission in the intestine, 314–21

532

INDEX

neurofilament protein (NFP), 398 neurokinin A (NKA), 103 neurokinin B (NKB), 195 neurokinin-3 (NK3), 195 neuromedin U (NMU), 262 neuromuscular transmission, 299–314 ATP, nitric oxide and vasoactive intestinal peptide, 304–7 autonomic neuromuscular junction, 299–300 acetylcholine, 300–1 endothelin, 303–4 intestinal smooth muscle contractility, 299 noradrenaline, 303 nitric oxide, 307–11 nitric oxide and intestinal cells of Cajal, 311–12 noradrenaline, 313–14 pituitary adenylate cyclase-activating peptide, 313–12 substance P, 301–3 neuronal intestinal dysplasia, 511, 514 neuropeptide Y (NPY) and peptide YY (PYY), 62, 255–6 effects on neuromuscular preparations, 256 electrophysiological effects of NPY and related peptides, 256 secretion, 320–1 vasoconstriction potentiation, 352 vasodilation, 352 neurotensin, 258–9 neurotransmitters, 213–65, 296–7, plasticity of expression of, 321–7 neurotrophins BDNF, 485 NGF, 484–6 NT-3 and development of the enteric nervous system, 484–90 NT4/5, 485 NT6, 485 nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, 197, 343 nicotinic receptors, 215–17 nitric oxide (NO), 36, 229–32, 379 effects on neuromuscular preparations, 230–1 effects on neurons and enteric smooth muscle cells, 231 inhibitory neuromuscular transmission, 307–12 role of endogenous NO, 231–2

nitric oxide synthase (NOS), 22 interneurons, 428–31 non-adrenergic non-cholinergic (NANC) nerves, 67, 227, 295–6 noradrenaline (NA), 61, 221–7, 303 inhibitory neuromuscular transmission, 313–4 secretion, 320 nuclear factor kappa-B (NF-κB), 485 nucleus tractus solitarius (NTS), 85 oncostatin M (OSM), 490 opioid peptides, 246–51 effects on neuromuscular preparations, 246–7 electrophysiological effects on neurons, 247–8 endogenous opioids, 248–51 neurotransmitter role, 445–6 opioid receptor like (ORL), 246 p-75 neurotrophin receptor, 485–6, 489 pancreatic polypeptide (PP), 62 parasympathetic nervous system, 171, 344 brainstem reflexes to stomach, 84–8 convergent vagal inputs in brainstem, 87–8 control of the colon, 173–8 control of the gall bladder, 193 modulation of vagal outflow to stomach, 86 nitric oxide, 174–6 prostaglandins, 176–8 spontaneous activity and gastric function, 85–6 peptide histidine isoleucine (PHI), 227 peptidergic afferent nerves, see spinal afferent peripheral nervous system (PNS), 245 peristalsis, 3, 6, 7, 40–4 opioids, 248–50 pharmacology adrenergic transmission, 221–7 amino acids as enteric neurotransmitters, 240–5 cholinergic transmission, 215–17 enteric nervous system, 213–65 histaminergic transmission, 245–6 NANC inhibitory transmission, 227–33 non-cholinergic excitatory transmission, 217–21

INDEX

peptides as enteric neurotransmitters, 246–65 serotonergic transmission, 233–40 phenylethanolamine-N-methyltransferase (PNMT), 65 piebald lethal mouse, 493–4 pilocarpine, 6 pituitary adenylate cyclase-activating peptide (PACAP), 35, 422 inhibitory neuromuscular transmission, 312–13 plasma protein extravasation, 121–2 leucocyte recruitment, 122–3 plasticity of the enteric nervous system, 295, 321–7, 450–2 associated with disease states, 451–2 developmental plasticity, 450 response to denervation, 450–1 platelet activating factor (PAF), 119, 378–9 pluripotent crest-derived neural precursors, 483–4 progenitor lineages in formation of ENS, 477–81 prostaglandins gastric vasodilation, 116 gastric mucosal protection, 140 prostaglandin D2 (PGD2), 378 prostaglandin E2 (PGE2), 116 purinergic transmission and receptors colon, 180–4 inhibitory neuromuscular transmission, 304–7 secretion, 319–20 ret proto-oncogene (c-ret), 483–4 rough endoplasmic reticulum (RER), 494 S/type I neurons, 10–12, 60 Schabadasch’s or Henle’s plexus, 396, 421, 440 Schwann cells, 469, 476, 485, 512 secretion acetylcholine, 315 ATP, 319–20 gastric and peptidergic afferent nerves, 123–7 gastric mediators, 127–8 histamine, 379–81 leukotrienes, 380 neurotransmitters, 315–20

533

substance P, 317–19 vasoactive intestinal peptide and related peptides, 315–17 secretomotor neurons, 436–8 NPY-immunoreactive neurons, 436–7 VIP-immunoreactive neurons, 436–7 sensory nerves, see IPANs, spinal nerves, vagus nerve sensory transduction, 38–40 chemoreceptors, 39–40 mucosal mechanoreceptors, 38–9 stretch, 38 serotonin (5-HT), 18, 234–8, 376 effects on neuromuscular preparations, 233–8 electrophysiological effects on neurons, 238 interneurons, 427–8 role of endogenous, 5-HT, 238–40 serotonergic transmission, 213, 233 Shah-Waardenburg syndrome, 512 signal, mast cells to the brain-in-the-gut, 371–9 simple reflexes, motor patterns and complex behavior, 40–4 muscle tension changes, effects when, 41–4 sodium nitroprusside (SNP), 230 somatostatin, 22, 32, 251–3 effects on neuromuscular preparations, 251–2 electrophysiological effects on neurons, 252 interneurons, 426–7 role of endogenous somatostatin, 252–3 sphincter of Oddi (SO) ganglia and neurons, 189 actions of CCK, 202–3 chemical coding, 203–4 morphological properties, 199–200 regulatory inputs, 201–3 structural and electrical properties, 199–201 sympathetic inputs, 201–2 synaptic inputs, 201 spinal afferent nerves blood flow, 108–21 mechanisms of gastric mucosal protection, 141–2 mediators of gastric mucosal protection, 138–40 gallbladder, 194–5 gastric motor activity, 128–32 gastric mucosal blood flow, 108–21, 144–9

534

INDEX

spinal afferent nerves (Continued) gastric secretion, 123–8 pathways in gastric mucosal protection, 138 resistance of gastric mucosal injury, 132–5 restitution and healing of gastric lesions, 143–4 stomach, 77, 105–8, 132–50 vasodilation, 346–8 vascular permeability and leucocyte recruitment, 121–3 spinal reflexes, 83–4 splanchnic nerves, 57, 84, 121, 145, 194, 201 subdiaphragmatic vagotomy, 104 sublineages of enteric neuronal precursors, 481–2 submucous plexus neurons, 436–41 colon, 438–9 inter-plexus interneurons, 438 secretomotor neurons, 436–7 species, not guinea-pig, 439–41 vasomotor neurons, 438 submucous interneurons and primary afferent neurons, 438 substance P (SP, see tachykinins) suramin, 24, 32, 34, 183, 233, 307, 313 sympathetic nervous system colon, 178–84 gall bladder, 194 noradrenaline and ATP as co-transmitters, 180–2 purine receptors in colon, 182–4 sphincter of Oddi, 201–2 sympathectomy effects, 322–3 vasoconstriction, 344–6 synaptic interactions ascending interneurons to motor neurons, 33–4 ascending interneurons, 32–3 descending interneurons and motor neurons, 35–6 descending interneurons, 34–5 IPANs and motor neurons, 28–30 IPANs to interneurons, 30–2 IPANs, 36–8, 383–4 presynaptic inhibition, 384–5 retrograde transmission from interneurons to IPANs, 36

synaptic neurotransmission, 1, 17, 20, 30, 31, 36, 60, 61, 62, 79, 129, 232, 246, 371, 385, 433 synaptic potentials, types of, 23–8 fast excitatory post-synaptic potentials (fEPSP), 60 inhibitory synaptic potentials, 201 slow, 381–2, 407–9 tachykinins, 1, 20, 139–40 electrophysiological effects, 219 gastric hyperaemia, 147 gastric mucosal protection, 139–40 gastric vasodilation, 115–16 neuromuscular preparations, 218, 301–3 neurokinin A (NKA), 103 neurokinin B (NKB), 195 receptors, 217–21 role of endogenous tachykinins, 219–21, 445 secretion, 317–19 tetraethylammonium (TEA), 25 tetrodotoxin (TTX), 73 thyrotropin-releasing hormone (TRH), 66 transforming growth factor-β (TGF-β), 483 Trichinella spiralis, 385–7 tumour necrosis factor α (TNFα), 371, 377–8 tyrosine hydroxylase (TH), 194 tyrosine kinase receptors, 485–90 TrkA, 485–6 TrkB, 486 TrkC, 486, 489–90 ulcerative colitis, 295, 326–7, 357, 367 vagus nerve afferent nerves, 77–80, 80–2 brainstem reflexes, 84–8 chemical coding, 66 convergent vagal inputs, 87–8 intraganglionic laminar endings (IGLE), 78, 402 mechanoreceptors, 80–2 modulation of vagal outflow, 86 mucosal receptors, 82 spontaneous activity, 85–6 vago-enteric interactions in stomach, 62–3 vascular supply, see blood vessels

INDEX

vascular tone of intestine, factors that influence, 343–4 vasoactive intestinal constrictor (VIC), 303 vasodilatation by afferent nerve stimulation, 108–13 vasomotor neurons, 438 calretinin-immunoreactive neurons, 438 VIP-like peptides, 22, 227–9 effects on neuromuscular preparations, 228 effects on neurons and enteric smooth muscle cells, 228–9

gastric mucosal protection by afferent nerves, 116 interneurons, 428–32 role of endogenous VIP-like peptides, 229 vasodilation, 351–2 viscerofugal neurons, 432–6

yohimbine, 182, 194, 222, 223, 225, 263, 320

535

Innervation of the Gastrointestinal Tract (Autonomic Nervous System)

Primer on the Autonomic Nervous System

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Ultrasound of the Gastrointestinal Tract

MRI of the Gastrointestinal Tract

Autonomic nervous system in old age

The integrative action of the autonomic nervous system

Integrative Action of the Autonomic Nervous System: Neurobiology of Homeostasis

Aging and the Gastrointestinal Tract

Development of the gastrointestinal tract, Volume 1

Gastrointestinal System

Primer on the Autonomic Nervous System, Third Edition

Primer on the Autonomic Nervous System (Third Edition)

The Autonomic Nervous System in Health and Disease

Disorders of the Nervous System

The Enteric Nervous System

Mobilisation of the Nervous System

Cytoskeleton of the Nervous System

The Nervous System

The Nervous System

The Enteric Nervous System

Physiology of the Gastrointestinal Tract, Volume 1-2, Fourth Edition

The Human Central Nervous System

Erythropoietin and the Nervous System

Toxicology of the Gastrointestinal Tract (Target Organ Toxicology Series)

Early cancer of the gastrointestinal tract: endoscopy, pathology, and treatment

Erythropoietin and the Nervous System

Central Nervous System Diseases

Infections of the Central Nervous System

Clinical Update on Inflammatory Disorders of the Gastrointestinal Tract (Frontiers of Gastrointestinal Research, Vol. 26)

Cells of the Nervous System (Gray Matter)

Innervation of the Gastrointestinal Tract (Autonomic Nervous System)

Primer on the Autonomic Nervous System

Physiology of the Gastrointestinal Tract

Ultrasound of the Gastrointestinal Tract

MRI of the Gastrointestinal Tract

Autonomic nervous system in old age

The integrative action of the autonomic nervous system

Integrative Action of the Autonomic Nervous System: Neurobiology of Homeostasis

Aging and the Gastrointestinal Tract

Development of the gastrointestinal tract, Volume 1

Gastrointestinal System

Primer on the Autonomic Nervous System, Third Edition

Primer on the Autonomic Nervous System (Third Edition)

The Autonomic Nervous System in Health and Disease

Disorders of the Nervous System

The Enteric Nervous System

Mobilisation of the Nervous System

Cytoskeleton of the Nervous System

The Nervous System

The Nervous System

The Enteric Nervous System

Physiology of the Gastrointestinal Tract, Volume 1-2, Fourth Edition

The Human Central Nervous System

Erythropoietin and the Nervous System

Toxicology of the Gastrointestinal Tract (Target Organ Toxicology Series)

Early cancer of the gastrointestinal tract: endoscopy, pathology, and treatment

Erythropoietin and the Nervous System

Central Nervous System Diseases

Infections of the Central Nervous System

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