Hypothalamic Integration of Circadian Rhythms

PROGRESS IN BRAIN RESEARCH VOLUME 139 VASOPRESSIN AND OXYTOCIN: FROM GENES TO CLINICAL APPLICATIONS

Other volumes in PROGRESS IN BRAIN RESEARCH Volume 111: Hypothalamic Integration of Circadian Rhythms, by R.M. Buijs, A. Kalsbeek, H.J. Romijn, C.M.A. Pennartz and M. Mirmiran (Eds.) – 1996, ISBN 0-444-82443-X. Volume 112: Extrageniculostriate Mechanisms Underlying Visually-Guided Orientation Behavior, by M. Norita, T. Bando and B.E. Stein (Eds.) – 1996, ISBN 0-444-82347-6. Volume 113: The Polymodal Receptor: A Gateway to Pathological Pain, by T. Kumazawa, L. Kruger and K. Mizumura (Eds.) – 1996, ISBN 0-444-82473-1. Volume 114: The Cerebellum: From Structure to Control, by C.I. de Zeeuw, P. Strata and J. Voogd (Eds.) – 1997, ISBN 0-444-82313-1. Volume 115: Brain Function in Hot Environment, by H.S. Sharma and J. Westman (Eds.) – 1998, ISBN 0-444-82377-8. Volume 116: The Glutamate Synapse as a Therapeutical Target: Molecular Organization and Pathology of the Glutamate Synapse, by O.P. Ottersen, I.A. Langmoen and L. Gjerstad (Eds.) – 1998, ISBN 0-444-82754-4. Volume 117: Neuronal Degeneration and Regeneration: From Basic Mechanisms to Prospects for Therapy, by F.W. van Leeuwen, A. Salehi, R.J. Giger, A.J.G.D. Holtmaat and J. Verhaagen (Eds.) – 1998, ISBN 0-444-82817-6. Volume 118: Nitric Oxide in Brain Development, Plasticity and Disease, by R.R. Mize, T.M. Dawson, V.L. Dawson and M.J. Friedlander (Eds.) – 1998, ISBN 0-444-82885-0. Volume 119: Advances in Brain Vasopressin, by I.J.A. Urban, J.P.H. Burbach and D. De Wied (Eds.) – 1999, ISBN 0-444-50080-4. Volume 120: Nucleotides and their Receptors in the Nervous System, by P. Illes and H. Zimmermann (Eds.) – 1999, ISBN 0-444-50082-0. Volume 121: Disorders of Brain, Behavior and Cognition: The Neurocomputational Perspective, by J.A. Reggia, E. Ruppin and D. Glanzman (Eds.) – 1999, ISBN 0-444-50175-4. Volume 122: The Biological Basis for Mind Body Interactions, by E.A. Mayer and C.B. Saper (Eds.) – 1999, ISBN 0-444-50049-9. Volume 123: Peripheral and Spinal Mechanisms in the Neural Control of Movement, by M.D. Binder (Ed.) – 1999, ISBN 0-444-50288-2. Volume 124: Cerebellar Modules: Molecules, Morphology and Function, by N.M. Gerrits, T.J.H. Ruigrok and C.E. De Zeeuw (Eds.) – 2000, ISBN 0-444-50108-8. Volume 125: Volume Transmission Revisited, by L.F. Agnati, K. Fuxe, C. Nicholson and E. Syková (Eds.) – 2000, ISBN 0-444-50314-5. Volume 126: Cognition, Emotion and Autonomic Responses: The Integrative Role of the Prefrontal Cortex and Limbic Structures, by H.B.M. Uylings, C.G. Van Eden, J.P.C. De Bruin, M.G.P. Feenstra and C.M.A. Pennartz (Eds.) – 2000, ISBN 0-444-50332-3. Volume 127: Neural Transplantation II. Novel Cell Therapies for CNS Disorders, by S.B. Dunnett and A. Björklund (Eds.) – 2000, ISBN 0-444-50109-6. Volume 128: Neural Plasticity and Regeneration, by F.J. Seil (Ed.) – 2000, ISBN 0-444-50209-2. Volume 129: Nervous System Plasticity and Chronic Pain, by J. Sandkühler, B. Bromm and G.F. Gebhart (Eds.) – 2000, ISBN 0-444-50509-1. Volume 130: Advances in Neural Population Coding, by M.A.L. Nicolelis (Ed.) – 2001, ISBN 0-444-50110-X. Volume 131: Concepts and Challenges in Retinal Biology, by H. Kolb, H. Ripps, and S. Wu (Eds.), – 2001, ISBN 0-444-50677-2. Volume 132: Glial Cell Function, by B. Castellano López and M. Nieto-Sampedro (Eds.) – 2001, ISBN 0-444-50508-3. Volume 133: The Maternal Brain. Neurobiological and neuroendocrine adaptation and disorders in pregnancy and post partum, by J.A. Russell, A.J. Douglas, R.J. Windle and C.D. Ingram (Eds.) – 2001, ISBN 0-444-50548-2. Volume 134: Vision: From Neurons to Cognition, by C. Casanova and M. Ptito (Eds.) – 2001, ISBN 0-44450586-5. Volume 135: Do Seizures Damage the Brain, by A. Pitkänen and T. Sutula (Eds.) – 2002, ISBN 0-444-50814-7. Volume 136: Changing Views of Cajal’s Neuron, by E.C. Azmitia, J. DeFelipe, E.G. Jones, P. Rakic and C.E. Ribak (Eds.) – 2002, ISBN 0-444-50815-5. Volume 137: Spinal Cord Trauma: Regeneration, Neural Repair and Functional Recovery, by L. McKerracher, G. Doucet and S. Rossignol (Eds.) – 2002, ISBN 0-444-50817-1. Volume 138: Plasticity in the Adult Brain: From Genes to Neurotherapy, by M.A. Hofman, G.J. Boer, A.J.G.D. Holtmaat, E.J.W. Van Someren, J. Verhaagen and D.F. Swaab (Eds.) – 2002, ISBN 0-444-50981-X.

PROGRESS IN BRAIN RESEARCH VOLUME 139

VASOPRESSIN AND OXYTOCIN: FROM GENES TO CLINICAL APPLICATIONS

EDITED BY

DOMINIQUE POULAIN STÉPHANE OLIET DIONYSIA THEODOSIS INSERM U.378, Neurobiologie Morphofonctionnelle, Institut François Magendie 1, Rue Camille St.-Saëns, 33077 Bordeaux Cedex, France

ELSEVIER AMSTERDAM – BOSTON – LONDON – NEW YORK – OXFORD – PARIS SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO 2002

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands © 2002 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier Science via their homepage (http://www.elsevier.com) by selecting ‘Customer support’ and then ‘Permissions’. Alternatively you can send an e-mail to: [email protected], or fax to: (+44) 1865 853333. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+1) 978 7508400, fax: (+1) 978 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London W1P 0LP, UK; phone: (+44) 207 631 5555, fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Global Rights Department, at the fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drugs dosages should be made.

First edition 2002 Library of Congress Cataloging in Publication Data World Congress on Neurohypophysial Hormones (2001: Bordeaux, France) Vasopressin and oxytocin / edited by Dominique Poulain, Stéphane Oliet, Dionysia Theodosis p. cm. – (Progress in brain research; v. 139) "The articles compromising this volume were first presented at the World Congress on Neurohypophysial Hormones, held in Bordeaux, France on September 8–12, 2001"--Pref. Includes bibliographical references index. ISBN 0-444-50982-8 1. Vasopressin--Physiological effect--Congresses. 2. Oxytocin--Physiological effect--Congresses. I. Poulain, Dominique. II. Oliet, Stéphane. III. Theodosis, Dionysia. IV. Series. QP376 P7 vol. 139 [QP572.V3] 612.8 2s--dc2l [612.4 92] 2002026512 British Library Cataloguing in Publication Data Vasopressin and oxytocin: from genes to clinical applications. - (Progress in brain research; v. 139) 1. Brain – Congresses 2. Vasopressin – Congresses 3. Oxytocin – Congresses I. Poulain, Dominique II. Oliet, Stéphane III. Theodosis, Dionysia 612.8’2 ISBN 0444509828

ISBN: ISBN: ISSN:

0-444-50982-8 (volume) 0-444-80104-9 (series) 0079-6123

∞ The paper used in this publication meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper).  Printed in The Netherlands.

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List of Contributors

K.C. Abbott, Nephrology Service, Walter Reed Army Medical Center, Washington, DC 20307, USA M. Åkerlund, Department of Obstetrics and Gynecology, University Hospital, S-221 85, Lund, Sweden J. Antunes-Rodrigues, Department of Physiology, School of Medicine, University of Sao Paulo, 14049 Ribeirao Preto, Sao Paulo, Brazil U. Bahner, Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum, Franz-JosefStrauss-Allee 11, 93053 Regensburg, Germany J.S. Bains, Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, Calgary, AB, Canada G.L. Bakris, Rush University Hypertension Center, Department of Preventive Medicine, Rush Presbyterian – St. Luke’s Medical Center, 1700 W. Van Buren, Suite 470, Chicago, IL 60612, USA C. Barberis, INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France C. Boudaba, Department of Cell and Molecular Biology, Neurobiology Division, Tulane University, 2000 Percival Stern Hall, New Orleans, LA 70118, USA C.W. Bourque, L7-216, Neurology Division, Centre for Research in Neuroscience, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada S.B. Bruno, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA A.B. Brussaard, Department of Experimental Neurophysiology, Vrije University Amsterdam, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands R.M. Buijs, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands K. Burger, Institute of Biochemistry, Johannes Gutenberg - University of Mainz, Becherweg 30, D-55099 Mainz, Germany Y. Chakfe, Neurobiology Unit, Montreal Neurological Institute, 3801 University Street, Montreal, QC H3G 2B4, Canada P. Coles, Departments of Medicine and Biochemistry, Division of Clinical and Molecular Endocrinology, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4951, USA J.M. Coulson, Departments of Physiology and Human Anatomy and Cell Biology, University of Liverpool, Liverpool, Crown Street, Liverpool, L69 3BX, UK J.T. Cunningham, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA G. Dayanithi, Department of Neurobiology, U432-INSERM, University of Montpellier II, Place Eugene Bataillon, F-34094 Montpellier, Cedex 5, France

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Y. De Keyzer, CNRS UPR 1524, Institut Cochin de Génétique Moléculaire, 24 rue du Faubourg Saint Jacques, 75014 Paris, France T.L. Dellovade, Neuroscience Department, Curis Inc., 61 Moulton Street, Cambridge, MA 02138, USA S. Di, Department of Cell and Molecular Biology, Neurobiology Division, Tulane University, 2000 Percival Stern Hall, New Orleans, LA 70118, USA T. Durroux, INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France C.A. Ecelbarger, Department of Medicine, Building D, Room 232, Division of Endocrinology and Metabolism, Georgetown University, 4000 Reservoir Road NW, Washington, DC 20007, USA F. Fahrenholz, Institute of Biochemistry, Johannes Gutenberg - University of Mainz, Becherweg 30, D-55099 Mainz, Germany R.L. Fields, Laboratory of Neurochemistry, Section on Molecular Neuroscience, NINDS/ NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA G. Gaibelet, AVIDIS S.A., Biopôle Clermont-Limagne, 63360 Saint-Beauzire, France H. Gainer, Laboratory of Neurochemistry, Section on Molecular Neuroscience, NINDS/ NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA G. Garcia, Exploratory Research Department, Sanofi-Synthelabo Recherche, 371 Rue du Professeur J. Blayac, 34184 Montpellier, Cedex 04, France G. Gimpl, Institute of Biochemistry, Johannes Gutenberg - University of Mainz, Becherweg 30, D-55099 Mainz, Germany R.J. Grindstaff, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA R.R. Grindstaff, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA J. Gutkowska, Laboratory of Cardiovascular Biochemistry, Research Centre CHUM –Hôtel Dieu, 3850 St. Urbain Street, Masson Pavilion, Montreal, QC H2W 1T8, Canada K.H.R. Higgs, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA M. Hirasawa, Neuroscience Research Group, and Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada S.B. House, Laboratory of Neurochemistry, Section on Molecular Neuroscience, NINDS/ NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA N. Hussy, CNRS-UMR 5101, CCIPE, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France M. Jankowski, Laboratory of Cardiovascular Biochemistry, Research Centre CHUM – Hôtel Dieu, 3850 St. Urbain Street, Masson Pavilion, Montreal, QC H2W 1T7, Canada I. Kächele, Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany A. Kalsbeek, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands M.A. Knepper, Laboratory of Kidney and Electrolyte Metabolism, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA J.-J. Koksma, Department of Experimental Neurophysiology, Research Institute Neurosciences, Vrije Universiteit Amsterdam, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands S.B. Kombian, Faculty of Pharmacy, Kuwait University, Kuwait

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K.J. Kovács, Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Szigony u. 43, H-1083 Budapest, Hungary G. Le Fur, Sanofi-Synthelabo Recherche, 174 Avenue de France, 75635 Paris, Cedex 13, France G. Leng, University of Edinburgh Medical School, Department of Biomedical Sciences, George Square, Edinburgh, EH8 9XD, UK Y. Li, Department of Pharmacology and Toxicology, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA M. Ludwig, Department of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, UK J.P. Maffrand, Exploratory Research Department, Sanofi-Synthelabo Recherche, 195 Route d’Espagne, 31036 Toulouse, Cedex, France D. Mazzella, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA S.M. McCann, Pennington Biomedical Research Center (LSU), 6400 Perkins Road, Baton Rouge, LA 70808-4124, USA I. Merchenthaler, The Women’s Health Research Institute, Wyeth Research, Collegeville, PA 19426, USA E. Mohr, Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany D. Mouginot, CHUL Research Center, Neuroscience Unit, Laval University, Saint-Foy, QC, Canada B. Mouillac, INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France F. Muders, Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum, Franz-JosefStrauss-Allee 11, 93053 Regensburg, Germany C. Mullin, Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany T. Murase, The First Department of Internal Medicine, Nagoya University, Nagoya, Japan I.D. Neumann, Institute of Zoology, University of Regensburg, 93040 Regensburg, Germany S. Nielsen, Department of Cell Biology, University of Aarhus, Aarhus, Denmark M. Palkovits, Laboratory of Neuromorphology, Semmelweis University, Budapest, Hungary I.F. Palm, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands M. Pascal, Exploratory Research Department, Sanofi-Synthelabo Recherche, 195 Route d’Espagne, 31036 Toulouse, Cedex, France M. Petersson, Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Institutet, S-171 77 Stockholm, Sweden Q.J. Pittman, Neuroscience Research Group, and Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada P. René, CNRS UPR 1524, Institut Cochin de Génétique Moléculaire, 24 rue du Faubourg Saint Jacques, 75014 Paris, France D.S. Richards, Department of Pharmacology and Toxicology, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA D. Richter, Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany

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G.A.J. Riegger, Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum, FranzJosef-Strauss-Allee 11, 93053 Regensburg, Germany M. Rusnak, Laboratory of Neurochemistry, Section on Molecular Neuroscience, NINDS/ NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA J.A. Russell, Department of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh, EH8 9XD, UK N. Sabatier, Department of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, UK J.-C. Schellenberg, School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland, Australia T. Sen, INSERM U469, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France C. Serradeil-Le Gal, Exploratory Research Department, Sanofi-Synthelabo Recherche, 195 Route d’Espagne, 31036 Toulouse, Cedex, France M. Shoham, Departments of Medicine and Biochemistry, Division of Clinical and Molecular Endocrinology, Case Western University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4951, USA P.J. Shughrue, Department of Neuroscience, Merck Research Laboratories, Sumneytown Pike and Broad Street, WP26A-3000, West Point, PA 19486, USA J.E. Stern, Department of Pharmacology and Toxicology, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA M.J. Sullivan, Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri–Columbia, Research Park, Columbia, MO 65211, USA J.G. Tasker, Department of Cell and Molecular Biology, Neurobiology Division, Tulane University, 2000 Percival Stern Hall, New Orleans, LA 70118, USA A. Thibonnier, University of Michigan, School of Engineering, Ann Arbor, MI, USA M. Thibonnier, Departments of Medicine and Biochemistry, Room BRB 431, Division of Clinical and Molecular Endocrinology, Case Western University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4951, USA Y. Tian, Department of Medicine, Building D, Room 232, Division of Endocrinology and Metabolism, Georgetown University, 4000 Reservoir Road NW, Washington, DC 20007, USA G. Valette, Exploratory Research Department, Sanofi-Synthelabo Recherche, 195 Route d’Espagne, 31036 Toulouse, Cedex, France J.G. Verbalis, Department of Medicine, Building D, Room 232, Division of Endocrinology and Metabolism, Georgetown University, 4000 Reservoir Road NW, Washington, DC 20007, USA D.L. Voisin, Laboratoire de Physiologie Oro-Faciale, Faculté de Chirurgie Dentaire, Clermont Ferrand, 63000, France J. Wagnon, Exploratory Research Department, Sanofi-Synthelabo Recherche, 371 Rue du Professeur J. Blayac, 34184 Montpellier, Cedex 04, France V. Wiegand, Institute of Biochemsitry, Johannes Gutenberg - University of Mainz, Becherweg 30, D-55099 Mainz, Germany M. Yamashita, Laboratory of Neurochemistry, Section on Molecular Neuroscience, NINDS/ NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA

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Preface

The articles comprising this volume were first presented at the World Congress on Neurohypophysial Hormones held in Bordeaux, France on September 8–12, 2001. This conference brought together more than 170 scientists from 18 countries who belong to the different fields of interest representing research in the hypothalamo–neurohypophysial system. This meeting follows that held in Edinburgh in 1999 and a series of International Meetings on oxytocin or vasopressin or on the neurohypophysis in general, held sporadically but very successfully over the past five decades, since the first meeting in Bristol in 1956. The next World Congress on Neurohypophysial Hormones will be held in Kyoto, Japan, and its organization, which is in the competent hands of Professor M. Kawata, is well underway. The two neurohypophysial neurohormones, oxytocin and vasopressin, exert a variety of central and peripheral actions and thus involve different scientific domains, which too often, even to-day, do not always find the appropriate occasion to interact. The meeting offered such an opportunity. This volume, therefore, is composed of chapters dealing with topics varying from basic and clinical neurosciences and neuroendocrinology, to reproductive, renal, cardiovascular physiology and pathology. It thus encompasses all areas of current neurohypophysial research and should be of vital interest as an integrative reference volume to specialized investigators and as an excellent introductory text to students, scientists and clinicians not yet closely familiar with the field. To ensure novelty and to make sure that all topics of current importance were covered, plenary and symposium speakers as well as poster presentations concentrated on recent advances made in the last few years. In the course of the conference, a particularly tragic event in modern history occurred, that will leave long-lasting unhappy memories. The quality and friendliness of the scientific and intellectual exchanges that took place between participants from so many different countries and cultural oulooks should remind us that it is always possible, albeit difficult, to overcome the dire consequences of such events. Dominique Poulain Stéphane Oliet Dionysia Theodosis

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Acknowledgements

The Local Organizing Committee, composed of Dominique Poulain (Cochair), Dionysia Theodosis (Cochair), Stéphane Oliet (Secretary), Frank Couillaud and Jean Marc Israel, wishes to express its thanks to the members of the International Scientific Advisory Board, that included W. Armstrong, C. Barberis, L. Burrell, P. Burbach, B. Chini, T. Day, A. Fergusson, F. Grant, T. Higuchi, J.J. Legros, J. Lemos, F. Moos, D. Murphy, I. Neumann, S. Nielsen, Q. Pittman, G. Robertson, W. Rosenthal, J. Russell, E. Szczepanska-Sadowska and K. Yagi, who put together an excellent Scientific Program and helped to shape the Conference. We also thank the French Neuroscience Society, CNRS, INSERM, ARMA, and Sanofi-Synthélabo for their financial support. We are particularly grateful to the members of the INSERM Unit 378 in Bordeaux and to Clémence Fouquet, Isabelle Conjat and Jean-Francis Renaudon who assisted with the scientific, secretarial and social work and contributed to make the Conference an international success.

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Contents

List of Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. The magnocellular neuronal phenotype: cell-specific gene expression in the hypothalamo-neurohypophysial system H. Gainer, M. Yamashita, R.L. Fields, S.B. House and M. Rusnak (Bethesda, MD, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

2. Estrogen modulates oxytocin gene expression in regions of the rat supraoptic and paraventricular nuclei that contain estrogen receptor-β P.J. Shughrue, T.L. Dellovade and I. Merchenthaler (Collegeville, PA, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Short-term modulation of GABAA receptor function in the adult female rat A.B. Brussaard and J.-J. Koksma (Amsterdam, The Netherlands) . . . .

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4. Cholesterol and steroid hormones: modulators of oxytocin receptor function G. Gimpl, V. Wiegand, K. Burger and F. Fahrenholz (Mainz, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. Central vasopressin systems and steroid hormones A. Kalsbeek, I.F. Palm and R.M. Buijs (Amsterdam, The Netherlands) 57 6. Regulation of renal salt and water transporters during vasopressin escape C.A. Ecelbarger, T. Murase, Y. Tian, S. Nielsen, M.A. Knepper and J.G. Verbalis (Washington, DC, Bethesda, MD, USA, Nagoya, Japan and Aarhus, Denmark) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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7. Stretch-inactivated cation channels: cellular targets for modulation of osmosensitivity in supraoptic neurons C.W. Bourque, D.L. Voisin and Y. Chakfe (Montreal, QC, Canada and Clermont Ferrand, France) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8. Glial cells in the hypothalamo-neurohypophysial system: key elements of the regulation of neuronal electrical and secretory activity N. Hussy (Montpellier, France) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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9. Functional synaptic plasticity in hypothalamic magnocellular neurons J.G. Tasker, S. Di and C. Boudaba (New Orleans, LA, USA) . . . . . . . . 113 10. Postsynaptic GABAB receptors in supraoptic oxytocin and vasopressin neurons J.E. Stern, Y. Li and D.S. Richards (Dayton, OH, USA) . . . . . . . . . . . . . 121 11. Neurohypophyseal hormones in the integration of physiological responses to immune challenges K.J. Kovács (Budapest, Hungary) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 12. Involvement of the brain oxytocin system in stress coping: interactions with the hypothalamo–pituitary–adrenal axis I.D. Neumann (Regensburg, Germany) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 13. Expression of human vasopressin and oxytocin receptors in Escherichia coli B. Mouillac, T. Sen, T. Durroux, G. Gaibelet and C. Barberis (Montpellier and Saint-Beauzire, France) . . . . . . . . . . . . . . . . . . . . . . . . . . 163 14. Molecular pharmacology and modeling of vasopressin receptors M. Thibonnier, P. Coles, A. Thibonnier and M. Shoham (Cleveland, OH and Ann Arbor, MI, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 15. Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a , V2 and V1b receptor ligands C. Serradeil-Le Gal, J. Wagnon, G. Valette, G. Garcia, M. Pascal, J.P. Maffrand and G. Le Fur (Toulouse, Montpellier and Paris, France) 197 16. Rat vasopressin mRNA: a model system to characterize cis-acting elements and trans-acting factors involved in dendritic mRNA sorting E. Mohr, I. Kächele, C. Mullin and D. Richter (Hamburg, Germany) 211 17. Dendritic action potentials in magnocellular neurons J.S. Bains (Calgary, AB, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 18. Modulation of synaptic transmission by oxytocin and vasopressin in the supraoptic nucleus S.B. Kombian, M. Hirasawa, D. Mouginot and Q.J. Pittman (Kuwait, Calgary, AB, and Sainte-Foy, QC, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . 235 19. The active role of dendrites in the regulation of magnocellular neurosecretory cell behavior M. Ludwig, N. Sabatier, G. Dayanithi, J.A. Russell and G. Leng (Edinburgh, UK and Montpellier, France) . . . . . . . . . . . . . . . . . . . . . . . . . . 247 20. Cardiovascular regulation of supraoptic vasopressin neurons J.T. Cunningham, S.B. Bruno, R.R. Grindstaff, R.J. Grindstaff, K.H.R. Higgs, D. Mazzella and M.J. Sullivan (Columbia, MO, USA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

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21. The central vasopressinergic system in experimental left ventricular hypertrophy and dysfunction F. Muders, G.A.J. Riegger, U. Bahner and M. Palkovits (Regensburg and Würzburg, Germany and Budapest, Hungary) . . . . . . . . . . . . . . . . . . 275 22. Cardiovascular effects of oxytocin M. Petersson (Stockholm, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 23. Treatment of the diabetic patient: focus on cardiovascular and renal risk reduction K.C. Abbott and G.L. Bakris (Washington, DC and Chicago, IL, USA) 289 24. Oxytocin in parturition of guinea pigs, humans, and other species J.-C. Schellenberg (Geneva, Switzerland) . . . . . . . . . . . . . . . . . . . . . . . . . . 299 25. Oxytocin, vasopressin and atrial natriuretic peptide control body fluid homeostasis by action on their receptors in brain, cardiovascular system and kidney S.M. McCann, J. Antunes-Rodrigues, M. Jankowski and J. Gutkowska (Baton, Rouge, LA, USA, Sao Paulo, Brazil and Montreal, QC, Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 26. Positive and negative regulators of the vasopressin gene promoter in small cell lung cancer J.M. Coulson (Liverpool, UK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 27. The vasopressin receptor of corticotroph pituitary cells P. René and Y. de Keyzer (Paris, France) . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 28. Involvement of oxytocin and vasopressin in the pathophysiology of preterm labor and primary dysmenorrhea M. Åkerlund (Lund, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 Published by Elsevier Science B.V.

CHAPTER 1

The magnocellular neuronal phenotype: cell-specific gene expression in the hypothalamo-neurohypophysial system H. Gainer ∗ , M. Yamashita 1 , R.L. Fields, S.B. House and M. Rusnak Section on Molecular Neuroscience, Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, MD 20892, USA

Abstract: The magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system are exceptional cell biological models to study mechanisms of cell-specific gene expression and neurosecretion of neuropeptides in the central nervous system. Single cell differential gene expression experiments have further defined these phenotypes by identifying novel and distinct regulatory molecules in these neurons. Transgenic mouse studies have led to the intergenic region (IGR) hypothesis, which states that the DNA sequences between the OT- and VP-genes contain critical enhancer sites for their cell-specific expression. The recent cloning and sequencing of the human IGR, and its comparison with the mouse IGR sequence has identified conserved sequences as putative, cell-specific enhancer sites which are now being evaluated by biolistic transfections of organotypic hypothalamic cultures. With these data, it is possible to target the gene expression of specific molecules to magnocellular neurons both in vivo and in vitro, in order to perturb and/or visualize neurosecretory and other processes. Keywords: Oxytocin; Vasopressin; Cell-specific gene expression; Magnocellular neuron; Apoptosis; Biolistics; Neurosecretion

Introduction The magnocellular neurons (MCNs) of the mammalian hypothalamo-neurohypophysial system (HNS) synthesize and secrete the nonapeptides, oxytocin (OT) and vasopressin (VP) at exceptionally high rates. Because of this property, these neuroendocrine cells have served as important model systems for the study of mechanisms of peptide biosynthesis and secretion in the central nervous system (CNS). In addition, the relatively easy access to the MCN cell

∗ Correspondence to: H. Gainer, Laboratory of Neurochemistry, NINDS/NIH, Building 36, Room 4D04, Bethesda, MD 20892-4130, USA. Tel.: +1-301-496-6719; Fax: +1-301-496-1339; E-mail: [email protected] 1 Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan.

bodies in the paraventricular (PVN) and supraoptic (SON) nuclei and axons in the median eminence by both stereotaxic and micropunch methods, and to their nerve terminals due to their presence in the posterior pituitary outside of the blood–brain barrier (Fig. 1), have made these neurons favorite subjects for many biochemical and physiological studies (Morris et al., 1978; Hatton, 1990, 1997a,b; Meister, 1993; Gainer and Wray, 1994; Armstrong, 1995; Burbach et al., 2001). Ever since it was first described in the fish central nervous system over 70 years ago (Scharrer, 1928, 1987), the HNS has been a veritable ‘rosetta stone’ for neuroendocrinology and neuroscience. Many seminal findings have been made using the HNS. These include the first discovery and characterization of neuropeptides (Du Vigneaud, 1954), the early development of peptide agonists and antagonists (Jard et al., 1987; Manning et al., 1987; Manning and Sawyer, 1989), and the first proposal of the prohormone con-

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Fig. 1. Oxytocin- (OT) and vasopressin- (VP) synthesizing neuronal systems in the hypothalamus. These include the MCNs in the paraventricular nuclei (PVN) and supraoptic nuclei (SON), constituting the hypothalamo-neurohypophysial system (HNS), parvocellular (CRH) neurons in the PVN projecting to the median eminence (ME), and in the suprachiasmatic nuclei (SCN). The inset shows immunocytochemically identified OT and VP neurons in the PVN stained gray and black, respectively. Abbreviations: A, anterior; I, intermediate; and P, posterior regions of the pituitary. Adapted from Brownstein et al. (1980).

cept (Sachs and Takabatake, 1964; Sachs et al., 1969). In addition, MCN studies provided the first full characterization of bursting pacemaker activity in CNS neurons (Leng, 1988), the first demonstration of neuropeptide secretion from dendrites (Ludwig, 1998), novel views of glial–neuronal interactive plasticity (Theodosis and Poulain, 1987; Hatton, 1997a) and glial influence on synaptic transmission (Oliet et al., 2001), and the demonstration that peptides can produce complex behaviors (Reijmers et al., 1998; Wang et al., 1998; Insel and Young, 2001). However, despite intense investigations by many laboratories over the past 25 years to address the central question of how MCNs produce such high rates of OT and VP transcription, biosynthesis and secretion in a cell-specific manner (Burbach et al., 2001), the answer remains elusive.

The MCN phenotype The MCNs in the HNS have been classified into two distinct phenotypes, the OT and VP neurons. This historic classification (Vandersande and Dierickx, 1975; Dierickx et al., 1978) has been reinforced by many immunocytochemical (ICC) and in situ hybridization histochemical (ISHH) studies. As a result, it became the generally accepted view that expression of the OT and VP genes was mutually exclusive and occurred in separate cells in the HNS (Mohr et al., 1988). These two phenotypes in rats have also been discriminated by their distinct electrical activity patterns in response to sustained physiological stimuli, i.e. OT cells increase their action potential frequencies with regular, continuous firing, whereas VP cells generate phasic bursting patterns in response to steady depolarization (Leng, 1988).

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These different properties have been attributed, in part, to higher levels of calcium-binding proteins (e.g. calbindin, calretinin, etc.) in OT neurons which prevent depolarizing after potentials considered critical for bursting, and to a novel non-activating outward potassium current found only in OT cells (Hatton, 1990, 1997b; Armstrong, 1995; Armstrong and Stern, 1998). The latter may underlie the milkejection high-frequency bursts observed only in OT cells. While the division of the MCNs into OT and VP phenotypes is generally accepted, these neurons also contain smaller amounts of other coexisting peptides (e.g. galanin, cholecystokinin, CRH, dynorphin, enkephalin, TRH, etc.) which can vary between cells depending on functional conditions (Meister, 1993), and suggesting a more subtle heterogeneity within the OT and VP neuronal phenotypes. Most interesting is that several laboratories, using ICC and ISHH, have reported coexistence between OT and VP peptides (Mezey and Kiss, 1991) and mRNAs (Kiyama and Emson, 1990; Glasgow et al., 1999; Xi et al., 1999) in about 1–3% of MCNs in the normal rat SON, and that this increases substantially to 17% after 2 days of lactation (Mezey and Kiss, 1991; Glasgow et al., 1999). Thus, while the segregation of OT and VP gene expression in separate cells in the HNS is the rule, it is clear that substantial coexistence of these two robustly expressed peptides can occur under some physiological conditions. Re-examination of the OT and VP MCN phenotypes by single-cell gene expression analysis In recent years, analyses of gene expression profiles in single neurons under various biological conditions (Eberwine et al., 1992) have provided new opportunities to examine the phenotypic diversity of cells in a complex organ such as the brain (Mackler and Eberwine, 1993). In a number of cases, cDNA libraries have been constructed from single neurons (Dulac and Axel, 1995; Merz et al., 1995; Korneev et al., 1996), and differential screening of cDNA libraries constructed from single olfactory neurons has led to the identification of pheromone receptors in mammals (Dulac and Axel, 1995). The magnocellular OT and VP neurons are excellent candidates for such a differential analysis. Because of their large size, and given their high rates of transcription, it

might be possible to identify unique mRNAs encoding specific transcriptional regulatory factors responsible for the cell-specific peptide gene expression in these cells by this approach. A strategy that we have used on the MCNs for this purpose is illustrated in Fig. 2. For the differential screening of OT- and VP-cell cDNA libraries, it is essential that the cDNAs being used be derived from an unequivocal cellular phenotype. We chose to use MCNs from lactating rats because OT and VP gene expression and the nonapeptide secretions are greatly upregulated in the HNS under this physiological condition. Since the chance that an isolated single cell taken at random from lactating rats is of the OT/VP coexistent phenotype is about 17% (Mezey and Kiss, 1991; Glasgow et al., 1999), we first evaluated whether the OT and VP expression phenotypes of single isolated MCNs from dissected rat SONs could be determined by RTPCR procedures. We found that even without PCR amplification, only 1–5% of a single MCN’s cDNA sample was necessary for an unequivocal assessment of the phenotypes, and the remaining cDNA could then be assayed for at least 25 other specific genes (Glasgow et al., 1999). For the preparation of cDNA libraries we amplified with PCR the cDNAs before the phenotype analysis (Fig. 2). Using the protocol described in Fig. 2, we successfully constructed separate cDNA libraries for the OT and VP neuronal phenotypes. Each library was derived from ten unequivocally characterized OT or VP MCN cDNAs which were pooled in order to minimize experimental variability. After completion of the differential screening of these cDNA libraries, we sequenced all the differentially expressed clones. A preliminary functional characterization of the clones that were obtained, based on southern blot analysis and DNA sequences, is shown in Table 1. OT and VP mRNAs were obtained only from the OT or VP cDNA libraries, respectively, thereby validating their specificities. Interestingly, most of the differentially expressed genes that we found were of unknown function, and preliminary in situ hybridization studies have shown that three of these are expressed exclusively in the HNS (not illustrated). In summary, the ability to generate single, identified, cell-specific (OT or VP neuron) cDNA libraries has allowed us an opportunity to differentially screen

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Fig. 2. Overview of single cell-differential hybridization strategy. Individual magnocellular neurons are isolated from dissociated rat supraoptic nuclei (see inset), their mRNAs reverse transcribed into cDNA which is then PCR amplified. The individual OT and VP MCN amplified cDNAs are phenotypically characterized, and then utilized for producing phenotypically specific cDNA libraries and finally used for differential screening. See text.

for uniquely and/or preferentially expressed genes in each neuronal phenotype. Some of these could be transcriptional-activating factors, but obviously genes encoding proteins involved in other cell functions (e.g. signal transduction, membrane receptors, etc.) would also be of interest.

Cell-specific expression of OT and VP genes in MCNs: the background The DNA sequences and structures of the OT and VP genes in several mammalian species has been known for more than a decade. The OT and VP genes each contain three exons and two introns, are found on the same chromosome (chromosome 2 in the mouse and chromosome 20 in the human) in close apposition,

5 TABLE 1 Differential analysis of single OT and VP magnocellular neuronal gene expression Oxytocin cell-specific genes Gene Oxytocin Vasopressin Secretion associated proteins Putative transfactors Unknown function

No. of hits (total = 43) 19 (44%) 0 6 1 17 (40%)

Vasopressin cell-specific genes Gene Oxytocin Vasopressin Secretion associated proteins Putative transfactors Unknown function

No. of hits (total = 51) 0 10 (20%) 2 1 38 (75%)

and in opposite transcriptional orientations (Burbach et al., 2001). Despite the availability of this information, very little is known about the mechanisms that are responsible for the cell-specific expression of these genes. This is, in large part, due to the lack of homologous cell lines that express these genes which then could serve as relevant experimental models for conventional deletion construct analyses. Extensive studies using heterologous cell lines that are derived from cells that do not normally express these genes have been very useful for the identification of various regulatory cis-elements in the OT and VP genes (Burbach et al., 2001). However, heterologous cells are by definition, irrelevant models for cell-specific gene expression analyses. Consequently, most of the data pertinent to the question of cell-specific gene expression of OT and VP genes has been obtained using transgenic models and by assaying expression in vivo in the MCNs (reviewed in Waller et al., 1998; Burbach et al., 2001; Gainer and Young, 2001). Transgenic mouse studies Experiments in transgenic mice and rats (Waller et al., 1998; Burbach et al., 2001; Gainer and Young, 2001) have led to the proposal that the cell-specific enhancers for OT and VP gene expression are not located on the 5 -upstream regions of these genes, but instead are present in the IGR downstream of the VP gene (Gainer, 1998; Gainer and Young, 2001).

This view is based on two general observations. The first is that constructs containing only 5 -flanking upstream regions of these genes (from 0.5–9 kb) connected either to intact, wild-type (usually heterologous) genes or to various reporter genes, do not produce cell-specific expression of these genes in MCNs of transgenic animals. Second, the inclusion of 3 -downstream flanking regions of the VP genes to these very same constructs can produce robust cell-specific gene expression in the magnocellular neurons. Subsequent experiments using deletion constructs of the VP component of the transgene suggested that the 3 -downstream region of the VP gene (about 0.6 kb of IGR) was the likely site of an enhancer for cell-specific expression of the OT gene. Further studies on the VP gene (Grant et al., 1993; Zeng et al., 1994; Waller et al., 1996, 1998; Jeong et al., 2001) showed that by extending the 3 downstream region of the VP gene to 2–3 kb, it was possible to get cell-specific expression of these transgenes in VP neurons only. These observations have led to a hypothesis, termed the ‘intergenic region’ (IGR) hypothesis (Gainer, 1998), which states that the cis-elements in the genomic DNA responsible for the cell-specific expression of OT- and VP-genes are located downstream of exon III of the VP gene. Insights from comparative genomics A useful theoretical approach is to compare the IGR sequences in different species, in order to identify conserved sequences that could be candidate regulatory elements. The recent demonstration that isotocin genes from fish can be expressed in a cell-specific manner in OT neurons in transgenic rats (Venkatesh et al., 1997) suggested a significant evolutionary conservation of the cell-specific determinants. In an earlier attempt to identify these enhancer elements, a comparison was made between the mouse and rat IGR sequences, but these two sequences, with the exception of a 6.4-kb LINE insert present only in the rat, were found to be too similar to allow for discrimination of conserved sequences that might be relevant for cell-specific gene expression (Ratty et al., 1996). The mouse and rat are separated by only one million years of evolution, and hence there appeared to be insufficient time for the divergence of non-essential sequences. In contrast, the human and

6 TABLE 2 Conserved sequences in mouse and human IGRs Motif

Position a in mouse IGR

Position a in human IGR

Putative OT-enhancer cluster AGGGGAG GTCGTG AGGAAGCGATC AGAGAGG GGACAA AACTGCTA CTTGACC

43 107 173 321 340 781 807

24 93 130 275 294 918 685

Putative VP-enhancer cluster TTCTCTCT GCCACA GGGATGCTCTGCA CTAAGGTCAGGGG CAGAGTC CTGGGT ACAGGGACCA CTCAGG GGCAGGT TTCAATCC GTGTATGGA TCCTCT AATCAGGCT GAGTCCC CAGCTACAGAGCCAT

1293 1358 1379 1396 1479 1552 1571 1618 1693 1717 1730 1777 1832 1921 1933

1097 1167 1444 1461 1316 1428 1447 1508 1597 1621 1894 1681 1739 1844 1857

Other conserved sequences AGGCCAG GCCCCCTCAACCTCT TCCCGTTTC CAATTAGACACCAGC

3211 3222 3541 3553

9592 9603 9994 10006

Analogous transfactor b GKLF, Ik-2, MZF1, GC BOX, SP1

V-Myb ER

Ik-2 RORα1, V-ErbA

Ik-2 TCF11, AP-1 δEF1 Gfi-1

RORα1 Ik-2 Ap-4, CCCAAT box

a

Position numbers start from the first base following exon III of the VP gene in the mouse and human IGRs (i.e position 43 for the mouse would be base 43 in the mouse IGR sequence accession no. U38901 and position 24 in human would be base 24 in the human IGR sequence accession no. AF254641). b The MatInspector V2.2 program was used to search for transcription factor consensus binding sequence matches. Abbreviation: GKLF, gut enriched Kruppel-like factor (Shields and Yang, 1998); Ik-2, Ikaros genezinc finger protein (Molnar and Georgopoulos, 1994); MZF1, myeloid zinc finger protein (Morris et al., 1994); GC BOX, GC box elements (Buche, 1990); SP1, stimulating protein; V-Myb, v-Myb protein (Grotewold et al., 1994); ER, estrogen receptor; V-ErbA, viral homolog of thyroid hormone receptor α1 (Subauste and Koenig, 1995) TCF11, CNC-bZIP factor (Johnsen et al., 1998); RORα1, novel orphan hormone nuclear receptor (Giguere et al., 1994); δEF1, δ-crystallin enhancer binding protein (Sekido et al., 1994); Gfi-1, zinc finger protein (Zweidler-McKay et al., 1996); Ap-4, activator protein 4; CCCAAT box, cellular and viral CCAAT box (Buche, 1990).

mouse are separated by about 100 million years, and we reasoned that this greater evolutionary distance might allow the conserved sequences in the IGR to be identified. We subsequently cloned and sequenced the human IGR (accession no. AF254641) and compared it to the mouse IGR sequence (Gainer et al., 2001). Twenty-six sequence motifs ranging from 6 to 16 bp in length were found to be conserved between the mouse and are located in three distinct clusters

in the mouse IGR. Twelve of the 26 sequences are similar to consensus motifs for known transcription factors and five of these are zinc finger proteins (Table 2). The conserved motifs found in the first two clusters in Table 2 are locations that are consistent with the available transgenic data which suggest that the elements required for cell specific expression of OT and VP reside 10 μM) attenuate or block the signaling of several GPCRs, including the OT receptor via a fast, reversible and non-genomic pathway. Progesterone is known to inhibit both cholesterol biosynthesis and the intracellular trafficking of cholesterol. We therefore test the hypothesis that progesterone affects the signal transduction and subdomain localization of receptors via its influence on cholesterol trafficking. Since cholesterol-rich subdomains (rafts) are considered to be organization centers for cellular signal transduction, changes of the level or distribution of cholesterol may have profound effects on receptor-mediated signaling in general. Using fluorescence recovery after photobleaching (FRAP) measurements with GFP-tagged oxytocin receptors the influence of steroids on the mobility and distribution of the oxytocin receptor in the plasma membrane was analyzed. Progesterone had no effect on the lateral mobility of the oxytocin receptor, but it led to marked inhibition of cellular motility such as vesicle trafficking and movements of filopodia. Non-genomic effects of progesterone and estradiol with respect to receptor signaling as well as the influence of cholesterol on signal transduction will be discussed in more detail. Keywords: Oxytocin receptor; Cholecystokinin receptor; Cholesterol; Progesterone; Steroids; Non-genomic effects; Signal transduction; Affinity state

∗ Correspondence to: G. Gimpl, Institute of Biochemistry, Johannes Gutenberg-University of Mainz, Becherweg 30, D-55099 Mainz, Germany. Tel.: +49-6131-3923829; Fax: +49-6131-3925348; E-mail: [email protected]

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Oxytocin receptor and cholesterol Cholesterol and its effect on the affinity state of the oxytocin receptor Oxytocin (OT) receptors require at least two essential components for high-affinity OT-binding, divalent cations such as Mg2+ and cholesterol. This is observed in different systems with both membraneembedded and solubilized receptors. Following solubilization with the detergent Chapso, OT receptors lose characteristic binding properties, the affinity for OT becomes lower and/or additional low-affinity state receptors appear in the extract. Solubilization with Chapso leads to a substantial cholesterol depletion of the soluble extract and we could demonstrate that substitution with cholesterol markedly enhanced the OT binding of soluble OT receptors (Fahrenholz et al., 1995; Klein et al., 1995). This became first evident in reconstitution of soluble OT receptors using liposomes of defined composition. A saturable high-affinity OT binding was obtained only with liposomes that contained a critical amount of cholesterol. Moreover, when OT receptors were expressed in insect cells, which naturally have plasma membranes with low cholesterol content, the receptors are mainly in a low-affinity state (K d > 100 nM). Following addition of cholesterol to the culture medium, a fraction of OT receptors is converted from a low- to a high-affinity state (K d ∼ 1 nM) (Gimpl et al., 1995). The low-affinity state was identified as a physiologically active receptor state and the conversion of the affinity states to each other is to a certain degree reversible. The interaction of cholesterol with OT receptors is of high specificity and is not due to mere changes of membrane fluidity (Gimpl et al., 1997). Furthermore, cholesterol stabilizes both membrane-associated and solubilized OT receptors against thermal denaturation (Gimpl and Fahrenholz, 2000). Taken together, the data provide evidence for a direct and cooperative molecular interaction of cholesterol with OT receptors: Cholesterol acts as an allosteric modulator and stabilizes the receptor in a high-affinity state for agonists and antagonists. In some cell systems, populations of highand low-affinity OT receptors have been observed (Pliska et al., 1986; Crankshaw et al., 1990; Di-Scala and Strosser, 1995; Gimpl and Fahrenholz, 2000).

This could reflect uneven cholesterol distributions within the plasma membrane of these cells. Highaffinity state OT receptors are expected to be preferentially localized in cholesterol-rich subdomains of the plasma membrane. In fact, in HEK293 fibroblasts stably expressing the human OT receptor the high-affinity state receptors were found to be about 2-fold enriched in cholesterol-rich plasma membrane domains (Gimpl and Fahrenholz, 2000). Receptor heterogeneity with respect to affinity states may be much more pronounced in cell systems with abundant cholesterol-rich microdomains such as lipid ‘rafts’ or caveolae structures. Interestingly, Broderick and Broderick (1990) noticed a conspicuous abundance of caveolae in myometrial cells at term. Cholesterol acts as an allosteric modulator of the oxytocin receptor Our previous studies suggest a direct molecular interaction between the oxytocin receptor and cholesterol. Since the ligand-binding activity of the OT receptor declined sharply when the cholesterol amounts were reduced below a critical level (about 50% of the cholesterol content found in untreated membranes), the molecular cholesterol–receptor interaction might be explained by a cooperative mechanism. Hill analysis of cholesterol content versus OT binding suggests that the OT receptor binds several molecules of cholesterol (n = 6) in a positive cooperative manner. Alternatively, at a critical cholesterol level, the distribution of the cholesterol molecules in the vicinity of the receptor could change significantly, e.g. by alterations in the formations of cholesterol dimers or by rearrangement of cholesterol between both leaflets of the membrane bilayer. Divalent metal ions like Mg2+ were found to increase both the OT binding capacity and the affinity state of the OT receptor (Soloff, 1990). This is surprisingly similar to what cholesterol does. Mg2+ increases the potency of OT analogs in stimulating uterine contractions and has been proposed to display its effect on the OT-receptor interaction by influencing positive cooperativity (Pliska and Kohlhauf, 1991). Conclusively, cholesterol and Mg2+ are essential allosteric modulators of the OT receptor and may be involved in the regulation of OT-mediated signaling functions.

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Do these allosteric modulators play a role for the regulation of OT-related physiological processes? Particularly in reproductive tissues, the steroid and cholesterol concentrations are highly dynamic. Using freeze-fracture cytochemistry with the cholesterolbinding filipin, marked increases in cholesterol have been found in rat uterine epithelial cells at the time of blastocyst implantation (Murphy and Dwarte, 1987). In the human placental syncytiotrophoblast basal membrane, Sen et al. (1998) observed a steady decrease in cholesterol/phospholipid ratio in correlation with an increase in membrane fluidity during placental development. At term, however, the cholesterol to phospholipids ratio in syncytiotrophoblast membranes was found to be increased as compared with the cholesterol/phospholipid ratio in early placentas (Mazzanti et al., 1994). We previously found that cholesterol can modulate receptor function by both changes of the membrane fluidity and direct binding effects, e.g. in case of the oxytocin receptor (Gimpl et al., 1997). Plasma membranes with lowered cholesterol content showed a decreased capacity (Bmax ) of binding sites and/or a decreased affinity (K d ) of ligand-receptor binding. Interestingly, Lopez et al. (1995) reported that pregnancy in humans was associated with increases in both density and affinity of OT receptors. However, to draw further conclusions, more studies are required. The cholesterol binding site(s) of the oxytocin receptor To obtain further information about cholesterol binding site(s) of the OT receptor, a molecular modeling approach was employed. Previous studies revealed that the cholecystokinin type B (CCKB) receptor, a G protein coupled receptor with some homology to the OT receptor, does not depend on cholesterol. We therefore used the CCKB receptor as a negative control in the modeling approach. Based on these assumptions, a candidate cholesterol docking domain has been identified in the OT receptor but was clearly absent in the corresponding region of the CCKB receptor. Within this cholesterol docking site key positions of the human OT receptor such as P197, Y200, W203, M296 and W297 have been suggested as important residues which may be putatively involved in cholesterol binding. This is illustrated in Fig. 1. Ac-

cording to these suggestions the human OT receptor has been mutated at single amino acid residues (e.g. W203) as well as by exchange of C-terminal receptor fragments for corresponding fragments of the CCKB receptor, i.e. producing receptor chimeras. The results showed that even the OTR-CCKBR chimeras which possess the C-terminus of the CCKBR including about half of the third intracellular loop, the two transmembrane regions and the cytosolic part behave like the OT receptor with respect to its cholesterol dependence. This suggests that the cholesterol binding site is not localized in this C-terminal part of the OT receptor. Further mutations resulted in an OT receptor which is functionally inactive due to misfolding or incorrect transport to the plasma membrane. Misfolding or mistranslocation of receptor mutants is a very common observation, e.g. with mutants of the vasopressin V2 receptor leading to diabetes insipidus. On the other hand, it is possible that the OT receptor requires cholesterol to be correctly folded and transported to the plasma membrane. According to this interpretation, the third transmembrane domain and/or the residue W203 form a cholesterol docking site as proposed by the molecular modeling approach. But this has to be substantiated by further mutations. We assume that the major cholesterol binding site which has to be occupied to create a stable and high-affinity receptor conformation is localized at or nearby the oxytocin binding site, i.e. in the N-terminal part of the OT receptor. Taken together, it seems likely that the receptor’s agonist binding site is formed in a stable conformation only when one or more cholesterol molecules including a Mg2+ ion are present. The employment of further technical strategies is necessary to prove this hypothesis. Cholesterol acts as stabilizer of the oxytocin receptor The high stability against thermal inactivation of the OT receptor in the presence of cholesterol is another interesting receptor property. This is illustrated in Fig. 2A. Oxytocin receptors localized in cholesterolpoor membrane domains (i.e. HDM fractions) were significantly less stable than receptors residing in the cholesterol-rich low-density membrane fractions (Cav-LDM). Receptors in HDM domains could be

46 Fig. 1. Schematic model of the human oxytocin receptor indicating amino acid residues which are putatively involved in ligand-binding, cholesterol-binding and associated signal transduction events. The glutamine and lysine residues highly conserved within the vasopressin/oxytocin receptor family may partly define an agonist-binding pocket which is common to all the different subtypes of this receptor family. According to a molecular modeling approach (Fanelli et al., 1999), an oxytocin docking site has been proposed (marked by arrows). In the inactive receptor conformation, the highly conserved arginine (R137) may be constrained in a pocket which is formed by polar residues (indicated by asterisks). Following agonist binding this arginine side chain may be shifted out of the ‘polar pocket’ thereby unmasking a G protein binding site. Receptor domains putatively interacting with oxytocin, peptide oxytocin antagonists and Gαq are marked by lines and dashed boxes as indicated in the symbol legend (Postina et al., 1996; Hoare et al., 1999; Breton et al., 2001). The amino acid residues marked by a circle with asterisk edge have been predicted to form a cholesterol docking domain according to molecular modeling (Gimpl et al., 2000). However, biochemical data do not fully support this prediction (see text).

47

(a)

(b)

Fig. 2. Effect of cholesterol on the stability of the oxytocin receptor against thermal inactivation (A) and proteolytical degradation (B). (A) Caveolin-enriched low-density membrane domains (Cav-LDM) and cholesterol-poor high-density membrane domains (HDM) were incubated at 37°C for indicated periods of time. In a parallel set of assays the HDM membranes were first pretreated for 10 min at 30°C with cholesterol-MβCD (0.3 mM). After that time, the membranes were pelleted and were washed once with binding buffer. Then, the membranes were incubated for various times at 37°C. The high-affinity oxytocin binding activity was determined in a radioligand-binding assay using 5 nM of [3 H]oxytocin. The data represent the remaining binding activity in percent of control binding at time t = 0. The data are given as means ± S.D. of three experiments. (B) Immunoblot analysis of the degradation process of the oxytocin receptor in membranes of HEK293 cells stably expressing the FLAG-tagged oxytocin receptor–GFP conjugate. The membranes were incubated at 37°C for 0 min or 4 h in the absence (lanes 1, 3) or presence (lanes 2, 4) of 0.3 mM Chol-MβCD. At the indicated time, the membranes were processed for SDS–PAGE (reducing conditions) and subsequent immunoblot. For specific receptor detection the anti-FLAG antibody M2 was used. The sharp band at ∼75 kDa corresponds to the deglycosylated receptor.

stabilized against thermal inactivation when additional cholesterol was supplied to the membranes (Fig. 2A). We further explored this receptor property and asked whether cholesterol is also capable to protect the receptor from proteolytic degradation. This is in fact the case as shown in Fig. 2B. The assumption of direct cholesterol–receptor binding is the most straightforward interpretation of the high resistance to proteolytic degradation of the oxytocin receptor in the presence of additional cholesterol. Does the proteolytical degradation of the oxytocin receptor play a physiological role, e.g. in case of normal receptor turnover or signaling offset as observed for the vasopressin 2 receptor (Kojro and Fahrenholz, 1995). If so, then oxytocin receptors residing in a cholesterol-rich microdomain are expected to show prolonged receptor signaling. We are engaged to prove this hypothesis.

Cholesterol and its effect on the mobility of OT receptors It is well established that cholesterol functions as the main regulator of membrane fluidity in eukaryotic cells. Alterations in the cholesterol content should therefore induce changes of the receptor mobility if the receptor floats passively in the lipid bilayer. Fluorescence recovery after photobleaching (FRAP) experiments were performed to address this question in more detail. After a brief 1-s spot bleaching of the GFP-tagged oxytocin receptor stably expressed in untreated HEK293 cells, the recovery normally occurs with a t1/2 of about 20 s with a mobile fraction of about 80% of the receptors (Table 1). This is about 10 times faster than the recovery process of oxytocin receptors localized in vesicles inside the cell. Cholesterol depletion below a threshold concentration (10 μM) are required to attenuate or block the signaling of the OT receptor (Burger et al., 1999). Using these high concentrations, however, progesterone is capable to attenuate or block the signaling function of all G protein coupled receptors that we have tested (Burger et al., 2000). The progesterone effects occurred within minutes, were reversible and could not be blocked by a protein synthesis inhibitor. Overall, the action of progesterone was found to be more cell type-specific than receptor-specific. Obviously, progesterone displays an inhibitory effect on receptor signaling via a more general mechanism. The question arises whether under physiological conditions those high micromolar concentrations of progesterone are found in cells in vivo. The progesterone levels in plasma or in nonsteroidogenic tissues are normally much lower. In steroidogenic tissues, however, very high amounts of progesterone have been measured. Near term, the human placenta secretes upward of 300 mg of progesterone daily. The progesterone content of this organ was shown to be 7 μg/g wet tissue (Simpson and Burkhart, 1980). In human corpus luteum, progesterone concentrations reached peak levels of about 25 μg/g tissue shortly after ovulation and in the early luteal phase (Swanston et al., 1977). Thus, in steroidogenic cells as well as in their environment, progesterone might be present at high doses which are able to attenuate the signaling of many receptors including the oxytocin receptor. What are the molecular mechanisms underlying this non-genomic progesterone action which is pre-

49

sumably operative in all cell types? Progesterone is long known to profoundly affect the intracellular trafficking of cholesterol. For example, cell biologists routinely use this steroid hormone and certain hydrophobic amines to induce a Niemann–Pick-type C-like distribution of cholesterol in cells. In this respect, progesterone is the most potent of natural steroids, with maximal effects occurring at 30 μM (Butler et al., 1992). Although it generally takes several hours of incubation with progesterone to induce this pathophysiological cholesterol distribution in cells, progesterone may start to disturb the cholesterol trafficking machinery within minutes. Presently, there are no adequate methods to detect these early changes of cholesterol trafficking. One candidate progesterone-binding protein is the multidrug resistance P-glycoprotein (Qian and Beck, 1990). This protein does not only play a role in detoxification, but appears also be involved in intracellular cholesterol transport. It has been demonstrated that progesterone inhibits both the cholesterol esterification and the transport of cholesterol to and from the plasma membrane. In particular, progesterone reduces the cholesterol pool residing in caveolae (Smart et al., 1996). Paradoxically, at the same time progesterone stimulates the activity of HMG-CoA reductase, the key enzyme of de novo cholesterol biosynthesis. Hence, cholesterol precursors like lanosterol begin to enrich in the membranes of the cell. On the basis of these progesterone-induced events the following scenario (see model in Fig. 3) may be operative. As mentioned above, the OT receptor needs a cholesterol-rich microenvironment to become stabilized in its high-affinity state. Since the cholesterol precursors, particularly lanosterol, are completely inactive to support the OT receptor in its high-affinity state (Gimpl et al., 1997), the responsiveness of the OT system may not be fully operative during the continuous presence of high progesterone concentrations. According to this scenario, progesterone withdrawal would restore the cholesterol transport so that the highly enriched amounts of cholesterol precursors would now become rapidly converted to cholesterol. This would lead to a sudden rise of cholesterol and should push the responsiveness to OT since low-affinity OT receptors could now be converted into their high-affinity state. According to this postulated mechanism, progesterone could af-

fect the signaling of all those receptors which are functionally dependent on cholesterol. Nevertheless, these events could only occur in cells which ‘see’ micromolar doses of progesterone, e.g. in steroidogenic tissues and their environment. Most likely, progesterone acts in these tissues via both genomic and non-genomic pathways together with other steroids to control receptor activity. General aspects of steroids and signaling Cholesterol in signal transduction Membrane cholesterol is considered to play a crucial role for signal transduction processes in multiple ways, e.g. (1) by its control of the fluidity state and biophysical properties of the lipid bilayer, (2) by formation of lipid rafts and caveolae, (3) in the hedgehog signaling pathway, (4) by activation of proteins via sterol-sensing domains, (5) as direct modulators of membrane proteins, e.g. GPCRs and ion channels. Caveolae are small invaginations of the plasma membrane and are considered to be a specialized form of rafts that contain the cholesterol-binding structural protein caveolin. Several membrane receptors such as platelet-derived growth factor, receptors for epidermal growth factor, adrenalin, bradykinin, endothelin, cholecystokinin, and oxytocin are enriched in caveolae or caveolae-like domains. In caveolae, receptors seem to be functionally connected to molecules of the signaling cascades, e.g. G protein α subunits, protein kinase Cα, mitogen-activated protein kinase, adenylyl cyclase and phosphoinositides (reviewed in Okamoto et al., 1998). Isshiki et al. (1998) demonstrated that in endothelial cells, calcium waves upon intracellular calcium release originate from caveolae-rich cell edges. Caveolin and caveolae are involved in intracellular cholesterol transport and cholesterol efflux to extracellular carriers (Bist et al., 1997; Liu et al., 1999). Thus, accumulating evidence suggests that dynamic cholesterol-dependent regulation of specific signal transduction pathways occurs within caveolae. Unlike caveolae, which are abundantly present in many but not in all cells, sphingolipid-cholesterol subdomains, often designated as rafts, may be present in every cell although there is some controversy on

50

Fig. 3. Schematic model of non-genomic inhibitory effects of progesterone. Progesterone inhibits both the signal transduction of Gq -coupled receptors (as shown here for the oxytocin receptor) and the intracellular trafficking of cholesterol. Principally, eukaryotic cells can obtain the required cholesterol (Chol, grey ellipses) by two sources: endogenously by de novo synthesis of cholesterol and exogenously by uptake of cholesteryl-ester (CE)-rich LDL (low-density lipoprotein) particles via receptor-mediated (R) endocytosis. De novo synthesized cholesterol first arrives at cholesterol-rich domains in the plasma membrane (caveolae and/or ‘lipid-rafts’), which may function as cholesterol-‘sorting centers’ within the plasma membrane. Progesterone blocks several intracellular transport pathways of cholesterol (black bars) except for the LDL receptor-mediated uptake of cholesterol. Moreover, cholesterol esterification does not occur in the presence of progesterone, presumably due to the lack of cholesterol substrate for acyl-CoA : cholesterol acetyltransferase (ACAT). As a consequence, unesterified cholesterol accumulates in lysosomes (or late endosomes) and lysosome-like compartments (designated as ‘lamellar bodies’). The key enzyme for the cholesterol de novo synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA Red), is stimulated in the presence of progesterone. But the cholesterol biosynthesis stops at the level of precursors. Overall, progesterone induces a state of cholesterol auxotrophy. However, following progesterone withdrawal, the accumulated precursors will be rapidly converted to cholesterol. Thus, cells will become overloaded with cholesterol for a certain period of time after which the cholesterol homeostasis will be reestablished. These reversible progesterone-induced changes of the cholesterol trafficking could have a strong influence on signal transduction processes, particularly in case of the OT receptor (OTRH and OTRL , high-affinity and low-affinity OT receptor). Adapted from Gimpl and Fahrenholz (2001).

their existence in vivo. They could provide platforms for efficient initiation of signaling cascades by bringing together receptors with their second messenger effectors (Incardona and Eaton, 2000; Simons and Toomre, 2000). As a component of the recently identified hedgehog pathway which is of fundamental importance, e.g. for the patterning of the nervous system, cholesterol plays a central role for ligand biogenesis and for signal transduction in receiving cells. Hedgehog proteins are morphogens posttranslationally modified with a covalently attached cholesterol molecule which anchors the protein in the outer leaflet of the

membrane bilayer and mediates its association with lipid rafts. The hedgehog receptor complex consists of Patched, a polytopic transmembrane protein with structural similarity (sterol sensing domain) to the Niemann–Pick C1 (NPC1) protein and a second protein, termed Smoothened, a heptahelical organized receptor like a GPCR. Patched acts as the ligand-binding subunit and regulates the activity of Smoothened, which functions as the signaling subunit. In the absence of ligand, Patched inhibits a tonic activity of Smoothened. The inhibition is released upon hedgehog binding thus activating a signal transduction cascade. Conclusively, in the hedgehog

51

signaling complex cholesterol participates in signal generation by controlling the expression and patterning of the hedgehog ligand, as well as in signal reception due to the relationship of Patched to NPC1 and other proteins with sterol sensing domains. Reduced hedgehog signaling dramatically manifests in holoprosencephaly, a severe brain malformation. In addition, other environmental and genetic causes of holoprosencephaly are related to sterols or sterol metabolism (Incardona and Eaton, 2000). Changes of the cholesterol level influence receptor-mediated signal transduction at several levels. Cholesterol depletion led to constitutively activation of the p42/44 MAP kinase cascade, and epidermal growth factor caused hyperactivation of ERK in cholesterol-depleted cells (Furuchi and Anderson, 1998; Galbiati et al., 1998). Similarly, Visconti et al. (1999) found that removal of cholesterol is able to initiate transmembrane signaling leading to subsequent stimulation of protein tyrosine phosphorylation and sperm capacitation. At the receptor level, there is evidence for a direct cholesterol interaction with a number of receptors such as the oxytocin receptor, galanin receptor (Pang et al., 1999), opioid receptor (Lagane et al., 2000), nicotinic acetylcholin receptor (Rankin et al., 1997), and rhodopsin (Albert et al., 1996). Dependent on the receptor type, cholesterol is able to modulate the affinity state, the binding capacity, the ligand-binding specificity, and/or the conformational stability of the receptor. Receptors which are not directly interacting with cholesterol may be affected in their binding properties by cholesterols’ effect on the membrane fluidity as shown for the cholecystokinin B receptor (Gimpl et al., 1997). Our recent analysis of ligand-induced calcium responses at different cholesterol contents of the cells revealed that many GPCRs require cholesterol for optimal signal transduction (Burger et al., 2000). Since both clathrin coat- and caveolae-mediated endocytosis require cholesterol, signal transduction is also expected to be cholesterol-dependent at the level of receptor internalization and downregulation (Subtil et al., 1999). Steroid hormones and signaling There is a growing number of examples of rapid, nongenomic signaling by steroids such as aldosteron-in-

duced increases in intracellular calcium in vascular smooth muscle cells, vitamin D-induced increases in intracellular calcium in osteosarcoma cells (Caffrey and Farach-Carson, 1989), and progesteronemediated maturation of amphibian and fish oocytes. 17-β-Estradiol has been found to induce Ca2+ dependent translocation of eNOS (Goetz et al., 1999) and might contribute to smooth muscle relaxation via direct binding to the β-subunit of the Maxi-K channel, which confers the channel with a higher Ca2+ sensitivity (Valverde et al., 1999). Valera et al. (1992) observed an inhibitory effect of progesterone on the neuronal nicotinic acetylcholine receptor. Administration of progesterone has been demonstrated to inhibit the transmembrane Ca2+ entry, to promote Ca2+ release from intracellular stores and membrane hyperpolarization subsequently activating K+ channels (Mironneau et al., 1981). As mentioned above, progesterone and closely related analogues inhibited the Ca2+ mobilization induced by stimulation of many GPCRs (Burger et al., 1999, 2000) in CHO and HEK293 cells. Oxytocin- and endothelin-1-induced increases in [Ca2+ ]i were also attenuated in progesterone-treated myometrial cells (Fomin et al., 1999). Table 2 gives a list of some selected presumably non-genomic effects induced by the steroid hormones progesterone and 17-βestradiol. A number of steroids act as important modulators in the central nervous system where several of them are de novo synthesized as neurosteroids. Modulatory effects of neurosteroids have been reported on the neuronal nicotinic acetylcholine receptor (see above), glycine receptors (Wu et al., 1990), nonNMDA glutamate receptors (Wu et al., 1991), the GABAA receptor (Majewska et al., 1986), and 5HT3 serotonin receptors (Wetzel et al., 1998). Thus, neurosteroids seem to function as general modulators of ligand-gated ion channels. On the other hand, there is still controversy about the specificity of these effects (Rupprecht and Holsboer, 1999). One crucial question is whether these various nongenomic actions of steroids are caused by interaction with the corresponding proteins or, alternatively, are due to the interference with a common and basic cellular mechanism. Administration of steroids may for example disturb the Ca2+ metabolism or directly interact with components of the cytoskele-

52 TABLE 2 Multiple actions of progesterone and estradiol presumably involving nongenomic mechanisms Steroid

Effect

References

Progesterone

Maturation of oocytes

Progesterone Progesterone Progesterone Progesterone Progesterone Progesterone Progesterone Progesterone Estradiol

Acrosome reaction of sperm Inhibitory modulation of nAChR Inhibitory effect on GPCR signaling Ligand of sigma receptor Cholesterol trafficking defects Cholesterol synthesis inhibitor Production of meiosis-activating sterols Counteracting of pregnenolone-induced stimulation of microtubuli assembly Neuropsychopharmacological actions (anesthetic, sedative, anxiolytic) Activation/translocation of eNOS

Estradiol Estradiol Estradiol Estradiol

Activating of MAP kinase Binding to Maxi-K channel β subunit Modulation fo GPCR signaling Neuroprotection

Blondeau and Baulieu (1984); Bayaa et al. (2000); Tian et al. (2000) Blackmore (1999) a Valera et al. (1992) Burger et al. (1999) Su et al. (1988) Butler et al. (1992) Metherall et al. (1996) Lindenthal et al. (2001) Murakami et al. (2000) Rupprecht and Holsboer (1999) a Goetz et al. (1999); Hisamoto et al. (2001) Russell et al. (2000) Valverde et al. (1999) Kelly and Wagner (1999) a Moosmann and Behl (1999)

a

Review articles summarizing many original findings to that topic.

ton. In this respect, pregnenolone and progesterone have been shown to affect microtubuli assembly and both steroids were identified as high-affinity ligands for microtubuli-associated protein 2 in neuronal hippocampal cells (Murakami et al., 2000). Recently, a connection between neurosteroids and the mysterious sigma receptor has been proposed. A number of findings suggest that sigma receptors mediate or at least influence the effects of systemically administered neurosteroids. Progesterone is to date the only natural ligand for the sigma1 receptor which surprisingly, exhibits a high homology to a fungal sterol isomerase (Hanner et al., 1996). The physiological significance of this homology is yet unclear. Perhaps sigma receptors are enzymatically involved in the synthesis of certain neurosteroid modulators. While the classical steroid hormones are produced in specialized steroidogenic cells starting from cholesterol, another group of steroids with putative reproductive functions have been identified as C29 intermediates of the de novo cholesterol synthesis pathway at the postsqualene level. Due to their effects these sterols are summarized as meiosis-activating sterols. Interestingly, Lindenthal et al. (2001) recently observed that administration of progesterone to a hepatoma cell line and rat testes

led to an accumulation of these meiosis-activating sterols via inhibition of certain postsqualene steps of cholesterol biosynthesis. These findings underline the putative importance of cholesterol precursors in reproductive tissue and a new possible role for progestins in this process. These few examples demonstrate that much effort will be necessary in the future to clarify the biochemical mechanisms of the multiple functions of steroids. Abbreviations Cav-LDM

caveolin-enriched low density membranes CCKBR cholecystokinin type B receptor eNOS endothelial NO synthase FRAP fluorescence recovery after photobleaching GPCR G protein coupled receptor HDM high-density membranes MAP kinase mitogen-activated protein kinase nAChR nicotinic acetylcholin receptor OT oxytocin OTR oxytocin receptor

53

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 5

Central vasopressin systems and steroid hormones Andries Kalsbeek ∗ , Inge F. Palm and Ruud M. Buijs Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands

Keywords: Circadian; Corticosterone; Estrogen; Luteinizing hormone; Paraventricular nucleus; Supachiasmatic nucleus; Sexual dimorphism; Vasopressin

Vasopressin systems in the brain The physiological effects of vasopressin (VP) as a peripheral hormone were first reported as early as 1895 (Oliver and Schäfer, 1895). However, it was almost 60 years before the probable source of VP released in the general circulation was described by Bargmann and Scharrer (1951). They proposed that VP was released from neurons residing in the supraoptic (SON) and paraventricular nucleus (PVN) that projected to the posterior pituitary. Only some 15 years after the localization of VP in the brain, a possible central function for VP was described for the first time by De Wied (1965). At first the central effects of VP were attributed to a ‘feedback’ action of VP released as a hormone from the neurohypophysis. The first immunocytochemical studies using VP antibodies were mainly intended to investigate in more detail the magnocellular VP neurons in the SON and PVN (Swaab and Pool, 1975; Vandesande and Dierickx, 1975; Vandesande et al., 1975), but in these initial studies VP was also detected in the suprachiasmatic nucleus (SCN). Subsequent, more sensitive immunocytochemical methods revealed an extensive distribution of VP-containing fibers outside the hypothalamus (Buijs, 1978; Buijs et al., ∗ Correspondence to: A. Kalsbeek, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. Tel.: +31-20-566-5500; Fax: +31-20-696-1006; E-mail: [email protected]

1978; Buijs and Swaab, 1979) and a neurotransmitter role for VP could be hypothesized. Initially the different intra- and extrahypothalamic VP projections were all thought to originate from the PVN and SON neurons and the SCN. However, using specific brain lesions or colchicine pretreatment to block axonal transport a considerable part of the VP innervation was found to be derived from additional VP-producing neurons located in the bed nucleus of the stria terminalis (BNST) and the medial amygdala (AME) (Hoorneman and Buijs, 1982; De Vries and Buijs, 1983; Van Leeuwen and Caffé, 1983). Consequently, using a wide variety of different techniques, the largest part of the VPergic innervation in the brain could be traced back to four major VP systems: (1) the sexually dimorphic system, with VP neurons localized in the BNST and the AME; (2) the autonomic and (3) endocrine system originating from parvocellular VP neurons located in the PVN; and (4) the biological clock system derived from VP neurons located in the SCN. It is still a matter of debate whether the VP system discovered first, i.e. the magnocellular VP neurons in the PVN and SON that produce VP as a hormone, also contribute to the central projections. Some double-labelled neurons in the PVN after tracer injections in both the neural lobe and spinal cord seem to indicate the latter. Steroid hormones and central VP systems have been found to interact in a variety of ways. The clearest example is presented by the sexually dimorphic system, which shows a complete disappearance

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in the absence of gonadal steroids. The VP neurons of the endocrine system show an opposite response in the sense that VP synthesis in these neurons is upregulated in the absence of the adrenal glucocorticoids. On the other hand, the VP neurons of the biological clock system and the autonomic system do not seem to be affected by circulating steroid hormones, but VP released from the biological clock system is involved in the control of corticosterone release and together with estrogen controls the release of luteinizing hormone. Next, the various interactions of steroid hormones and VP will be discussed in more detail for each of the central VP systems described above. Steroid hormone effects on vasopressin systems The magnocellular VP system A large number of experiments have indicated an effect of gonadal steroids on the VP system that projects to the bloodstream. For instance, under basal conditions, plasma VP and 24-h urinary excretion of VP are higher in male than in female rats (Stone et al., 1989). Immobilization stress results in a marked stimulation of plasma VP concentrations in females, whereas in males there is no significant VP response (Carter and Lightman, 1986). In vitro, the osmotically stimulated VP release from a hypothalamic slice and the VP mRNA increase are inhibited by testosterone (Swenson and Sladek, 1997). In the female rat the activity of the neurohypophysial VP system is affected by the estrous cycle (Swaab and Jongkind, 1970; Skowsky et al., 1979; Forsling et al., 1991). Also the intrahypothalamic release of VP in response to the elevation of plasma osmolality is greater in female rats than in males (Ota et al., 1994). During the 48 h preceding and following parturition dramatic changes occur in the magnocellular VP neurons of the PVN and SON of the rat as reflected in its activity and mRNA levels (Swaab and Jongkind, 1970; Thomas et al., 1996). Also in humans a sexual dimorphism in the control of plasma VP has been demonstrated (Rhodes and Rubin, 1999). The effects of glucocorticoids on the magnocellular VP system are less clear. VP release from hypothalamic slices containing both the PVN and

SON is inhibited in a dose-dependent manner by corticosterone (Liu and Chen, 1995), but in vivo, blockade of glucocorticoid receptors resulted in decreased VP mRNA levels in the SON and decreased plasma VP levels (Pesonen et al., 1992). On the other hand, it has been demonstrated several times that adrenalectomy does not alter magnocellular VP activity (Herman, 1995). The effects, described above, on the magnocellular VP system may either result from the direct influence of steroid hormones on magnocellular VP neurons or involve indirect effects of steroid hormones affecting the central regulation of VP secretion from the posterior pituitary. Indeed, initially no receptors for either gonadal steroids (Axelson and Van Leeuwen, 1990; Zhou et al., 1994) or adrenal steroids (Morimoto et al., 1996) could be detected on magnocellular VP neurons. Recently, however, the presence of estrogen receptor β was reported in magnocellular VP neurons (Alves et al., 1998). The sexually dimorphic VP system The effects of gonadal steroids on the VP systems in the BNST and the AME (Fig. 1) are among the most dramatic reported for a neurotransmitter system. Several other neuropeptide systems also show dramatic fluctuations in their expression under the influence of gonadal steroids; however, a complete elimination of the expression of a particular neuropeptide by gonadectomy has been reported only for the VP cells of the BNST and AME (De Vries et al., 1986). Those pronounced effects of sex steroids have also resulted in some very clear sexual dimorphisms, with male rats having twice as many VP-ir cells in the BNST than females. Since these VP neurons project to many forebrain areas, such as the lateral habenular nucleus and the lateral septum, consequently these target areas also contain many more VP-ir fibers in males than in females (Fig. 2; De Vries et al., 1981; De Vries and Buijs, 1983; Van Leeuwen et al., 1984; Bamshad et al., 1993; Wang et al., 1996). Following gonadectomy of adult male or female rats, VP fiber density in the lateral septum and lateral habenular nucleus decreases gradually until, after about 10 weeks, virtually no fibers can be detected anymore (De Vries et al., 1984; Dubois-Dauphin et al., 1994). Similar changes

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Fig. 1. The vasopressin sexually dimorphic system as illustrated by a sagittal scheme of the main vasopressin pathways arising from the BNST and AME. LS, lateral septum; VDBB, diagonal band of Broca; LH, lateral habenula; HIP, hippocampus; CG, central gray; DR, dorsal raphe; LC, locus coeruleus; OT, olfactory tubercle.

are observed when there is a natural cause for the decreased gonadal activity, i.e. during aging (Fliers et al., 1985; Goudsmit et al., 1988) or during the non-breeding season in seasonal species (Buijs et al., 1986; Hermes et al., 1990; Lakhdar-Ghazal et al., 1995; Bittman et al., 1996). In addition to altering the activity of VP cells in the adult rat, gonadal steroids also have a perinatal ‘organizational’ effect, which results in a sexual dimorphism of these extrahypothalamic VP systems (De Vries et al., 1984; Wang et al., 1993). Concomitant with VP-ir also VP mRNA levels decline, indicating that gonadal steroids do not only affect VP peptide content but also VP gene expression (Miller et al., 1989; Carter and Murphy, 1993; Szot and Dorsa, 1994). Disappearance of VP-ir cells and fibers and VP mRNA is prevented in females by estrogen and in males by testosterone replacement. In contrast with the gonadal steroids, adrenal steroids do not seem to be a strong signal for the sexually dimorphic VP system. Although dexamethasone

is able to suppress VP gene expression in the BNST and AME, it does so apparently by its suppressive effect on circulating testosterone levels (Urban et al., 1991). An opposite effect is seen in Sprague–Dawley rats, i.e. removal of circulating glucocorticoids causes a decrease of circulating testosterone levels and a subsequent decrease of VP mRNA in the AME (Viau et al., 2001); apparently the sensitivity of the gonadal system to adrenal manipulation is strain-dependent. The endocrine VP system The parvocellular VP neurons in the PVN belong to either the endocrine or the autonomic VP system as described above (Fig. 3). Vasopressin from neurons of the endocrine VP system is released in the median eminence and in most of these neurons VP is co-localized with corticotropin-releasing hormone (CRH). It has previously been shown that various stimuli causing an activation of the hypothalamic– pituitary–adrenal axis, such as immobilization, nov-

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Fig. 2. Sex difference in the vasopressin innervation of the lateral septum. Males (B) have many more vasopressin-immunoreactive fibers than females (A). Modified from De Vries et al. (1981).

elty, insulin and high doses of colchicine, go along with a depletion of the CRH and VP content in the external zone of the median eminence (Berkenbosch et al., 1989; De Goeij et al., 1992; Bartanusz et al., 1993; Romero et al., 1993). The co-release of VP strongly potentiates the ACTH-releasing effect of CRH (Rivier and Vale, 1983; Rivier et al., 1984). The emerging view is that VP is the principal regulated variable that puts a situation-specific drive on the axis, whereas CRH serves mainly to impose a stimulatory tone (Kovacs et al., 2000). Under resting conditions the parvocellular VP neurons of the PVN

do not contain detectable amounts of VP immunoreactivity or mRNA. However, in the early 80s it was shown that both CRH and VP expression are strongly upregulated after adrenalectomy (Sawchenko et al., 1984; Whitnall, 1988). The increased expression of VP and CRH in the parvocellular PVN neurons apparently is due to a disappearance of glucocorticoid feedback, since corticosterone replacement or dexamethasone administration prevented these effects of adrenalectomy (Kovacs et al., 1986; Carter et al., 1993; Herman, 1995). Although local implants of dexamethasone into the region of the PVN are suffi-

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Fig. 3. The autonomic and endocrine vasopressin system as illustrated by a sagittal scheme of the main vasopressin pathways arising from the parvocellular PVN neurons. ME, median eminence; PBN, parabrachial nucleus; A1, A1 group; DVC, dorsal vagal complex; IML, intermediolateral column of the spinal cord.

cient to block adrenalectomy-induced CRH and AVP increases, and glucocorticoid receptors are present on CRH-containing neurons (Liposits et al., 1987; Ceccatelli et al., 1989), local effects of glucocorticoids on parvocellular neurons may not be the sole source of inhibitory feedback. Several brain regions other than the PVN, such as the lateral septum and the hippocampus, also contain high levels of glucocorticoid receptors and exert a profound inhibitory effect on the activity of the HPA axis (Moberg et al., 1971; Seggie et al., 1974; Baldino et al., 1988). The adrenalectomy-induced increase of VP (but not CRH) synthesis in the PVN is abolished by a concomitant gonadectomy (Viau et al., 1999, 2001), suggesting a permissive role for gonadal steroids on VP synthesis that is revealed in the absence of glucocorticoids. A stimulatory effect of estrogen on VP and/or CRH synthesis might be part of the explanation for the well-known sexual dimorphism in HPA activity (Nicholson et al., 1985; Handa et al., 1994; Atkinson and Waddell, 1997), with higher

plasma corticosterone levels consistently reported in females. Indeed, females show higher bioassayable levels of hypothalamic CRH (Hiroshige et al., 1973) and increased levels of CRH mRNA in the PVN (Watts and Swanson, 1989). However, since androgen receptors in the PVN are essentially located in those neurons in the PVN that give rise to the descending projections (Simerly et al., 1990; Zhou et al., 1994), the feedback actions of the gonadal steroids on the endocrine VP system are probably neurogenic in nature, mediated upstream from the PVN (Viau and Meaney, 1996). The autonomic VP system Vasopressin-containing parvocellular neurons in the PVN also project to the brainstem and spinal cord, where they terminate within the nucleus of the solitary tract, the dorsal motor nucleus of the vagus and the intermediolateral column (Swanson, 1977; Buijs, 1978; Lang et al., 1983; Sawchenko, 1987), i.e.

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nuclei containing either the parasympathetic or sympathetic preganglionic neurons. Therefore, this part of the PVN VP system may play an important role in the regulation of autonomic mechanisms and is labeled the autonomic VP system (Fig. 3). Double retrograde tracing studies have established that the populations of cells in the PVN that project to the median eminence are almost completely separated from those that give rise to the long descending projections (Swanson and Kuypers, 1980; Swanson et al., 1980). The absence of increased immunostaining after adrenalectomy in PVN neurons with descending projections (Sawchenko, 1987) affirms this clear separation of neurons belonging to either the endocrine or the autonomic VP system in the PVN. Both glucocorticoid and gonadal steroid receptors have been found in the parvocellular PVN neurons (Sar and Stumpf, 1980; Rhodes et al., 1982; Ceccatelli et al., 1989; Jirikowski et al., 1993; Morimoto et al., 1996); however, only for the gonadal steroid receptors a co-localization within neurons with descending projections could be shown (Corodimas and Morrell, 1990; Wagner et al., 1993; Zhou et al., 1994). Most of them apparently contain oxytocin (Axelson and Van Leeuwen, 1990; Zhou et al., 1994; Simonian and Herbison, 1997; Alves et al., 1998). Clear effects of either glucocorticoids or gonadal steroids on the autonomic VP system have not been described. The biological clock VP system The main component of the mammalian biological clock system, i.e. the endogenous pacemaker, is localized in the SCN. The SCN is a bilaterally paired nucleus in the ventral part of the anterior hypothalamus, situated next to the third ventricle and on top of the optic chiasm. It consists of several classes of neurons which can be identified by their electrophysiological properties (Pennartz et al., 1998a,b) or their transmitter content (Romijn et al., 1996, 1997). Vasopressin-containing neurons constitute an important subpopulation of this heterogeneous nucleus, i.e. between 10% and 30% of the ∼10,000 SCN neurons may contain VP (Sofroniew and Weindl, 1980; Moore and Speh, 1993; Madeira et al., 1997), a feature which is maintained across many mammalian species (Sofroniew and Weindl, 1980; Cassone et al., 1988; Reuss et al., 1989; Goel et al., 1999; Smale

and Boverhof, 1999). SCN neurons contain an internal pacemaker producing an endogenous rhythm in electrical activity with peak values found during subjective daytime even when the SCN neurons are completely isolated from the surrounding brain tissue (Inouye and Kawamura, 1979; Groos and Hendriks, 1982; Bos and Mirmiran, 1990). Interestingly, VP secretion follows the general pattern of electrical and metabolic activity of the SCN neurons (Gillette and Reppert, 1987). Amongst others the medial preoptic area (MPOA), the periventricular PVN and the dorsomedial hypothalamus (DMH) have been identified as projection areas of the SCN VP neurons (Fig. 4) by using different combinations of lesion, tracer and immunocytochemical techniques (Hoorneman and Buijs, 1982; Watts and Swanson, 1987; Kalsbeek et al., 1993; Leak and Moore, 2001). In contrast with the projection areas of the VP neurons in the BNST and the AME, VP fiber densities in SCN projection areas do not respond to changes in the level of circulating gonadal steroids (Fig. 5; De Vries et al., 1984; Viau et al., 2001). This lack of effect of gonadal steroids on the biological clock VP system is in good agreement with the relative paucity of either estrogen or androgen receptors in the SCN (Simerly et al., 1990; Zhou et al., 1994; Shughrue et al., 1997; Alves et al., 1998). On the other hand, sexual dimorphisms of its projections have been reported (Crenshaw et al., 1992; LakhdarGhazal et al., 1992; Horvath et al., 1998) and several additional data suggest that gonadal steroids are able to affect the (output of the) biological clock (Daan et al., 1975; Morin et al., 1977; Albers, 1981; Kow and Pfaff, 1984; Krajnak et al., 1998). At present, however, it seems most probable that these changes are neurogenic, i.e. mediated by steroid-sensitive afferent inputs to the SCN (De La Iglesia et al., 1999; Horvath et al., 1999). Like gonadal steroid receptors, glucocorticoid receptors, too, show a relative paucity in the adult mammalian SCN (Van Eekelen et al., 1987; Yi et al., 1994) and the effects of glucocorticoids on the SCN VP system are at best ambiguous (Vandesande et al., 1974; Carter and Murphy, 1989; Gozes et al., 1994; Larsen et al., 1994; Maurel et al., 2000; Viau et al., 2001). The apparent insensitivity of the biological clock to adrenal steroids has recently been strengthened further by its lack of effect on circadian clock

63

Fig. 4. Photomicrographs of the thalamic area around the third ventricle (III) in a control (B) and castrated rat (A). Note that castration eliminates the staining of vasopressin fibers in the lateral habenula (LH) that derive from the BNST, but not the staining of fibers in the paraventricular nucleus of the thalamus (PV), which are derived from the SCN. Modified from De Vries et al. (1984).

genes in the SCN as opposed to those in peripheral tissues (Balsalobre et al., 2000). Vasopressin effects on steroid hormones Vasopressin and the corticosterone rhythm The prominent rhythm of VP release from SCN terminals and the close proximity of its PVN projections to the CRH-containing neuroendocrine motorneurons led us to investigate a possible causal relation (Kalsbeek and Buijs, 1992). Our initial experiments revealed a strong inhibitory effect of SCNderived VP on corticosterone release when delivered at the level of the PVN/DMH area in SCN-lesioned animals (Kalsbeek et al., 1992). Subsequent studies

using the application of VP and a VP antagonist in the PVN/DMH area of SCN-intact animals proved the inhibition to be caused by endogenous VP, released from SCN terminals (Fig. 6). Additional experiments using timed VP antagonist administration at different times of the L/D cycle further clarified the control mechanism of the daily corticosterone surge (Kalsbeek et al., 1996b). The increased release of VP during the first part of the light period ensures basal corticosterone levels during the initial part of the sleep period. Subsequently, the concomitant arrest of VP release and the increased release of an additional SCN transmitter stimulating corticosterone release during the second part of the light period results in the daily corticosterone surge just before awakening. After the onset of the dark period also

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Fig. 5. The biological clock vasopressin system as illustrated by a sagittal scheme of the main projections arising from the vasopressinergic SCN neurons. MPOA, medial part of the preoptic area; Pe, periventricular part of the PVN; DMH, dorsomedial nucleus of the hypothalamus; PV, paraventricular nucleus of the thalamus.

Fig. 6. Effects of a 1-h administration of vasopressin, vasopressin antagonist or Ringer (all from t = 0 onwards) at the level of the PVN/ DMH area on plasma corticosterone values. Note the pronounced dis-inhibitory effect of the vasopressin antagonist, whereas vasopressin and Ringer do not notably affect basal corticosterone levels. The shaded area delineates corticosterone levels of control animals without hypothalamic infusions. Modified from Kalsbeek et al. (1996a).

65

the release of the stimulatory SCN transmitter will vanish and corticosterone levels will slowly decline. Although in the meantime the inhibitory effect of VP on corticosterone release as just described has been replicated by different laboratories (Wotjak et al., 1996; Gomez et al., 1997) its direction is completely opposite to the previously described and well-known stimulatory effect of VP on ACTH and corticosterone release (Gillies et al., 1982; Rivier and Vale, 1983; Rivier et al., 1984). The logical explanation of course is that the different effects are caused by VP derived from two completely different systems. The inhibitory effect concerns VPergic SCN neurons that synapse at the level of the PVN and DMH, the stimulatory effect is due to VP derived from parvocellular PVN neurons and acting at the level of the median eminence. At present it is not clear which are the exact target neurons for the inhibitory VPergic projections. Since VP generally has a stimulatory effect on its postsynaptic target neuron (Raggenbass, 2001) it is unlikely that the VP-containing fibers directly contact CRH-containing neurons. Indeed, a direct projection is also not supported by the sparse SCN projections to the hypophysiotropic neurons (Buijs et al., 1993; Vrang et al., 1995). Most probably the inhibitory effect of VP is mediated by GABAergic interneurons in the DMH and subparaventricular PVN (Hermes et al., 2000). However, when comparing the corticosterone and ACTH responses induced by the hypothalamic administrations of the VP antagonist it is clear that the SCN control of corticosterone release cannot be explained solely by its effect on the HPA axis, i.e. via the subsequent release of CRH and ACTH. Comparing the response of ACTH with that of corticosterone in different experimental conditions reveals that the circadian release of corticosterone does not depend heavily on the release of ACTH, contrary to, for instance, the stress-related release of corticosterone (Kalsbeek et al., 1996b; Buijs et al., 1997). Instead of a stimulation of ACTH release it seems that the daily corticosterone peak is mainly caused by an increased sensitivity of the adrenal cortex to ACTH (Jasper and Engeland, 1994; Buijs et al., 1999). Indeed, using the transneuronal virus tracing technique a neuronal pathway between SCN, dorsal PVN, spinal cord and adrenal cortex could be established (Buijs et al., 1999). Therefore, via the GABA-containing interneurons in the DMH

and the subparaventricular PVN, the VPergic projection from the SCN seems to contact not only the endocrine CRH-containing neurons but also the autonomic neurons in the dorsal PVN. Therefore, via its effect on the GABAergic interneurons the daytime peak of VP release from SCN terminals in the PVN/DMH area might ensure basal corticosterone levels via two separate mechanisms: (1) it inhibits the release of ACTH via its effect on the endocrine CRH neurons, and (2) it inhibits the sensitivity of the adrenal cortex for ACTH via its effect on the autonomic PVN neurons. Evidently, the purpose of this rhythm is not only to drive the daily corticosterone peak in mammals but also to maintain, for example, stress-induced corticosterone activations within the physiological range appropriate for the stress and appropriate for that moment of the L/D cycle (Buijs et al., 1997). Vasopressin and the luteinizing hormone surge Daily rhythms in plasma levels of gonadal steroids are usually not very pronounced, except during human puberty (Norjavaara et al., 1996; Mitamura et al., 1999; Gupta et al., 2000). Nevertheless there is a clear relation between the mammalian biological clock and many aspects of sexual behavior: for instance the temporal organization of pulsatile activity in the hypothalamic–pituitary–adrenal (HPG) axis is essential for successful reproduction. Lesion studies have shown that two brain structures are indispensable for generating the preovulatory surge of luteinizing hormone (LH): firstly, the MPOA containing the dense concentration of estrogen receptors necessary for the positive estrogen feedback and, secondly, the SCN providing the timing signal for the LH surge on the day of proestrus. Early anatomical studies already indicated a dense VP innervation in the MPOA, which probably derives from the SCN since it was not sensitive to gonadal hormones (Hoorneman and Buijs, 1982; De Vries et al., 1984). More recent studies showed that estrogen receptor containing neurons in the MPOA receive direct synaptic contacts from SCN fibers, probably containing VP as a neurotransmitter (De La Iglesia et al., 1995; Watson et al., 1995) and that VP receptor mRNA is expressed in MPOA neurons (Ostrowski et al., 1994; Funabashi et al., 2000b). In addition,

66

Fig. 7. Daily levels of plasma LH in intact (shaded area) and ovariectomized and estrogen-treated animals. The ovariectomized and estrogen-treated animals were administered vasopressin in the MPOA during 5 h. Vasopressin-treated animals were divided in animals with a high LH surge amplitude (•; ∼1/3 of the animals) and those with an LH surge comparable to that of control animals (◦; ∼2/3 of the animals). Modified from Palm et al. (2001).

some early works of Södersten et al. (1983, 1985, 1986) indicated an interesting relationship between female sexual behavior and SCN-derived VP, although the effect had not been localized to a specific SCN target area. We hypothesized that the MPOA functions as an intermediate brain area for the transmission of circadian information from the SCN to the HPG axis. Indeed, an increase in extracellular VP levels (by reverse microdialysis) in the MPOA of SCN-intact animals had a stimulatory effect on the LH surge (Fig. 7), whereas it did not affect plasma corticosterone levels (Palm et al., 2001). The stimulatory effect of VP was restricted to a specific time-period that coincided with the sensitive time window for a daily neuronal signal prior to the LH surge (Everett and Sawyer, 1950), and also with the peak of VP secretion by SCN neurons (Schwartz et al., 1983; Schwartz and Reppert, 1985; Gillette and Reppert, 1987; Kalsbeek et al., 1995). The important role of SCN-derived VP in the initiation of the LH surge was further emphasized by our experiments in SCN-lesioned animals. The complete absence of any circadian output from the SCN induces basal, nonfluctuating LH levels, but a 2-h administration of VP in the MPOA is sufficient to reinstate a complete LH surge that is comparable to the estrogen-induced

surges in SCN-intact animals, both in shape and amplitude (Palm et al., 1999). Therefore, in our view the high VP secretion by SCN terminals in the MPOA, occurring during the sensitive time window prior to the surge, is the circadian signal essential for the generation of an LH surge. So, by the simultaneous secretion of VP in different target areas, the SCN may reduce the stress response in one area (PVN/DMH), while it stimulates sexual receptivity in another (MPOA). In addition, it seems physiologically relevant that the stimulatory role of VP on the HPG axis coincides with an inhibitory role of SCN-derived VP on stress hormone secretion. Once every 4 or 5 days the combination of a positive estrogen feedback and circadian VP input, probably by MPOA neurons bearing both estrogen and VP receptors, enables the MPOA to trigger a concerted action of gonadotropin-releasing hormone (GnRH) containing neurons. The resulting massive release of GnRH in the median eminence will initiate the next LH surge. Using a completely different experimental setup a similar conclusion was reached by Funabashi et al. (2000a). Next to this indirect control of the SCN direct projections from the SCN to the endocrine GnRH motorneurons, although sparse, have also been reported (Van Der Beek et al., 1993,

67

1997), i.e. an anatomical situation very much comparable with the circadian control of the HPA axis. The use of intermediate brain structures such as the DMH and MPOA, of course, has the great advantage of enabling the integration of information from different sources (such as circadian, hormonal, metabolic, stress, etc.) before a definitive signal is sent to the endocrine motorneuron.

Pe PV PVN

Conclusion

References

The present overview shows that there are a number of interactions between central VP systems and circulating steroid hormones. For understanding the type of interaction, however, it is essential not to consider the central VP system as a uniform entity, but to appreciate its variation. In addition to the fact that the distribution of steroid receptors may be very site-specific, also the neurotransmitter functions of VP are closely linked to its site of origin and production. Finally, of course, the function of VP in the target area will depend heavily on the function of that region. Abbreviations ACTH AME BNST CG CRH DMH DR DVC GABA GnRH HIP HPA HPG IML ir LC L/D LH ME MPOA OT PBN

adrenocorticotropic hormone medial amygdala bed nucleus of the stria terminalis central gray corticotropin-releasing hormone dorsomedial nucleus of the hypothalamus dorsal raphe dorsal vagal complex gamma aminobutyric acid gonadotropin-releasing hormone hippocampus hypothalamic–pituitary–adrenal hypothalamic–pituitary–gonadal intermediolateral column of the spinal cord immunoreactivity locus coeruleus light/dark luteinizing hormone median eminence medial preoptic area olfactory tubercle parabrachial nucleus

SCN SON VDBB VP

periventricular area paraventricular nucleus of the thalamus paraventricular nucleus of the hypothalamus suprachiasmatic nucleus supraoptic nucleus diagonal band of Broca (ventral part) vasopressin

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 6

Regulation of renal salt and water transporters during vasopressin escape Carolyn A. Ecelbarger 1,∗ , Takashi Murase 2, Ying Tian 1 , Soren Nielsen 3 , Mark A. Knepper 4 and Joseph G. Verbalis 1 1

Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University, Washington, DC 20007, USA 2 The First Department of Internal Medicine, Nagoya University, Nagoya, Japan 3 Department of Cell Biology, University of Aarhus, Aarhus, Denmark 4 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Abstract: Hyponatremia, defined as a serum sodium < 135 mmol/l, is one of the most commonly encountered and serious electrolyte disorders of clinical medicine. The predominant cause of hyponatremia is an inappropriate elevation of circulating vasopressin levels relative to serum osmolality or the ‘syndrome of inappropriate antidiuretic hormone secretion’ (SIADH). Fortunately, the degree of the hyponatremia is limited by a process that counters the water-retaining action of vasopressin, namely ‘vasopressin escape’. Vasopressin escape is characterized by a sudden increase in urine volume with a decrease in urine osmolality independent of circulating vasopressin levels. Until recently, little was known about the molecular mechanisms underlying escape. In the 1980s, we developed an animal model for vasopressin escape in which male Sprague–Dawley rats were infused with dDAVP, a V2-receptor-selective agonist of vasopressin, while being fed a liquid diet. Rats drank a lot of water in order to get the calories they desired. Using this model, we demonstrated that the onset of vasopressin escape (increased urine volume coupled to decreased urine osmolality) coincided temporally with a marked decrease in renal aquaporin-2 (water channel) protein and mRNA expression in renal collecting ducts. This protein reduction was reversible and correlated to decreased water permeability of the collecting duct. Studies examining the mechanisms underlying AQP2 decrease revealed a decrease in V2-receptor mRNA expression and binding, as well as a decrease in cyclic AMP production in response to acute-dDAVP challenge in collecting duct suspensions from these escape animals. Additional studies showed an increase in sodium transporters of the distal tubule. These changes, hypothetically, might help to attenuate the hyponatremia. Future studies are needed to fully elucidate systemic, intra-organ, and cellular signaling responsible for the physiological phenomenon of vasopressin escape. Keywords: Aquaporins; SIADH; Thiazide-sensitive Na-Cl cotransporter; Epithelial sodium channel; Hyponatremia; Bumetanide-sensitive Na-K-2Cl cotransporter

∗ Correspondence to: C.A. Ecelbarger, Building D, Room 232, 4000 Reservoir Road NW, Georgetown University, Washington, DC 20007, USA. Tel.: +1-202-687-0453; Fax: +1-202-687-2040; E-mail: [email protected]

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Background Hyponatremia and vasopressin escape Hyponatremia, defined as a serum sodium 10 h. In the case of the human V1a receptor, the Nterminal methionine is followed with Arg and Leu residues at positions 2 and 3, respectively. Based on the N-end rule these two residues are supposed

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Fig. 3. Screening of the bacterial clones by Coomassie blue and 6 × His Western blotting. The human V1a receptor and its version fused with the short T7 tag were expressed in BL21(DE3) or BL21(DE3)pLysS bacterial strains. The culture was performed in standard conditions with vigorous shaking at 37°C in LB medium supplemented with ampicillin 100 μg/ml (or its analog carbenicillin). At an A600 of 0.6, protein expression was induced by addition of 1 mM IPTG and growth continued for 3 h. Cells were harvested by centrifugation and the pellets resuspended in Laemmli buffer. After sonication, a few microliters of each sample were loaded onto SDS–polyacrylamide gels. After running the gels, screening of the samples was done directly by staining the proteins with Coomassie blue (A,B). In parallel, chemiluminescence detection of the receptors was performed by Western blotting using an antibody (1 : 2000 dilution) directed against the 6 × His tag and directly coupled to horseradish peroxidase (C,D). In A and B, three clones transformed with the wild-type or the T7-tagged V1a receptors were compared to a control BL21(DE3) clone transformed with the empty vector. In C, BL21(DE3) cells were transformed with or without pET 21b+ containing or not the human V1a cDNA sequence. In D, BL21(DE3)pLysS cells were transformed with or without pET 21a+ containing or not the human V1a cDNA sequence.

to be very bad, and their presence could be responsible for the poor expression level and rapid degradation of the recombinant receptor. We thus decided to mutate Arg and Leu residues with Gly and Ala residues, respectively, which are considered as two stabilizing amino acids. Introduction of the

point mutations was verified by dideoxynucleotide sequencing and the mutated receptor was expressed in BL21(DE3) or BL21(DE3)pLysS bacterial strains. As seen in the left panel of Fig. 4, the mutated receptor was easily detectable using the Western blot and chemiluminescence procedures. However, the level

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Fig. 4. Effect of point mutations in the receptor sequence and of glucose addition in the culture medium. The human V1a receptor and its mutant combining Gly and Ala residues at positions 2 and 3 respectively, were expressed in the BL21(DE3)pLysS bacterial strain. The culture was performed in standard conditions with vigorous shaking at 37°C in LB medium supplemented with ampicillin 100 μg/ml (or carbenicillin), with or without glucose 0.4%. At an A600 of 0.6, protein expression was induced by addition of 1 mM IPTG and growth continued for 3 h. Chemiluminescence detection of the receptors was realized as described in legend to Fig. 3. In the left panel, BL21(DE3)pLysS cells were transformed with or without pET 21a+ vector containing or not the mutant receptor. In the central panel, the same bacterial clone expressing the mutant receptor was treated with glucose 0.4%. Increasing quantities of material were put into wells II and III. In the right panel, BL21(DE3) cells expressing the wild-type receptor were treated or not with glucose 0.4%. Increasing quantities of material were put into wells III and IV.

of expression was still low and did not allow direct detection of the protein with Coomassie blue (not shown). The protein band, detected using the 6 × His tag, appeared at approximately 42 kDa. This molecular mass is smaller than the one expected for the receptor which should be visualized around 50 kDa. A 42-kDa protein is thus probably the result of proteolytic degradation of the receptor. In bacterial strains like BL21(DE3) or BL21(DE3) pLysS, expression of the T7 RNA polymerase is un-

der the control of the lacUV5 promoter. Sometimes, this enzyme can be produced even in the absence of the IPTG inducer. Consequently, if the target protein, like the V1a receptor, is sufficiently toxic to E. coli, this basal level (leakiness) of T7 RNA polymerase is negative to the growth of the cells. This derepression of the lacUV5 promoter is mediated by cyclic adenosine monophosphate (cAMP) (Grossman et al., 1998). It has been demonstrated that this derepression can be effectively avoided by including glucose

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in the culture medium. Glucose inhibits cAMP production and acts as a lac repressor. In order to verify this effect, we added 0.4% of glucose in the bacterial LB medium before IPTG induction and expressed the mutant V1a receptor (containing Gly and Ala residues at positions 2 and 3). As illustrated in the central panel of Fig. 4, the addition of glucose allowed us to visualize the receptor protein with more ease by Western blot and chemiluminescence methods. The immunoreactive band at around 42 kDa was prominent but another band at around 55 kDa could also be detected. Interestingly, this upper band roughly corresponds to the expected size of the receptor protein. The calculated molecular weight of the protein is 49 kDa including the spacer arm between the natural Cterminal amino acid and the 6 × His tag. However, it is well known that some proteins with 6 × His tags attached run more slowly on SDS gels than equivalent untagged proteins, and may appear to be several kilodaltons larger than expected. This encouraging result prompted us to check whether it could be possible to detect the wild-type receptor when adding glucose in the bacterial culture medium. As seen in the right panel of Fig. 4, the human V1a receptor was easily detectable using the Western blot and chemiluminescence procedures. Once again, the receptor appeared as a unique band at around 42 kDa. This size is compatible with that of a degraded product. In conclusion, adding glucose in the culture medium or mutating the destabilizing residues located at the N-terminus end of the V1a receptor greatly improves the level of expression of the recombinant protein in E. coli. Combining both approaches allowed us to visualize the undegraded receptor at approximately 55 kDa. Expression of the receptors as fusion proteins High-level expression of membrane proteins in bacteria, and especially GPCRs, is very difficult, probably due to the fact that the cells cannot tolerate large amounts of the foreign protein within their membranes. Besides the toxicity, the expression is extremely low as a result of degradation as well. Factors which influence the success of E. coli expression include the host, the nature of the promoter employed, the growth conditions, the origin and the nature of the recombinant protein and its intracel-

lular location. The problem of low abundance has been successfully side-stepped in the case of soluble proteins by the recombinant route. In contrast, the production of large quantities of active membrane proteins by overexpression is not routine. Therefore, the development of strategies to overcome this barrier should significantly boost membrane protein structural research. In this regard, membrane proteins expressed as fusion proteins with different partners, as well as targeted to inclusion bodies (IBs), are potentially useful. Firstly, a few GPCRs have been expressed as maltose binding protein or thioredoxin fusions and purified in microgram amounts in the presence of detergent in functional forms (Tucker and Grisshammer, 1996). Schistosomal glutathioneS-transferase (GST) is also commonly used as a fusion partner when expressing proteins in E. coli. The usefulness of GST has been demonstrated by overexpressing first the rat olfactory receptor OR5 as a fusion protein (Kiefer et al., 1996) and later with other different GPCRs (Kiefer et al., 2000). Secondly, IBs seem to form when highly expressed recombinant proteins cannot be tolerated as soluble proteins in the cell cytoplasm. These IBs are amorphous electrondense structures seen as refractile particles under phase contrast microscopy (Williams et al., 1982). Although IBs have often been considered as undesirable, dead end products of protein expression, their formation can be an advantage as their isolation from cell homogenates is a convenient and effective first purification step. Proteins in IB form are insoluble aggregates that lack functional activity, but could represent an excellent starting point for producing them in large amounts, provided that procedures can be developed to reconstitute them in vitro. Moreover, proteins accumulated in IBs are relatively pure and are protected from proteolytic degradation. In addition, toxic proteins may not inhibit cell growth when present in inactive form as IBs. Obviously, the aggregated protein has to be refolded to its native state, but there has been substantial progress in the last several years in methodologies for the refolding of several α-helical membrane proteins and two GPCRs derived from IBs (Grigorieff et al., 1996; Kiefer et al., 1996). Finally, purification of 6 × Histagged proteins is compatible with solubilization of IBs using strong denaturing agents such as urea or guanidine hydrochloride.

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Fig. 5. Construction of the cDNA coding for the GST-V1a 6 × His fusion protein. Incorporating a MluI/SpeI fragment from the commercial pET 42b+ plasmid into the pET 21a+ plasmid cut with MluI/NheI resulted in the obtention of a new vector containing the GST-fused V1a or OT cDNA sequences. In both cases, receptors expressed from these new constructions are still tagged at their C-terminus end with the 6 × His epitope.

We thus decided to produce the human V1a /OT receptors as fusion proteins with GST being the partner. As depicted in Fig. 5, we have used a combination of the available pET 21a+ and pET 42b+ plasmids, containing the optimized gene of V1a receptor and GST, respectively, to construct a new vector in which the fusion protein GST-V1a 6 × His is under the control of the strong T7 promoter. This new fusion protein was expressed by transformation of the vector into BL21(DE3) and BL21(DE3)pLysS strains. The clones were screened by Coomassie blue-stained SDS gels as well as Western blot using chemiluminescence and antibodies against the 6 × His tag. The result is shown on Fig. 6. The GST-fused receptor was easily detectable using the Western blot and chemiluminescence procedures but the level of production was still low and did not

allow direct detection of the protein with Coomassie blue (not shown). The protein appeared as a unique intense band with a molecular mass around 62 kDa. This size is smaller than the one expected for this new construct, approximately 80 kDa. Once again, the results suggested that the fusion protein was degraded. Interestingly, considering that Western blot procedure against the 6 × His tag allows detection of receptors which are only totally translated (the 6 × His tag is located at the C-terminus of the receptor), the band at 62 kDa should contain the entire V1a receptor. In that context, the fusion protein is probably degraded in the GST part. Engineering a specific cleavage site for thrombin or factor Xa between GST and the V1a receptor should lead to complete removal of the fusion partner and generate the undegraded purified receptor for subsequent structural studies. In

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Fig. 6. Detection of the GST-V1a 6 × His fusion protein by western blot. BL21(DE3)pLysS cells were screened for expression of the GST-V1a 6 × His fusion protein using the Western blot and chemiluminescence procedures as described in legend to Fig. 3. The culture was performed in standard conditions with vigorous shaking at 37°C in LB medium supplemented with ampicillin 100 μg/ml (or carbenicillin). At an A600 of 0.6, protein expression was induced by addition of 1 mM IPTG and growth continued for 3 h. Two representative clones are shown.

conclusion, doing a fusion with GST did not allow us to significantly improve the level of expression of the receptor, when compared to that obtained for the mutant receptor or that observed in the presence of glucose in the culture medium. However, this construction is much more promising because of the protective effect of GST against proteolysis.

Purification of the V1a receptor by immobilized metal affinity chromatography Immobilized metal affinity chromatography (Porath et al., 1975) is a powerful method for purifying proteins with engineered 6 × His tags. The purification is usually done in one-step procedure and is

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based on the affinity between the six neighboring His residues of the tag and an immobilized metal ion (usually nickel). The nickel is held by chelation with reactive groups covalently attached to a solid support (agarose for example). The Ni-NTA resin uses nitrilotriacetic acid (NTA) as the chelator. After unbound proteins are washed away, the target protein is recovered by elution with imidazole. This system is versatile and allows proteins to be purified under gentle, non-denaturing conditions, or in the presence of either high concentrations of strong denaturing agents urea or guanidine. We have applied this purification method to the human V1a /OT receptors expressed in bacteria. A flow chart describing the steps for purification of the recombinant receptors is detailed in Fig. 7. The method

Fig. 7. Overview of the V1a /OT receptor purification procedure.

has been performed for isolating the mutant V1a receptor (containing Gly and Ala residues at positions 2 and 3, respectively) and the GST-fused V1a receptor from a 100-ml starting culture volume. Samples taken from each step of the procedure have been checked for the presence of the receptors by Western blot and chemiluminescence approaches. As illustrated in Fig. 8, the results demonstrated that both mutant and GST-fused V1a receptors could be isolated in the centrifugation pellet after cell lysis, although a low amount of the GST-fused protein was still in the supernatant. We first deduced that the receptors were not cytoplasmic and were thus localized in a particulate fraction, probably membranes or IBs. After solubilization of IBs (and membranes), incubation of the samples with Ni-NTA agarose resin and elution in

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Fig. 8. Purification of the mutant and GST-fused V1a receptors using immobilized metal affinity chromatography. For each construct, a 100-ml culture was started in standard conditions with vigorous shaking at 37°C in LB medium supplemented with ampicillin 100 μg/ml (or carbenicillin). At an A600 of 0.6, protein expression was induced by addition of 1 mM IPTG and growth continued for 3 h. As detailed in Fig. 7, extracts from bacterial cultures, expressing the mutant or the fusion protein, were prepared for immobilized metal affinity chromatography. Samples (a few microliters) taken from different steps of the purification procedure were checked for the presence of the receptors. Chemiluminescence detection of the receptors was realized as described in the legend to Fig. 3. For each panel, the legend is the following: TPE, total protein extract; S1, supernatant of centrifugation after cell sonication (lysis); S2, supernatant of centrifugation after inclusion bodies solubilization; FT1 and FT2, flow through after incubation of the Ni-NTA resin with receptors from S1 and S2 supernatants, respectively; W1 and W2, washed material of the Ni-NTA columns from S1 and S2 samples, respectively; E1 and E2, eluates from S1 and S2 extracts, respectively.

the presence of 250 mM imidazole, we were able to purify both the mutant and the GST-fused V1a receptors. As expected, the mutant was isolated as a major band around 42 kDa and a minor band at around 55 kDa, representing a degraded product and the entire receptor, respectively. The GST-fused V1a receptor was isolated as a unique band with a molecular mass around 62 kDa, probably a degraded product as well but containing the entire V1a receptor. We estimated the quantity of purified receptors to be very low, less than 0.5 mg/l of bacterial culture. For comparison, to date, only a few receptors have been produced in E. coli at a level high enough for the purification of milligram quantities (Kiefer et al., 1996, 2000; Grisshammer and Tucker, 1997).

Concluding remarks Determination of the structure of integral membrane proteins, such as GPCRs, requires a suitable overexpression system and an efficient purification procedure. The high level expression of these seven transmembrane domain proteins is still a major problem which has not yet been solved. In the present study, we have investigated the usefulness of a bacterial expression system for the production of the AVP/OT receptors. In order to increase the level of expression, we have first optimized the nucleotide sequence of V1a and OT receptors for bacterial expression, fused these sequences with different N-terminal tags useful for expression, and added a 6 × His tag at

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the C-terminus end for their one-step purification using immobilized metal affinity chromatography. Expressing the wild-type V1a receptor in BL21(DE3) or BL21(DE3)pLysS bacterial strains was very toxic to the cell and totally prevented the visualization of the protein either by Coomassie blue-stained gels or by Western blotting. The mutant carrying Gly and Ala residues at positions 2 and 3, respectively, was expressed and detected. The effect of the mutations could be mimicked by the addition of glucose in the culture medium. In all cases, proteins that have been detected by Western blot and chemiluminescence procedures were proteolysed. Only the addition of the GST tag to the wild-type receptor allowed to produce a protein species containing the entire receptor. Purification of the receptors using the 6 × His tag/Ni-NTA interaction was very fast and efficient. The notion that lack of expression of the wildtype receptor may be due to improper insertion of the protein in the bacterial membrane was supported by finding that the GST-fused and the mutant V1a receptors were targeted to IBs and could be better expressed. The present results confirm that insertion of overexpressed eukaryotic membrane proteins into the bacterial membrane is toxic to the cells and thus significantly decreases the level of expression. Producing the receptors directly into IBs is consequently very promising. Interestingly, these insoluble particles protect the recombinant target protein from proteolytic degradation. Moreover, although the receptors are expressed as an aggregated and misfolded state into IBs, it has been demonstrated in several cases that refolding of membrane proteins from IBs can be achieved by detergent exchange and reconstitution into liposomes. Because biophysical studies require large quantities of purified receptors, the ease of large scale fermentation still makes E. coli very attractive. However, this expression system needs substantial improvement before routine high level expression of these transmembrane proteins. The results described in this chapter, are promising, especially those obtained with the GST fusion protein. In the future, we should optimize AVP/OT receptor expression using new fusion partners that facilitate targeting of the recombinant proteins to IBs and/or protection against proteolytic degradation.

Abbreviations 3D AVP cAMP GPCR GST IBs IPTG LB medium NTA OT PCR SDS

three dimensional arginine-vasopressin adenosine 3 :5 -cyclic monophosphate G protein-coupled receptor glutathione-S-transferase inclusion bodies isopropyl-β-D-thiogalactopyranoside Luria–Bertani medium nitrilotriacetic acid oxytocin polymerase chain reaction sodium dodecyl sulfate

Acknowledgements This work was supported by grants from INSERM, the program Molécules et Cibles Thérapeutiques from INSERM and CNRS, the Action Incitative Physique et Chimie du Vivant from the CNRS, and the fellowship program Poste Vert from INSERM (to T.S.). We are grateful to Drs. J. Parello and J.-L. Banères for helpful discussions. B.M. wishes to thank N. Delaunay for the introduction to Powerpoint software. References Bachmair, A., Finley, D. and Varshavsky, A. (1986) In vivo halflife of a protein is a function of its amino-terminal residue. Science, 234: 179–186. Barberis, C., Mouillac, B. and Durroux, T. (1998) Structural bases of vasopressin/oxytocin receptor function. J. Endocrinol., 156: 223–229. Barberis, C., Morin, D., Durroux, T., Mouillac, B., Guillon, G., Seyer, R., Hibert, M., Tribollet, E. and Manning, M. (1999) Molecular pharmacology of AVP and OT receptors and therapeutic potential. Drug News Persp., 12: 279–292. Bockaert, J. and Pin, J.-P. (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J., 18: 1723–1729. Breton, C., Chellil, H., Kabbaj-Benmansour, M., Carnazzi, E., Seyer, R., Phalipou, S., Morin, D., Durroux, T., Zingg, H.H., Barberis, C. and Mouillac, B. (2001) Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor. J. Biol. Chem., 276: 26931–26941. Chini, B., Mouillac, B., Ala, Y., Balestre, M.-N., Trumpp-Kallmeyer, S., Hoflack, J., Elands, J., Hibert, M., Manning, M., Jard, S. and Barberis, C. (1995) Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor. EMBO J., 14: 2176–2182.

176 Cotte, N., Balestre, M.-N., Phalipou, S., Hibert, M., Manning, M., Barberis, C. and Mouillac, B. (1998) Identification of residues responsible for the selective binding of peptide antagonists and agonists in the V2 vasopressin receptor. J. Biol. Chem., 273: 29462–29468. Cotte, N., Balestre, M.-N., Aumelas, A., Mahé, E., Phalipou, S., Morin, D., Hibert, M., Manning, M., Durroux, T., Barberis, C. and Mouillac, B. (2000) Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding. Eur. J. Biochem., 267: 4253–4263. Dillon, P.J. and Rosen, C.A. (1993) Use of polymerase chain reaction for the rapid construction of synthetic genes. In: B.A. White (Ed.), Methods in Molecular Biology, PCR protocols: current methods and applications. Vol. 15, Humana Press, Totowa, NJ, pp. 263–268. Fanelli, F., Barbier, P., Zanchetta, D., De Benedetti, P.G. and Chini, B. (1999) Activation mechanism of human oxytocin receptor: a combined study of experimental and computersimulated mutagenesis. Mol. Pharmacol., 56: 214–225. Freidinger, R.M. and Pettibone, D.J. (1997) Small molecule ligands for oxytocin and vasopressin receptors. Med. Res. Rev., 17: 1–16. Gimpl, G. and Fahrenholz, F. (2001) The oxytocin receptor system: structure, function and regulation. Physiol. Rev., 81: 629–683. Gribskov, M., Devereux, J. and Burgess, R.R. (1984) The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res., 12: 539–549. Grigorieff, N., Ceska, T.A., Downing, K.H., Baldwin, J.M. and Henderson, R. (1996) Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol., 259: 393– 421. Grisshammer, R. and Tate, C.G. (1995) Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys., 28: 315–422. Grisshammer, R. and Tucker, J. (1997) Quantitative evaluation of neurotensin receptor purification by immobilized metal affinity chromatography. Protein Express. Purif., 11: 53–60. Grossman, T.H., Kawasaki, E.S., Punreddy, S.R. and Osburne, M.S. (1998) Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability. Gene, 209: 95–103. Hawtin, S.R., Wesley, V.J., Parslow, R.A., Patel, S. and Wheatley, M. (2000) Critical role of a subdomain of the N-terminus of the V1a vasopressin receptor for binding agonists but not antagonists: functional rescue by the oxytocin receptor Nterminus. Biochemistry, 39: 13524–13533. Hirel, P.-H., Schmitter, J.-M., Dessen, P., Fayat, G. and Blanquet, S. (1989) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. USA, 86: 8247–8251. Kane, J.F. (1995) Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol., 6: 494–500.

Kiefer, H., Krieger, J., Olszewski, J.D., Von Heijne, G., Prestwich, G.D. and Breer, H. (1996) Expression of an olfactory receptor in Escherichia coli: purification, reconstitution and ligand binding. Biochemistry, 35: 16077–16084. Kiefer, H., Vogel, R. and Maier, K. (2000) Bacterial expression of G protein-coupled receptors: prediction of expression levels from sequence. Receptors Channels, 7: 109–119. Manning, M., Cheng, L.L., Stoev, S., Sawyer, W.H., Tribollet, E., Barberis, C., Wo, N.C. and Chan, W.Y. (1995) Novel potent and selective antagonists and radioiodinated ligands for oxytocin and vasopressin receptors. In: T. Saito, K. Kurokawa and S. Yoshida (Eds.), Neurohypophysis: Recent Progress of Vasopressin and Oxytocin Research. Elsevier Science, Amsterdam, pp. 21–38. Mendre, C., Dufour, M.-N., Le Roux, S., Seyer, R., Guillou, L., Calas, B. and Guillon, G. (1997) Synthetic rat V1a vasopressin receptor fragments interfere with vasopressin binding via specific interaction with the receptor. J. Biol. Chem., 272: 21027–21036. Mirzabekov, T., Bannert, N., Farzan, M., Hofmann, W., Kolchinsky, P., Wu, L., Wyatt, R. and Sodroski, J. (1999) Enhanced expression, native purification, and characterization of CCR5, a principal HIV-1 coreceptor. J. Biol. Chem., 274: 28745– 28750. Mouillac, B., Chini, B., Balestre, M.-N., Elands, J., TrumppKallmeyer, S., Hoflack, J., Hibert, M., Jard, S. and Barberis, C. (1995) The binding site of neuropeptide vasopressin V1a receptor: evidence for a major localization within transmembrane regions. J. Biol. Chem., 270: 25771–25777. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M. and Miyano, M. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science, 289: 739–745. Pawate, S., Schey, K.L., Meier, P., Ullian, M.E., Mais, D.E. and Halushka, P.V. (1998) Expression, characterization and purification of C-terminally hexahistidine-tagged thromboxane A2 receptors. J. Biol. Chem., 273: 22753–22760. Phalipou, S., Seyer, R., Cotte, N., Breton, C., Barberis, C., Hibert, M. and Mouillac, B. (1999) Docking of linear peptide antagonists into the human V1a vasopressin receptor: identification of binding domains by photoaffinity labeling. J. Biol. Chem., 274: 23316–23327. Porath, J., Carlsson, J., Olsson, I. and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 258: 598–599. Postina, R., Kojro, E. and Fahrenholz, F. (1996) Separate agonist and peptide antagonist binding sites of the oxytocin receptor defined by their transfer into the V2 vasopressin receptor. J. Biol. Chem., 271: 31593–31601. Serradeil-LeGal, C., Lacour, C., Valette, G., Garcia, G., Foulon, L., Galindo, G., Bankir, L., Pouzet, B., Guillon, G., Barberis, C., Chicot, D., Jard, S., Vilain, P., Garcia, C., Marty, E., Raufaste, D., Brossard, G., Nisato, D., Maffrand, J.-P. and Le Fur, G. (1996) Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J. Clin. Invest., 98: 2729–2738.

177 Strosberg, A.D. and Marullo, S. (1992) Functional expression of receptors in microorganisms. Trends Pharmacol. Sci., 13: 95–98. Thibonnier, M., Coles, P., Conarty, D.M., Plesnicher, C.L. and Shoham, M. (2000) A molecular model of agonist and nonpeptide antagonist binding to the human V1 vascular vasopressin receptor. J. Pharmacol. Exp. Ther., 294: 195–203. Thibonnier, M., Coles, P., Thibonnier, A. and Shoham, M. (2001) The basic and clinical pharmacology of nonpeptide vasopressin receptor antagonists. Annu. Rev. Pharmacol. Toxicol., 41: 175–202. Tobias, J.W., Shrader, T.E., Rocap, G. and Varshavsky, A. (1991)

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CHAPTER 14

Molecular pharmacology and modeling of vasopressin receptors M. Thibonnier 1,∗ , P. Coles 1 , A. Thibonnier 2 and M. Shoham 1 1

Departments of Medicine and Biochemistry, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, OH, USA 2 University of Michigan School of Engineering, Ann Arbor, MI, USA

Abstract: AVP receptors represent a logical target for drug development. As a new class of therapeutic agents, orally active AVP analogs could be used to treat several human pathophysiological conditions including neurogenic diabetes insipidus, the syndrome of inappropriate secretion of AVP (SIADH), congestive heart failure, arterial hypertension, liver cirrhosis, nephrotic syndrome, dysmenorrhea, and ocular hypertension. By immunoprecipitation and immunoblotting, we elucidated the phosphorylation pattern of green fluorescent protein-tagged AVP receptors and showed interactions with the specific kinases PKC and GRK5 that are agonist-, time- and receptor subtype-dependent. The tyrosine residue of the NPWIY motif present in the 7th helix of AVP receptors is rapidly and transiently phosphorylated after agonist stimulation. This phosphorylation is instrumental in the genesis of the mitogenic cascade linked to the activation of this receptor, presumably by establishing key intramolecular contacts and by participating in the creation of a scaffold of proteins that produce the activation of downstream kinases. The random screening of chemical entities and optimization of lead compounds recently resulted in the development of orally active non-peptide AVP receptor agonists and antagonists. Furthermore, the identification of the molecular determinants of receptor–ligand interactions should facilitate the development of more potent and very selective orally active compounds via the approach of structure-based drug design. We developed three-dimensional molecular docking models of peptide and non-peptide ligands to the human V1 vascular, V2 renal and V3 pituitary AVP receptors. Docking of the peptide hormone AVP to the receptor ligand binding pockets reflects its dual polar and non-polar structure, but is receptor subtype-specific. The characteristics of non-peptide AVP analogs docking to the receptors are clearly distinct from those of peptide analogs docking. Molecular modeling of the results of site-directed mutagenesis experiments performed in CHO cells stably transfected with the human AVP receptor subtypes revealed that non-peptide antagonists establish key contacts with a few amino acid residues of the receptor subtypes that are different from those involved in agonist binding. Moreover, these interactions are species-specific. These findings provide further understanding of the signal transduction pathways of AVP receptors and new leads for elucidation of drug–receptor interactions and optimization of drug design. Note to the reader: The recent cloning and molecular characterization of AVP/OT receptor subtypes call for the revision of their nomenclature. For the sake of clarity and in reference to their main site of expression, we call the V1a receptor the V1 vascular receptor, the V2 receptor the V2 renal receptor and the V1b or V3 receptor the V3 pituitary receptor in the present review. Keywords: Vasopressin; Vasopressin receptor; Nonpeptide; Antagonists; 3-D modeling; Kinase; Receptor phosphorylation

∗ Correspondence to: M. Thibonnier, Room BRB431, Division of Clinical and Molecular Endocrinology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4951, USA. E-mail: [email protected]

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Introduction The neurohypophysial hormone arginine vasopressin (AVP) is actively involved in the regulation of free water reabsorption, body fluid osmolality, blood volume, blood pressure, cell contraction, cell proliferation, and ACTH secretion via the stimulation of specific G protein-coupled membrane receptors (GPCRs) classified into V1 vascular (V1 R), V2 renal (V2 R), and V3 pituitary (V3 R) subtypes having distinct pharmacological profiles and intracellular second messengers (Thibonnier et al., 1998b). Following agonist stimulation, GPCRs develop a reduction in responsiveness called desensitization that involves phosphorylation of the receptor by several types of kinases including tyrosine kinases, kinases activated by second messengers (PKA, PKC), and serine/threonine G protein-coupled receptor kinases (GRKs) (Lefkowitz, 1998). The mode of interaction of AVP receptors with intracellular kinases remains to be demonstrated. The examination of the amino acid composition of AVP receptors indicates the presence of several consensus motifs for PKC and GRK. Using immunoprecipitation and immunoblotting techniques, we studied the direct interaction between green fluorescent protein (GFP)tagged AVP receptors and PKC and GRKs. AVP receptors represent a logical target for drug development. However, the potential usefulness of AVP receptor antagonists in treating human diseases still remains an unanswered question because of the lack of currently FDA-approved orally active agents. However, orally active AVP receptor antagonists could be used in the treatment of various human diseases. Potential therapeutic applications may include: • The blockade of V1 vascular AVP receptors in arterial hypertension, congestive heart failure, and peripheral vascular disease. Moreover, the blockade of the V1 vascular AVP receptors present in the non-pregnant uterus may alleviate the symptoms of primary dysmenorrhea, a major cause of lost wages in the female population. • The blockade of V2 renal AVP receptors in the syndrome of inappropriate secretion of AVP, congestive heart failure, liver cirrhosis, nephrotic syndrome and any state of excessive retention of free water and subsequent hyponatremia.

•

The blockade of V3 pituitary AVP receptors in ACTH-secreting tumors.

Phosphorylation pattern of AVP receptors Agonist activation of GPCRs triggers a cascade of events including G protein coupling, phosphorylation of intracellular domains, internalization of the occupied receptor, and activation of intracellular messengers. Indeed, AVP activation of the V1 R elicits a rapid internalization and phosphorylation of the receptor (Innamorati et al., 1998). The family of AVP receptors is interesting in that its various members are coupled to different G proteins and exert opposite effects on cell growth and proliferation: the V1 R is coupled to Gq and produces a mitogenic response whereas the V2 R is coupled to Gs and produces an antimitogenic response (Thibonnier et al., 1998a). We recently showed that in CHO cells transfected with the wild-type V1 R, AVP induced a progression through both the S and G2 –M phases of the cell cycle (Thibonnier et al., 2000b) whereas no progression through the cell cycle was observed for the wild-type V2 R stimulation. When compared to the wild-type V1 R, AVP no longer produced progression through the cell cycle for a V1 R lacking its C-terminus (V1 R359X truncated receptor). AVP produced a normal progression through the cell cycle in mutants displaying the proximal portion of the V1 R C-terminus (V1 R399X, V1 R406X, and V1 R409X truncated receptors). AVP stimulation of the V1 RS382G-R384G mutant receptor (inactivation of the GRK-specific motif) produced a progression from the G0 –G1 phase to the S phase but no further progression to the G2 –M phase. Study of the cell cycle progression in the chimeric V1 R/V2 R and V2 R/V1 R clones revealed patterns similar to those of the corresponding wild-type receptors, i.e., cell cycle progression for the V1 R/V2 R clone, but no cell cycle progression for the V2 R/V1 R chimera. These data suggest that, like for IP production and DNA synthesis, the C-terminus of the V1 R participates in the effect of AVP on cell cycle progression, but cannot elicit such response by itself. Serine/threonine phosphorylation We explored the structural elements of the human V1 R mediating the mitogenic properties of this recep-

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Fig. 1. Structure of the C-termini of the wild-type human V1 vascular, V2 renal and V3 pituitary AVP receptors. The PKC motifs [(S/T)X(R/K)] are in enlarged italic characters and the diacidic motifs are enlarged and underlined.

tor (Berrada et al., 2000; Thibonnier et al., 2000b,c). Phosphorylation of intracytoplasmic residues of GPCRs plays an obligatory role in signal transmission (Pitcher et al., 1998). The examination of the amino acid composition of the human V1 R, V2 R and V3 R reveals the presence of several serine and threonine potential phosphorylation sites at the level of the intracytoplasmic loops of all receptors. In contrast, the V1 R and V3 R C-termini contain one proximal G protein-coupled receptor kinase (GRK) consensus motif (diacidic motif upstream of serine residues) and two to three distal protein kinase C (PKC) consensus motifs ([S/T]X[R/K]) whereas the human V2 R C-terminus contains one GRK consensus motif but no PKC motif (Fig. 1). These observations suggest that the C-terminus of AVP receptors plays a determinant role in receptor phosphorylation and signal transduction. We studied the physical association of AVP receptors with GRK and PKC in wild-types and mutated forms of the receptors fused to green fluorescent protein (Berrada et al., 2000). After agonist stimulation, PKC dissociates from the V1 R, associates with the V3 R, but does not interact with the V2 R. Furthermore, AVP activation of the receptors leads to a brief association of all the receptor subtypes with GRK5 following a time-course that varies with the receptor subtype. Exchanging the V1 R and the V2 R C-termini alters the time course of PKC and GRK5 association. Mutation of the GRK site present in the proximal portion of the V1 R Cterminus dramatically reduces the extent of receptor phosphorylation. Thus, agonist stimulation of AVP receptor subtypes triggers receptor subtype-specific interactions with GRK and PKC through specific serine-containing motifs present in the C-termini of the receptors. Our direct assessment on the physi-

cal association between the human V1 R and kinases indicates that PKC phosphorylates the unoccupied receptor whereas GRK5 is responsible for agonistinduced phosphorylation of the receptor within seconds. These findings suggest that a dynamic interaction exists between the V1 R, PKC and GRK5 in an agonist-dependent fashion. Upon agonist activation, PKC is released from the receptor C-terminus, allowing GRK5 to associate with the proximal portion of this C-terminus. The phosphorylation pattern of the V2 R is clearly different from that of the V1 R. Phosphorylation of the V2 R occurs at a slower rate, lasts longer, and involves GRK5 but not PKC. Tyrosine phosphorylation The AVP receptors share among them a NPWIY motif present in their seventh helix. An NPX2–3 Y motif is present in virtually all members of the GPCR family and is positioned to be part of the conformational changes induced by agonist binding. For instance, the NPVIY motif of the rhodopsin receptor may form a specific interaction with methionine257 of the 6th helix that stabilizes the inactive receptor conformation (Han et al., 1998). Asparagine302 and tyrosine306 of the bovine rhodopsin NPVIY motif project inside the molecule and the OH group of tyrosine306 is close to asparagine73 of the 2nd helix, facilitating hydrogen-bonding constraints between helices II and VII (Palczewski et al., 2000). The tyrosine residue present within this NPX2–3 Y motif is instrumental in several events including receptor internalization and signal transduction (Barak et al., 1994, 1995; Laporte et al., 1996). We investigated the role played by the tyrosine residue of the NPWIY motif present in the sev-

182

enth transmembrane domain of the human V1 R in ligand binding characteristics, internalization kinetics, phosphorylation pattern, and signal transduction. As we reported previously, addition of a GFP tag at the carboxy terminus of the receptor did not interfere with its membrane insertion and ligand characteristics (Berrada et al., 2000). Mutation of the tyrosine348 residue of the NPWIY motif present in the 7th helix of the V1 R did not hamper the proper insertion of the receptor inside the cell membrane and did not alter the affinity for the native ligand AVP, as K d values were similar to that of the wildtype receptor. Moreover, competition binding experiments with AVP, the non-peptide V1 R antagonist SR 49059, and the non-peptide V2 R antagonist SR 121463A indicated that the mutation of tyrosine348 did not alter the ligand subtype selectivity of the V1 R. V1 Rs internalize once they are occupied by AVP (Fishman et al., 1985; Thibonnier, 1992). As the conserved NPXn Y motif of GPCRs does not seem to be a general sequestration sequence, we explored the role of the V1 R NPWIY motif in the receptor internalization in stably transfected CHO cells expressing the V1 R Y348A mutated clone. The wildtype V1 R was quickly internalized at 37°C with an internalization half-life of 5 min and a maximal level of internalization of 89%. The extent and rate of internalization of the V1 R-Y348A mutant were similar, with an internalization half-life of 6 min and a maximal level of internalization of 91%. Thus the NPWIY motif of the V1 R does not play a role in the receptor internalization. The cellular localization of the fluorescent wild-type and mutated V1 Rs was also studied by epifluorescent microscopy before and after AVP stimulation. As reported previously, non-transfected CHO cells did not produce any fluorescence (Berrada et al., 2000). Before AVP stimulation, both the wild-type and Y348A mutant V1 Rs displayed a diffuse and homogenous fluorescent pattern at the cell surface (Fig. 2). We have previously shown that within minutes after agonist stimulation of CHO cells transfected with the wild-type AVP or OT receptors, the pattern of fluorescence changed and became granular, reflecting the internalization and aggregation of the receptors inside the cytosolic compartment (Berrada et al., 2000). A similar effect of agonist binding on the cellular distribution of the

fluorescent receptors was noted for the V1 R-Y348A mutant, thus confirming the lack of involvement of this motif in the membrane distribution and internalization process of the receptor (Fig. 3). Binding of the non-peptide V1 R antagonist SR49059 to the Y348A mutant receptor did not modify the diffuse fluorescent pattern of the receptor, thus confirming that the antagonist-occupied receptor is not internalized (Fig. 3). The V1 Rs is rapidly phosphorylated after agonist stimulation, presumably at the level of serine/ threonine residues (Innamorati et al., 1998). In fact, we mentioned above that the mutation of a GRK motif (SRR sequence downstream of a ED diacidic motif) present in the C-terminus of the V1 R prevented the association with GRK5 and reduced the extent of receptor phosphorylation (Berrada et al., 2000). However, tyrosine phosphorylation of the V1 R has not been demonstrated so far. Thus, we used immunoprecipitation and immunoblotting techniques to look at the possibility of agonistinduced tyrosine phosphorylation of the wild-type and Y348A mutant V1 Rs with a C-terminus GFP tag. We have previously shown that the addition of a GFP tag to the C-terminus of AVP/OT receptors allows their efficient and specific immunoprecipitation and immunoblotting without interfering with signal coupling (Berrada et al., 2000). On both direct immunoblotting and immunoprecipitation/immunoblotting autoradiograms, the glycosylated full length wild-type and Y348A mutated V1 R-GFP complexes both presented as a broad band of molecular mass of 105–110 kDa (Fig. 4). Minor bands of molecular mass of 65–70 kDa were detected and may represent unglycosylated or degraded forms of the receptor (Phalipou et al., 1997a). Indeed, omission of the protease inhibitors in the lysis buffer increased the intensity of these smaller bands (data not shown). Immunoprecipitation of the V1 R with an antiGFP monoclonal antibody followed by immunoblotting with an anti-phosphotyrosine antibody revealed that the wild-type V1 R underwent tyrosine phosphorylation after agonist stimulation (Fig. 5A). Tyrosine phosphorylation was rapid, occurring within 30 s after agonist stimulation, and transient, peaking within 5 min after agonist stimulation. When the same blots were reprobed with an anti-GFP polyclonal antibody, the V1 R-GFP was detected in equal amounts in all

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Fig. 2. Cellular distribution of fluorescent wild-type and NPWIY348A mutated V1 Rs. CHO cells expressing the wild-type and NPWIY348A mutant V1 Rs fused to GFP at their C-terminus (and control non-transfected cells) were studied by fluorescent microscopy. Left panels represent direct microscopic appearance of cells, while right panels represent fluorescent patterns of the same cells.

lanes (data not shown). Immunoprecipitation and immunoblotting of extracts from cells expressing the GFP protein alone revealed no signal, thus ruling out non-specific tyrosine phosphorylation of the GFP tag (Fig. 5B). Furthermore, sequential immunoprecipitation, first with the anti-phosphotyrosine antibody, then with the anti-GFP monoclonal antibody, followed by immunoblotting with the anti-GFP poly-

clonal antibody revealed the same 105–110-kDa band (Fig. 5C), thus confirming that indeed the wildtype V1 R is tyrosine-phosphorylated in response to agonist stimulation. Conversely, immunoprecipitation with the antiphosphotyrosine antibody followed by immunoblotting with the anti-GFP antibody isolated the same band (Fig. 6). The extent of receptor phosphoryla-

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Fig. 3. Effects of agonist and antagonist binding on the cellular distribution of the fluorescent NPWIY348A mutated V1 R. CHO cells expressing the NPWIY348A mutant V1 R fused to GFP at its C-terminus were studied by fluorescent microscopy. The distribution of the fluorescent receptors was assessed before and after agonist (1 μM AVP) or after V1 R nonpeptide antagonist (1 μM SR490590) binding for 15 min at 37°C.

tion was dramatically reduced in the V1 R-Y348A mutant, thus indicating that the tyrosine residue of the NPWIY motif of the 7th helix is the target of a tyrosine kinase (Fig. 6). As the C-terminus of the human V1 R contains also a tyrosine residue in position 388 that could be the target of tyrosine kinases after agonist stimulation, immunoblotting with an anti-phosphotyrosine antibody was performed with a truncated form of the human V1 R whose C-terminus has been deleted at position 359. This truncated V1 R underwent agonist-dependent tyrosine phosphorylation to the same degree as the wild-type receptor (data not shown), thus ruling out the involvement of the receptor C-terminus in tyrosine phosphoryla-

Fig. 4. Immunoprecipitation and immunoblotting of the GFPtagged wild-type and Y348A mutated V1 Rs. CHO cells expressing GFP-tagged wild-type and Y348A mutant V1 Rs were lyzed, immunoprecipitated (IP) with an anti-GFP monoclonal antibody and immunoblotted (IB) with an anti-GFP polyclonal antibody. Total cell lysate (TCL) samples were directly immunoblotted. A peroxidase-labeled secondary antibody was used for detection of the chemiluminescent signal.

Fig. 5. AVP-induced tyrosine phosphorylation of the GFP-tagged wild-type V1 R. Transfected cells were grown to subconfluence in 100-mm dishes then serum-starved and stimulated by AVP (1 μM at 37°C up to 10 min). The cells were lyzed and cell extracts were immunoprecipitated (IP) with an anti-GFP monoclonal antibody or an anti-phosphotyrosine monoclonal antibody. Immunoprecipitates were resolved by SDS–PAGE followed by immunoblotting (IB) with an anti-phosphotyrosine monoclonal antibody or an anti-GFP polyclonal antibody. A peroxidaselabeled secondary antibody was used for detection of the chemiluminescent signal. (A) IP and IB of CHO cells expressing GFP-tagged wild-type V1 Rs. (B) CHO cells expressing the GFP tag alone. (C) Double IP of CHO cells expressing GFP-tagged wild-type V1 Rs.

185

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Fig. 6. Reduction of AVP-induced tyrosine phosphorylation of the GFP-tagged Y348A V1 R. CHO cells expressing GFP-tagged wild-type and Y348A mutant V1 Rs were grown to subconfluence, then serum-starved and stimulated by AVP (1 μM at 37°C up to 5 min). The cells were lyzed and cell extracts were immunoprecipitated (IP) with an anti-phosphotyrosine monoclonal antibody. Immunoprecipitates were resolved by SDS–PAGE followed by immunoblotting (IB) with an anti-GFP polyclonal antibody. A peroxidase-labeled secondary antibody was used for detection of the chemiluminescent signal.

tion. In CHO cells transfected with the pEGFP-N3 vector alone, phosphotyrosine proteins did not coimmunoprecipitate with an anti-GFP antibody (data not shown). We recently reported that AVP stimulation of the V1 R produced the simultaneous activation of several kinases, including PI 3-kinase, p42/p44 MAP kinase and calcium/calmodulin kinase II that led to a mitogenic response (Thibonnier et al., 2000b). We tested the influence of the V1 R-Y348A mutation on the activation of the kinases linked to V1 R activa-

tion. As shown in Fig. 7, the extent and duration of activation of p42/p44 MAP kinase and p70 kinase (downstream of the PI 3-kinase) were dramatically reduced in cells transfected with the mutated V1 R. The activation of calcium/calmodulin kinase II by the V1 R-Y348A mutant was not altered (data not shown). Finally, assessment of tyrosine phosphorylation of total cell lysates after AVP stimulation revealed a major reduction of the level of protein phosphorylation in the presence of the Y348A mutation, thus indicating that Tyr348 is instrumental in linking the V1 R to tyrosine kinases after AVP stimulation (Fig. 8). We investigated the role of the NPWIY motif phosphorylation in the signal transduction cascade of the V1 R. V1 Rs are coupled to phospholipase C with a subsequent increased production of inositol phosphates (IP). In CHO cells transfected with the wildtype V1 R, AVP produced a dose-dependent increase in the formation of IP (Fig. 9) that was blocked by the non-peptide V1 R antagonist SR 49059, but not by the V2 R antagonist SR 121643A (data not shown). AVP could still stimulate IP production in cells expressing the Y348A mutated V1 R, but the maximum response was only 45% of that of the wild-type V1 R ( p < 0.01). These data suggest that the NPWIY motif of the V1 R participates in the stimulation of IP production. One major cellular event resulting from the stimulation of V1 Rs is cell growth and proliferation, which can be assessed by measuring nucleic acid synthesis through [3 H]thymidine uptake. Thus, AVP-induced [3 H]thymidine uptake was measured in CHO cells transfected with the wild-type or the mutated form of the human V1 R. We have previously shown that AVP induced a dose-dependent increase in [3 H]thymidine uptake in CHO cells transfected with the wild-type V1 R (Thibonnier et al., 1998b). AVP stimulation of DNA synthesis was dramatically reduced in the Y348A mutant, whereas this mutated clone was normally responsive to stimulation by 10% FBS (Fig. 10). These data suggest that the NPWIY motif of the V1 R participates in the nucleic acid synthesis step of the mitogenic response. We have previously shown that AVP induced a dose-dependent increase of cell proliferation in CHO cells transfected with the wild-type V1 R (Thibonnier et al., 1998b). As shown in Fig. 11, AVP stimulation of cell proliferation was dramatically reduced in the

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p42/p44 p70 βActin 0 ' 10' 20' 30' 40' 60' 90' 90' AVP stimulation (min.) 10% FBS

10% FBS

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Fig. 7. Effect of the Y348A mutation on AVP-induced kinase phosphorylation. Transfected CHO cells were grown to sub-confluence, serum-starved, then stimulated by 1 μM AVP for 0–90 min or by 10% FBS for 90 min. Cells were lysed, protein was resolved by gel electrophoresis followed by immunoblotting with p42/p44 and p70 phosphospecific antibodies. The same blots were stripped and reprobed with an anti-actin antibody to verify homogeneity of protein transfer.

Fig. 8. Effect of the Y348A mutation on AVP-induced protein phosphorylation. Transfected CHO cells were grown to sub-confluence, serum-starved, then stimulated by 1 μM AVP for 0–30 min. Cells were lyzed (TCL, total cell lysate), protein were resolved by gel electrophoresis followed by immunoblotting (IB) with an anti-phosphotyrosine monoclonal antibody. The same blots were stripped and reprobed with an anti-actin antibody to verify homogeneity of protein transfer.

Y348A mutant, thus confirming that the NPWIY motif of the V1 R is required to elicit a mitogenic response.

In conclusion, as expected for a typical GPCR, the V1 R becomes phosphorylated after agonist stimulation. We and others have shown that agonist stim-

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AVP Concentration (M) Fig. 9. AVP-induced inositol phosphate production by wild-type and mutated AVP receptors. CHO cells expressing the wild-type and Y348A mutant V1 Rs were grown to confluence in 12-well dishes and incubated in inositol-free DMEM buffer with myo[23 H]inositol. Formation of [3 H]inositol phosphates was measured after addition of increasing concentrations of AVP for 30 min at 37°C (n = 6 for each data point).

Fig. 10. AVP-induced [3 H]thymidine uptake in CHO cells transfected with the wild-type or mutated receptor cDNA clones. CHO cells expressing the wild-type and Y348A mutant V1 Rs were grown to sub-confluence in 24-well dishes and incubated in serum-free F12 medium for 72 h. The cells were subsequently stimulated overnight by increasing concentrations of AVP (or 10% FBS as a positive control), followed by incubation with [3 H]thymidine for 45 min, DNA precipitation and liquid scintillation counting (n = 8 for each data point).

ulation of the AVP/OT receptors led to receptor subtype-specific interactions with GRK and PKC through serine residues of specific motifs present in the C-termini of the receptors (Innamorati et al., 1998; Berrada et al., 2000). In addition to the GRK-dependent serine phosphorylation, we directly

Fig. 11. AVP-induced cell proliferation in CHO cells transfected with the wild-type or mutated receptor cDNA clones. CHO cells expressing the wild-type and Y348A mutant V1 Rs were grown to sub-confluence in 96-well dishes and incubated in serum-free F12 medium for 72 h. The cells were stimulated by AVP for 24 h, followed by addition of the dye MTS for 2 h. Absorbance was recorded at 490 nm (n = 12 for each data point).

demonstrated for the first time that the V1 R is also phosphorylated at the level of a conserved tyrosine residue present in its 7th helix. Similarly, mutation of Tyr326 of the NPLIY motif of the β2 -adrenergic receptor to an alanine residue resulted in a dramatic reduction of the receptor phosphorylation in response to agonist stimulation (Barak et al., 1995). The phosphorylation of GPCR serine/threonine residues by GRKs has been shown to trigger the binding of arrestins with subsequent interaction with clathrin, internalization and desensitization of the receptors (Laporte et al., 1999). The results presented here demonstrate that tyrosine phosphorylation of the NPWIY motif of the V1 R serves other purposes as no role in ligand binding and receptor internalization was found. As a matter of fact, prevention of Tyr348 phosphorylation in response to AVP stimulation severely hampers the mitogenic signaling cascade linked to activation of the V1 R. Similarly, alanine mutations of the residues of the NPLFY motif of the angiotensin II type 1 receptor were shown to impair the agonistinduced stimulation of IP formation (Hunyady et al., 1995). These findings suggest that agoniststimulation of the V1 R triggers at least two distinct pathways: one is governed by the GRK-dependent phosphorylation of serine/threonine residues present in the cytoplasmic C-terminus of the receptor that leads to the receptor internalization and subsequent desensitization. The other involves the tyrosine phos-

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phorylation of the NPWIY motif of the 7th helix that plays a crucial role in the transmission of the mitogenic signal. Protein tyrosine phosphorylation regulates protein–protein interaction and enzyme activation involved in mitogenic signal transduction following activation of cytokine and growth factor receptors. Furthermore, phosphotyrosine binding (PTB) domains of Shc and insulin receptor substrate 1 preferentially bind to tyrosine-phosphorylated NPXY-containing insulin and interleukin-4 receptors. The member of the insulin receptor family leukocyte tyrosine kinase contains two NPXY motifs that equally contribute to activation of the Ras pathway and generation of mitogenic signals. Mitogenic signaling by GPCRs involves phosphorylation reactions and assembly of protein complexes. At least three types of scaffolds of GPCR– protein complexes have been identified (Luttrell et al., 1999). They include transactivated receptor tyrosine kinases (RTKs), integrin-based focal adhesions, and GPCRs themselves. Indeed, GPCRs can serve themselves as signaling platforms after agonist stimulation, recruiting GRKs, subunits of G proteins, arrestins, and other kinases (e.g. c-Src). Recruitment of β-arrestin and activation of c-Src initiates the assembly of a protein scaffolding that leads to MAPK recruitment and mitogenic signaling. The assembly of such scaffolding leads to the mitogenic response. The determination of the crystal structure of rhodopsin at a 2.8 Å resolution has identified a set of residues that mediate interaction between the helices and the cytoplasmic surface, where G protein activation occurs (Palczewski et al., 2000). Among them, the OH group of the tyrosine residue of the NPXXY motif present in the 7th helix is close to a conserved asparagine residue present in the 2nd helix, suggesting the presence of additional interhelical constraints between helices 2 and 7. We hypothesize here that the phosphorylation of the Tyr348 residue of the V1 R upon agonist stimulation produces a conformational change that activates the mitogenic cascade. In a model similar to that described for growth factor receptors, agonist activation of the V1 R triggers tyrosine phosphorylation of the NPWIY motif within the 7th helix that allows binding of proteins containing phosphotyrosine binding domains and further assembly of a scaffold of the various components of the mitogenic cascade.

Three-dimensional molecular docking of peptide AVP analogs to AVP receptors Cloning of human and animal AVP/OT receptor subtypes and their stable expression in immortalized cell lines has allowed several investigators to begin defining the molecular determinants of AVP receptor subtypes peptide ligand selectivity (Chini et al., 1995; Mouillac et al., 1995; Ufer et al., 1995; Postina et al., 1996; Phalipou et al., 1997b; Cotte et al., 1998). As shown in Fig. 12a, we built a 3-D model of the human V1 R and successfully docked AVP within the structure of this receptor (Thibonnier et al., 2000a). The 3-D model of AVP was docked onto V1 R by initially placing it in the upper portion of the transmembrane region (the expected binding pocket), and searching for the binding site with the program LIGIN within a 20×20×20 Å box around the original ligand position. In the docking of AVP, some steric overlap (1–3 residues) was allowed between the ligand and receptor. Energy minimization with program XPLOR relieved these short contacts. AVP has a polar as well as a non-polar surface. The exocyclic tripeptide Pro7 –Arg8 –Gly9 and one side of the hormone ring (Gln4 , Asn5 ) are mainly hydrophilic, whereas the other part of the ring (Cys1 , Cys6 , Tyr2 , and Phe3 ) is essentially hydrophobic in nature. This dual surface property is reflected in the nature of the binding pocket that is formed by residues from transmembrane segments (TMSs) 1, 3, 4, 5, 6, and 7, as well as the first extracellular loop (Fig. 13). The bottom of the cleft is mainly hydrophobic, closed by the aromatic and hydrophobic residues Met135, Phe136 , Phe179 , Phe307 and Ile330. The entrance to the binding pocket and one side of it contain predominantly hydrophilic residues. The Arg8 guanido group at the entrance to the cleft forms a salt bridge with Asp112 located on the first extracellular loop. Trp111 forms van der Waals contacts with the hydrophobic part of Arg8 . The ε-amino group of Lys128 forms a hydrogen bond to the amide side chain nitrogen of Asn5 . Other hydrogen bonds are formed between the side chain moieties of Gln185 and Ser182 with Gln4 , and Ser213 Oγ with Tyr2 OH. Another wall of the pocket is lined with the hydrophobic residues Ile55 and Ile330 . Some amino acid residues that are common to

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Fig. 12. Docking of AVP to the human V1 vascular (a) and V3 pituitary (b) AVP receptors. The loops are labeled il1, il2, and il3 for the intracellular loops and el1, el2 and el3 for the extracellular loops. The transmembrane segments are labeled H1–H7. The different orientation of AVP binding to the receptor subtypes is clearly shown.

all AVP/OT receptor subtypes are important for peptide agonist binding. They are D207 , Q214 , Q218 , K308 , Q311 , Q413 , and Q620 . None of these residues is involved in peptide and non-peptide antagonist binding (Mouillac et al., 1995). The presence of a disulfide bond between two conserved cysteine residues present in exoloops 1 and 2 is required to maintain the integrity of the receptor structure. Studies performed recently with the V1 vascular, V2

renal AVP and the OT receptors from several species revealed that a few key residues determine peptide ligand selectivity for a given receptor subtype. For instance, residue Tyr115 located in the first extracellular loop is crucial for high affinity binding of peptide agonists and confers V1 vascular receptor subtype specificity (Chini et al., 1995). The use of natural small synthetic peptides mimicking segments of the V1 R revealed that the N-terminal part of the V1 R is

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Fig. 12 (continued).

not involved in peptide agonist binding (Mendre et al., 1997). At variance, natural peptides mimicking the external loops of the V1 R, especially one peptide mimicking the 205–218 portion of the 2nd extracellular loop, were able to inhibit specific AVP binding to the V1 R. Site-directed mutagenesis experiments of the cloned bovine and porcine V2 Rs revealed that Asp103 in the first extracellular loop is responsible for high affinity binding of the V2 R peptide agonist dDAVP (Ufer et al., 1995). Similarly, residues responsible for selective binding of peptide agonists and antagonists to the V2 renal receptor were identified (Cotte et al., 1998). Residues 202 (Arg vs. Leu) in the second extracellular loop and 304 (Gly vs. Arg) in the 7th transmembrane domain are responsible for species-selective cyclic peptide antagonists binding in an independent and additive manner. Residue 100

(Lys vs. Asp) in the second transmembrane domain plays a similar role for peptide agonist discrimination. For peptide agonist binding and selectivity for the OT receptor subtype, the first three extracellular domains are most important (Postina et al., 1996). The N-terminal domain and the first extracellular loop of the OT receptor interact with the linear Cterminal tripeptidic part of the ligand OT, whereas the second extracellular loop of the OT receptor interacts with the cyclic part of OT. The molecular determinants of peptide antagonist binding to the OT receptor are different, i.e. the transmembrane helices 1, 2, and especially 7. Introduction of just seven amino acids of the upper part of the 7th TMS of the OT receptor into the V2 R sequence is sufficient to introduce high affinity binding for an OT peptide antagonist into the V2 R.

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Fig. 13. Docking of the non-peptide antagonist OPC-21268 onto the model of the human V1 vascular AVP receptor. (a) Top view. (b) Stabilizing effect of the G337A, I224V, and I310V mutations on antagonist binding.

We created a three-dimensional molecular model of the human V3 R. As basis for our model building of the V3 R receptor we used a model of the

seven TMSs of V3 R generated by G. Vriend with the program WHATIF (Vriend, 1990) based upon the crystal structure of bacteriorhodopsin (Rodriguez

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et al., 1998). The three extracellular and three intracellular loops of the V3 R were subsequently constructed with program Look v3.5 (Molecular Applications Group, Palo Alto, CA 94304), using the spatial constraints for the ends of each loop provided by the coordinates of the helical bundle. Look v3.5 is a protein-modeling program that segmentally builds a protein by aligning short stretches of its sequence with homologous peptides of known structure, and also performs a full energy refinement of the model. As the N-terminal and C-terminal domains of the V3 R are not involved in the binding of agonists or antagonists, they were not included in this model. A disulfide bridge exists between cysteines 107 and 186 located on the second and third extracellular loops, respectively. Disruption of this disulfide bridge is known to cause a significant drop in binding affinity of ligands. Thus, it was necessary to ascertain that these cysteine residues were close enough in the model and that the sulfhydryl groups had the proper orientation in order to be able to form the disulfide bridge. This was achieved by performing an energy refinement in program X-PLOR with the constraint of forming this particular disulfide bridge. The sulfur–sulfur distance refined to a value of 2.01 Å, consistent with the formation of a disulfide bridge. The rest of the structure was not significantly altered by this refinement procedure. The 3-D appearance of the human V3 R was compared to that of the human V1 R (Fig. 12). There is a tight overlap at the level of all TMSs with four TMSs (1st, 5th, 6th, and 7th) perpendicular to the membrane plan and three TMSs (2nd, 3rd, and 4th) slightly tilted. On the other hand, the respective extra- and intracellular loops are of different length and seem to have different conformations in these two receptors. The maximum deviation and rms values between the models we have built for the two receptor subtypes are excellent, only 3.8 and 1.4 Å for all common α carbon atoms, respectively. We docked the 3-D model of AVP that we developed previously to the human V3 R with the program LIGIN (Fig. 12b). Energy minimization with program X-PLOR relieved the short contacts. As shown in Fig. 12, it appears that the docking pattern of AVP to the human V3 R is strikingly different from its docking pattern to the human V1 R: instead of being docked in a slanted fashion like in the V1 R, AVP docked inside the

ligand binding pocket of the V3 R in a vertical fashion. The AVP hydrophilic tail is in close contact with the upper portion of the ligand binding pocket, especially the 3rd extracellular loop whereas the hydrophobic ring is buried deep inside the TMSs where it makes close contact with several residues of the 6th TMS. This unique docking pattern of AVP to the V3 R that is distinct from its docking to the V1 R but also to the V2 R, is a major finding that readily explains why ligands that are specific for the V1 R and the V2 R have a poor affinity for the V3 R and that V3 R-selective ligands have yet to be discovered. Thus, these studies suggest that the molecular determinants of peptide agonists and antagonists binding to AVP/OT receptors are distinct. Three-dimensional molecular docking of non-peptide antagonists to AVP receptors Examination of the 3-D structure of the non-peptide AVP receptor antagonists indicates that this is a rather heterogeneous class of compounds (Thibonnier et al., 2001). As there was no knowledge of the molecular determinants of AVP receptors involved in nonpeptide antagonist binding, we studied this issue by site-directed mutagenesis and molecular modeling techniques. The first non-peptide AVP V1 R antagonist found by random screening and optimization of chemical entities, OPC-21268 has an excellent affinity for the rat V1 R (25 nM), but has a poor affinity for the human V1 R (8800 nM) (Thibonnier et al., 1998b). The human and rat V1 Rs share a high degree of structural homology with 96% sequence identity. The differing residues are presumably involved in species-related variations in antagonist binding. Comparison of the human and rat V1 R sequences revealed that only 20 amino acid differences are present in the extracellular loops and the upper portions of the transmembrane segments. We reasoned that these interspecies differences in amino acid sequence modulate the receptor affinity for non-peptide compounds. Thus, we produced a series of reverse mutations in which corresponding rat amino acids were introduced by site-directed mutagenesis into the human V1 R sequence (Thibonnier et al., 2000a). The influence of these interspecies amino acid differences on nonpeptide antagonist binding was subsequently tested. The introduction of rat amino acids in positions 224,

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310, 324, or 337 of the human V1 R sequence dramatically altered OPC-21268 affinity for the receptor whereas binding of AVP, the peptide V1 R antagonist d(CH2 )5 Tyr(Me)AVP, and the non-peptide V1 R antagonist SR49059 was not altered by these mutations. In order to gain information about the location of the OPC-21268 binding site, a model of this compound was docked onto a homology-built threedimensional model of the human V1 R (Thibonnier et al., 2001). Very little direct structural information is available for GPCRs, and for many years molecular models of these receptors have been built based upon the crystal structure of bacteriorhodopsin. Although bacteriorhodopsin consists of the seven transmembrane helices by which GPCRs are characterized, it shares very little sequence homology with any of the GPCRs. Still, the use of bacteriorhodopsin to establish the orientation of the transmembrane domains of AVP receptors is the only way to build a model based on an experimentally determined highresolution structure (Henderson et al., 1990). Coordinates of bovine rhodopsin are also available, but only for the seven TMSs without any loops. As basis for our model, building of the V1 R receptor we used a model of the seven TMS of V1 R generated by G. Vriend with the program WHATIF (Vriend, 1990) based upon the crystal structure of bacteriorhodopsin (G Protein-Coupled Receptor Data Base at http:// swift.emblheidelberg.de/7tm/htmls/consortium.html) (Rodriguez et al., 1998). The three extracellular and three intracellular loops of the V1 R were subsequently constructed with program Look v3.5 (Molecular Applications Group, Palo Alto, CA 94304), using the spatial constraints for the ends of each loop provided by the coordinates of the helical bundle. Look v3.5 is a protein-modeling program that segmentally builds a protein by aligning short stretches of its sequence with homologous peptides of known structure, and also performs a full energy refinement of the model (Levitt, 1992). As the N-terminal and C-terminal domains of the V1 R are not involved in the binding of agonists or antagonists, they were not included in this model. A disulfide bridge exists between cysteines 124 and 203 located on the second and third extracellular loops, respectively. Disruption of this disulfide bridge is known to cause a significant drop in binding

affinity of ligand. Thus, it was necessary to ascertain that these cysteine residues were close enough in the model and that the sulfhydryl groups had the proper orientation in order to be able to form the disulfide bridge. This was achieved by performing an energy refinement in program X-Plor with the constraint of forming this particular disulfide bridge. The sulfur– sulfur distance refined to a value of 2.03 Å, consistent with the formation of a disulfide bridge. The rest of the structure was not significantly altered by this refinement procedure. Models of the non-peptide AVP receptor antagonists were constructed with the program Alchemy 2000 (Tripos Inc., St. Louis, MO). First, the compounds are drawn in two dimensions, and then extended into a three-dimensional model by a 2D-to3D builder incorporated in Alchemy 2000. Conformations with the lowest energy and devoid of any short contacts were saved. Finally, the most stable conformations are subjected to an optimization using the program Gaussian 98 (Gaussian, Inc., Pittsburgh PA, 1998). Docking of the non-peptide ligand OPC-21268 to the receptors was done with the program LIGIN based on a built-in complementarity function (Sobolev et al., 1996). This function is a sum of the surface areas of atomic contacts. These contacts are weighted according to the types of atoms in contact, and another term is included to prevent short contacts. After maximizing the complementarity function, LIGIN optimizes the lengths of possible hydrogen bonds. In order to take into account possible movements of the receptor upon ligand binding, steric overlap between the ligand and a specified number of residues in the receptor can be allowed without energy penalty. The location of the bound antagonist OPC-21268 is distinct from the AVPbinding pocket with only partial overlap near the extracellular surface (Fig. 13). The hydrophobic part is embedded in the transmembrane region far deeper than AVP does, whereas the polar part is located on the surface of the extracellular side. The binding pocket is formed by residues from TMSs 4, 5, 6, 7 as well as the third extracellular loop. The 27fold increase in the affinity of the Gly337Ala mutant is explained by the formation of two van der Waals contacts of the methyl carbon with carbon atoms C22 and C28 of the bicyclic ring structure of OPC-21268

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at the bottom of the cleft (Fig. 13b). The Glu324Asp mutant has an indirect effect. It enables the formation of a hydrogen bond of the carboxylate side chain with the amide side chain atom of Gln311 . This causes a polarization of this amide nitrogen atom and enables it in turn to form another hydrogen bond to the N57 nitrogen atom of OPC-21268. The Ile310Val mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. The Ile224Val mutant relieves overcrowding in a hydrophobic binding site involving the aromatic residues Trp175 , Phe179 , Phe307 and Trp304 . The smaller valine side chain allows for better positioning of the aromatic residues to interact with the bicyclic ring structure of OPC-21268. Finally, the Ile310Val mutant reduces the hydrophobicity in the vicinity of the polar oxygen atom of the antagonist. Thus, the model explains all the mutations that significantly increase the affinity towards OPC-21268. The combination of site-directed mutagenesis and 3-D modeling in our study identified key residues involved in binding of the non-peptide antagonist OPC-21268 to the V1 R. Our data clearly identified a single residue in the 7th TMS explaining the different affinities of the human and rat V1 R for OPC-21268. The docking model developed for the present study confirmed the importance of this single residue, Ala337 . Furthermore, the model predicts that a serine residue at this position should cause an even tighter binding due to the formation of a hydrogen bond between the serine Oγ atom with the quinoline oxygen atom of OPC-21268 in addition to the van der Waals interaction of the serine β-carbon with carbon atoms 22 and 28 of this antagonist. This study also suggests modifications to the antagonist to increase the affinity for the receptor. For example, elimination of the quinoline oxygen atom should stabilize the interactions with the hydrophobic pocket deep inside the transmembrane region. However, this may cause adverse solubility problems. A similar situation exists for residue 310 of the receptor and oxygen 47 of the antagonist. A hydrophobic residue in the vicinity of this polar atom is clearly unfavorable. A valine at this position, as found in the human sequence, is better than an isoleucine, the corresponding rat residue, but a threonine would be even better. Alternatively, replacement of oxygen 47 of the antagonist with a carbon atom should also

increase the affinity. With respect to residue 224, a valine at this position seems to be optimal. This residue is located in a rather crowded hydrophobic environment into which a valine seems to fit better than the bulkier isoleucine. Combination of the three mutations in positions 224, 324, and 337 did not improve further the affinity of the V1 R for OPC-21268 when compared to the two double mutations, thus suggesting that alterations of the structure of the non-peptide antagonist will be required to increase further the affinity of this compound. The field of GPCRs suffers from a lack of experimentally determined structures. Therefore, molecular modeling is a very useful tool to derive structural information for the V1 R. It provides a framework to design and test new drugs as well as site-specific mutations in a rational way. However, one has to keep in mind the limitations of molecular modeling. The approach is based on the assumption that the seven transmembrane segments are similar in structure to bacteriorhodopsin. The ‘Achilles’ heel’ of this approach are the loops connecting the helical regions as well as the N- and C-terminal non-helical segments. The former were built by sequence similarity to known protein segments from a database within the program LOOK, whereas the N- and C-terminal stretches were left out altogether from the model because they are not involved in ligand binding. The validity of the model is supported by the experimentally determined affinities for the drugs. The model explains very well all of our findings. It does not prove that the model is correct but the model is certainly consistent with the data, and it provides a tool for designing new drugs and mutants. In conclusion, our study provided for the first time the structural basis of species-selective binding of a non-peptide antagonist to the V1 R. These findings should generate new ideas for drug development of non-peptide AVP receptor antagonists and for optimizing drug–receptor interactions. Conclusions In the near future, results of ongoing clinical studies testing the new orally active non-peptide AVP receptor antagonists will tell us if these medications live up to their potential therapeutic indications. V1 R

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antagonists may turn out to be an effective treatment of dysmenorrhea. V2 R antagonists or aquaretics will undoubtedly facilitate the treatment of hyponatremia. However, because of their potency and the risk of rapid and excessive correction of serum sodium with potential neurologic complications, their use will require caution, especially in patients who do not have free access to water. Dual V1 R/V2 R antagonists in a ratio that remains to be established may provide useful adjuvant treatment of arterial hypertension and congestive heart failure. Finally, the design of specific V3 R antagonists may offer diagnostic tools and medical treatment for ACTH-secreting tumors. Acknowledgements We would like to thank Doreen Conarty, Christine Plesnicher, Xiang Luo and Karim Berrada for their significant contribution to the work summarized in this review. This chapter was supported by grants from the National Institutes of Health to M.T. and from the National Science Foundation to M.S. The Ireland Cancer Research Center Flow Cytometry Core Facility is supported by NIH Grant P30 CA43703 and the Cystic Fibrosis Video Microscope Core Facility is supported by NIH Grant P30 DK2651. M.T. has or has had research contracts with Otsuka, Parke-Davis, Sanofi, and Wyeth-Ayerst. References Barak, L.S., Tiberi, M., Freedman, N.J., Kwatra, M.M., Jelkowitz, R.J. and Caron, M.G. (1994). J. Biol. Chem., 269: 2790– 2795. Barak, L., Ménard, L., Ferguson, S.S.G., Colapietro, A.M. and Caron, M.G. (1995). Biochemistry, 34: 15407–15414. Berrada, K., Plesnicher, C.L., Luo, X. and Thibonnier, M. (2000). J. Biol. Chem., 275: 27229–27237. Chini, B., Mouillac, B., Ala, Y., Balestre, M.N., Trumpp-Kallmeyer, S., Hoflack, J., Elands, J., Hibert, M., Manning, M., Jard, S. and Barberis, C. (1995). EMBO J., 14: 2176–2182. Cotte, N., Balestre, M.N., Phalipou, S., Hibert, M., Manning, M., Barberis, C. and Mouillac, B. (1998). J. Biol. Chem., 273: 29462–29468. Fishman, J.B., Dickey, B.F., Bucher, N.L.R. and Fine, R.E. (1985). J. Biol. Chem., 260: 12641–12646. Han, M., Smith, S.O. and Sakmar, T.P. (1998). Biochemistry, 37: 8253–8261. Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E. and Downing, K.H. (1990). J. Mol. Biol., 213: 899– 929.

Hunyady, L., Bor, M., Baukal, A.J., Balla, T. and Catt, K.J. (1995). J. Biol. Chem., 270: 16602–16609. Innamorati, G., Sadeghi, H. and Birnbaumer, M. (1998). J. Biol. Chem., 273: 7155–7161. Laporte, S.A., Servant, G., Richard, D.E., Escher, E., Guillemette, G. and Leduc, R. (1996). Mol. Pharmacol., 49: 89–95. Laporte, S.A., Oakley, R.H., Zhang, J., Holt, J.A., Ferguson, S.S.G., Caron, M.G. and Barak, L.S. (1999). Proc. Natl. Acad. Sci. USA, 96: 3712–3717. Lefkowitz, R.J. (1998). J. Biol. Chem., 273: 18677–18680. Levitt, M. (1992). J. Mol. Biol., 226: 507–533. Luttrell, L.M., Daaka, Y. and Lefkowitz, R.J. (1999). Curr. Opin. Cell Biol., 11: 177–183. Mendre, C., Dufour, M.N., Le Roux, S., Seyer, R., Guillou, L., Calas, B. and Guillon, G. (1997). J. Biol. Chem., 272: 21027– 21036. Mouillac, B., Chini, B., Balestre, M.N., Elands, J., TrumppKallmeyer, S., Hoflack, J., Hibert, M., Jard, S. and Barberis, C. (1995). J. Biol. Chem., 270: 25771–25777. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C., Motoshima, H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M. and Miyano, M. (2000). Science, 289: 739–745. Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, D., Barberis, C. and Mouillac, B. (1997a). J. Biol. Chem., 272: 26536–26544. Phalipou, S., Cotte, N., Carnazzi, E., Seyer, R., Mahe, E., Jard, D., Barberis, C. and Mouillac, B. (1997b). J. Biol. Chem., 272: 26536–26544. Pitcher, J.A., Freedman, N.J. and Lefkowitz, R.J. (1998). Annu. Rev. Biochem., 67: 653–692. Postina, R., Kojro, E. and Fahrenholz, F. (1996). J. Biol. Chem., 271: 31593–31601. Rodriguez, R., Chinea, G., Lopez, N., Pons, T. and Vriend, G. (1998). CABIOS, 14: 523–528. Sobolev, V., Wade, R.C., Vriend, G. and Edelman, M. (1996). Proteins Struct. Funct. Genet., 25: 120–129. Thibonnier, M. (1992). Regul. Pept., 38: 1–11. Thibonnier, M., Berti-Mattera, L.N., Dulin, N., Conarty, D.M. and Mattera, R. (1998a) In: I.J.A. Urban, J.P.H. Burbach and D. De Wied (Eds.), Progress in Brain Research, Vol. 119. Elsevier Science, Amsterdam, pp. 143–158. Thibonnier, M., Conarty, D.M., Preston, J.A., Wilkins, P.L., Berti-Mattera, L.N. and Mattera, R. (1998b). Adv. Exp. Med. Biol., 449: 251–276. Thibonnier, M., Coles, P., Conarty, D.M., Plesnicher, C.L. and Shoham, M. (2000a). J. Pharmacol. Exp. Ther., 294: 195–203. Thibonnier, M., Conarty, D.M. and Plesnicher, C.L. (2000b). Am. J. Physiol., 279: H2529–H2539. Thibonnier, M., Plesnicher, C., Berrada, K. and Berti-Mattera, L. (2000c). Am. J. Physiol., 281: E81–E92. Thibonnier, M., Coles, P., Thibonnier, A. and Shoham, M. (2001). Annu. Rev. Pharmacol. Toxicol., 41: 175–202. Ufer, E., Postina, R., Gorbulev, V. and Fahrenholz, F. (1995). FEBS Lett., 362: 19–23. Vriend, G. (1990). J. Mol. Graph., 8: 52–56.

D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 15

Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands C. Serradeil-Le Gal 1,∗ , J. Wagnon 2 , G. Valette 1 , G. Garcia 2 , G. Simiand, M. Pascal 1 , J.P. Maffrand 1 and G. Le Fur 3 1

2

Exploratory Research Department, Sanofi-Synthélabo Recherche, 195 Route d’Espagne 31036 Toulouse Cédex, France Exploratory Research Department, Sanofi-Synthélabo Recherche, 371 Rue du Professeur J. Blayac 34184 Montpellier Cédex 04, France 3 Sanofi-Synthélabo Recherche, 174 Avenue de France, 75635 Paris Cédex 13, France

Abstract: The involvement of vasopressin (AVP) in several pathological states has been reported recently and the selective blockade of the different AVP receptors could offer new clinical perspectives. During the past few years, various selective, orally active AVP V1a (OPC-21268, SR49059 (Relcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA-985 (Lixivaptan), SR121463, VP-343, FR-161282) and mixed V1a /V2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, Phase IIb clinical trials for some of them. For many years now, our laboratory has focused on the identification of nonpeptide vasopressin antagonists with suitable oral bioavailability. Using random screening on small molecule libraries, followed by rational SAR and modelization, we identified a chemical series of 1-phenylsulfonylindolines which first yielded SR49059, a V1a receptor antagonist prototype. This compound displayed high affinity for animal and human V1a receptors and antagonized various V1a AVP-induced effects in vitro and in vivo (intracellular [Ca2+ ] increase, platelet aggregation, vascular smooth muscle cell proliferation, hypertension and coronary vasospasm). We and others have used this compound to study the role of AVP in various animal models. Recent findings from clinical trials show a potential interest for SR49059 in the treatment of dysmenorrhea and in Raynaud’s disease. Structural modifications and simplifications performed in the SR49059 chemical series yielded highly specific V2 receptor antagonists (N-arylsulfonyl-oxindoles), amongst them SR121463 which possesses powerful oral aquaretic properties in various animal species and in man. SR121463 is well-tolerated and dose-dependently increases urine output and decreases urine osmolality. It induces free water-excretion without affecting electrolyte balance in contrast to classical diuretics (e.g. furosemide and hydrochlorothiazide). Notably, in cirrhotic rats with ascites and impaired renal function, a 10-day oral treatment with SR121463 (0.5 mg/kg) totally corrected hyponatremia and restored normal urine excretion. This compound also displayed interesting new properties in a rabbit model of ocular hypertension, decreasing intraocular pressure after single or repeated instillation. Thus, V2 receptor blockade could be of interest in several water-retaining diseases such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), liver cirrhosis and congestive heart failure and deserves to be widely explored. Finally, further chemical developments in the oxindole family have led to the first specific and orally active V1b receptor antagonists (with SSR149415 as a representative), an awaited class of drugs with expected therapeutic interest mainly in ACTH-secreting tumors and various emotional diseases such as stress-related disorders, anxiety and depression. However, from the recently described tissue locali-

∗ Correspondence to: C. Serradeil-Le Gal, Exploratory Research Department, Sanofi-Synthélabo Recherche, 195 Route d’Espagne 31036 Toulouse Cédex, France. Tel.: +33-5-6116-2384; Fax: +33-5-6116-2586; E-mail: claudine.serradeil@ sanofi-synthelabo.com

198 zation for this receptor, we could also speculate on other unexpected uses. In conclusion, the development of AVP receptor antagonists is a field of intensive pharmacological and clinical investigation. Selective and orally active compounds are now available to give new insight into the pathophysiological role of AVP and to provide promising drugs. Keywords: Vasopressin; V1a receptor; V1b receptor; V2 receptor; Nonpeptide antagonist; SSR149415; SR49059; SR121463; Aquaretics; ACTH

Introduction AVP, functions and receptors Vasopressin (AVP) is a cyclic nonapeptide which exerts a variety of biological effects in mammals. The primary role of AVP involves the regulation of water and solute excretion by the kidney. However, this hormone is also actively involved in a number of other physiological functions including blood pressure control, platelet aggregation, liver glycogenolysis and neoglucogenesis, uterus contraction, cell proliferation, adrenocorticotropin (ACTH) release by the adenohypophysis, aldosterone secretion by the adrenals and clotting factor release (Barberis et al., 1999). Together with oxytocin (OT), another structurally related nonapeptide, AVP is also implicated in interneuronal communication in the central nervous system (CNS) and modulates several behavioral functions such as feeding, memory, thermoregulation and the control of adaptative, social and sexual processes (Dreifuss et al., 1991). These central and peripheral effects of AVP are based upon a local or systemic release pattern into the organism and occur via interaction with specific seven transmembrane G-protein-coupled receptors. Three AVP receptors (V1a , V1b (or V3 ) and V2 ) and one type of OT receptor have been cloned in animal species and in man and have been clearly identified by their primary structure, gene localization, mRNA distribution, pharmacology and functions (Lolait et al., 1995; Thibonnier et al., 1998). Briefly, the AVP V1 (V1a and V1b ) receptors mediate phospholipase C activation and intracellular calcium mobilization. AVP V1a receptors have a ubiquitous central and peripheral localization (liver, platelet, uterus, brain etc). The AVP V1b receptors are involved mainly in the stimulating effect of AVP on ACTH secretion in the pituitary but, as recently demonstrated, V1b receptors have a wide distribution in the rat brain and also an endocrine role

in other organs such as the pancreas and the adrenals (Lee et al., 1995; Grazzini et al., 1996). Finally, to complete the review of this field, two putative receptors for AVP (or related peptides) have been reported. First, the AVP-activated Ca2+ mobilizing (VACM-1) receptors, unlike other AVP receptors, appear to possess only one transmembrane domain and reportedly bind AVP activating a Ca2+ second messenger pathway. VACM-1 also modulates the cAMP response of V2 receptors when expressed in CHO cells. The cDNA for VACM-1 has been cloned from rabbit, rat and human tissues and mRNA is present in numerous, if not all, tissues, including brain, kidney and endothelial cells. Recently, it has been shown that the VACM-1 protein is identical to cullin-5, which belongs to a newly discovered family of proteins implicated in the cell cycle, clearly different from the AVP/OT receptor family (Burnatowska-Hledin et al., 1995, 2000). Secondly, a novel dual angiotensin II (Ang II)/AVP receptor with an AT1/V2 receptor profile has revealed specific binding for both Ang II and AVP with positive coupling to adenylyl cyclase in response to both hormones. It has been cloned in the rat and in man. This receptor localized in the renal thick ascending limb tubules and collecting ducts, may be involved in renal tubular Na+ and fluid reabsorption. Recent data have also shown a wide distribution in the rat CNS (Ruiz-Opazo et al., 1995; Hurbin et al., 2000). However, clear characterization of this 7-TM receptor using reference peptide and nonpeptide AVP/OT ligands is needed to further explore this entity. At present, the biological role of these atypical proteins, VACM-1 and the mixed AVP/Ang II receptor, remains to be defined. Design of AVP receptor antagonists In the 1980s, a number of cyclic and linear peptide receptor antagonists and agonists derived from

199

the natural hormone and exhibiting various selectivity profiles for AVP and OT receptors were designed by M. Manning in collaboration with W. Sawyer. Amongst them, d(CH2 )5 Tyr(Me)AVP, a selective AVP V1a receptor antagonist still used as a V1a reference; d(CH2 )5 [Tyr (Et)2 , Val4 , D-Arg8 ]VP, a potent V2 /V1a antagonist with aquaretic properties in the rat; d(CH2 )5 [D-Ile2 , Ile4 ]AVP, the first selective V2 peptide ligand and Aaa– D-Tyr(Et)–Phe– Val–Asn–Abu–Pro–Arg–Arg(NH2 ), the first linear V2 /V1a antagonist, represented key steps in the story of peptide AVP receptor antagonists (Manning and Sawyer, 1989, 1991; Jard, 1998). They offered the first valuable pharmacological tools for the classification of AVP/OT receptors and for the acute characterization of their role. They also represented the first generation of radiolabeled ligands for mapping the distribution of AVP and OT receptors. Of note, the team from SmithKline and Beecham Laboratories was particularly active in developing peptide V2 receptor antagonists for clinical purposes. Unfortunately, due to profound species differences well-known in the field of AVP, molecules which were potent V2 receptor antagonists in several animal models turned out to be V2 receptor agonists in humans (Allison et al., 1988). More recently, in the 1990s, besides the molecular cloning of the 4 AVP/OT receptor subtypes, the first nonpeptide AVP V1a /V2 receptor antagonists appeared and allowed, due to their oral bioavailability, clinical evaluation of their therapeutic uses. Chemical structures selective for V1a receptors or V2 receptors or exhibiting a mixed V1a /V2 antagonist profile became available and were intensively studied in various animal models and evaluated in clinical trials (Albright and Chan, 1997; Thibonnier et al., 2001). Finally, very recently in the new millennium, the first selective V1b ligands were discovered (Derick et al., 2000; Serradeil-Le Gal et al., 2002). In the present chapter, we will first review the different orally active, nonpeptide AVP receptor antagonists reported up to now and their potential therapeutic indications. Since our laboratory has focused for many years on the identification of nonpeptide AVP antagonists with suitable oral bioavailability, we will present some recent pharmacological and clinical data supporting their main therapeutic indications. For more details of the development of

orally active V1a , V2 AVP receptor antagonists in general, the reader is referred to the many excellent and accurate reports that have been published recently (Albright and Chan, 1997; Freidringer and Pettibone, 1997; Mayinger and Hensen, 1999; Thibonnier, 1999; Paranjape and Thibonnier, 2001; Thibonnier et al., 2001). The second part of this chapter focuses on the pharmacological characterization of SSR149415, the first selective V1b receptor antagonist described so far (Serradeil-Le Gal et al., 2002). This molecule constitutes a unique probe for exploring the poorly known V1b receptor and the interest of V1b blockade in several pathological states. Nonpeptide AVP V1a and/or V2 receptor antagonists and their potential clinical indications Resulting from high throughput screening (HTS) of thousands of small nonpeptide molecules (molecular weight ≈ 500) belonging to a variety of chemical libraries, the first orally active AVP receptor antagonists were reported in the 1990s and today, an impressive number of patents covers the field of nonpeptide AVP ligands. Only some of them, with different selectivity profiles (V1a , V2 , V1a and V2 ) are undergoing clinical trials (Phase IIb for the most advanced) and the results are eagerly awaited to evaluate both the benefit and the safety of this class of drugs. The term ‘Vaptan’ has been coined to officially name all the members of this new class of drugs (e.g. Relcovaptan (SR49059), Tolvaptan (OPC-41061), Lixivaptan (VPA 985) and Conivaptan (YM-087)). Nonpeptide V1a receptor antagonists The first V1a receptor antagonists, OPC-21268 and SR49059 (Relcoptan) were described in 1991 and 1993, respectively, and have been involved in various clinical trials (Yamamura et al., 1991; Serradeil-Le Gal et al., 1993). They are still the major players identified in this field. These molecules, belonging to different chemical series (quinolinone and indoline series, respectively), were obtained following chemical optimization and SAR of a lead compound found by random screening. It is important to underline that the OPC-quinolinone series was the starting point for the design of many AVP antago-

200 TABLE 1 Orally active, nonpeptide AVP V1a receptor antagonists reported in 2001 and their chemical structures

(1) (2) (3) (4) (5) (6)

Name

Company

Chemical series

Development status

OPC-21268 SR49059 (Relcovaptan) None None None None

Otsuka Sanofi-Synthélabo Eli Lilly Yamanouchi Fujisawa Yamanouchi

Quinolinone derivative N-Sulfonyl-indoline derivative Azetidinone derivatives Benzazepine derivatives Benzazepine derivatives Triazole derivatives

Phase II Japan; stopped US/Europe Phase II/stopped Preclinical Preclinical Preclinical Preclinical

(2)

(4) N

OPC-21268 -

O

O

N

F O O

(1)

N H

NH

O

NH

(5)

CH3O

O

OCH O 3

O

H3 C

(3)

OCH3

O O

(CH3 ) 3

CH 3

SR49059

OC H3 CH 3

N

O

NH2

N

CON H CONH 2

O

N

O O

SO2

N

CH 3

O

N

H

F

N

O N

HO

Cl

N

O

O

Cl

N

(6)

N

N

N N

CH3

nists reported by several competitors (Tables 1–3). Of note, structure–activity relationships among analogues of OPC-21268 yielded also derivatives with marked affinity for the human OT receptor further developed by Merck as selective OT receptor antagonists for the treatment of preterm labor (Freidringer and Pettibone, 1997). In addition, rational modifications of these selective V1a structures (OPC-21268 and SR49059) further yielded potent selective V2 , mixed V1 and V2 , and more recently, pure V1b receptor antagonists (in the case of SR49059) with oral bioavailability, as illustrated in Tables 2, 3 and 5. More recently, two other chemical series of V1a receptor ligands described in patents by Eli Lilly as azetidine derivatives and by Yamamouchi as triazole derivatives, have emerged (see illustration with compounds 3 and 6, respectively, in Table 1). However, up to now, no specific lead compounds have been identified and no pharmacological studies are reported with these new derivatives.

Clinical indications for nonpeptide V1a receptor antagonists Due to the ubiquitous localization of V1a (brain, vessels, platelet, uterus, adrenals. . . ) and to their various central and peripheral biological effects (see Section 1), V1a blockade could be of interest in a large number of diseases. AVP may be increased in several pathological situations such as hypertension, congestive heart failure, dysmenorrhea, brain edema, small cell lung cancers and various CNS disorders (depression, anxiety. . . ) (Table 4). Due to profound interspecies variability in AVP/ OT receptors (Allison et al., 1988; Pettibone et al., 1992), the affinity of OPC-21268 for the human V1a receptor was very weak and this compound failed to prevent AVP-induced contractions in various human vascular preparations in vitro (Serradeil-Le Gal et al., 1993; Burrell et al., 1994). If some clinical trials have been reported with this compound, its loss of affinity for the human V1a receptors has severely precluded clinical developments. At variance with OPC-21268, SR49059 exhibited a constant high affinity for animal and human V1a receptors and

201 TABLE 2 Orally active, non-peptide AVP V2 receptor antagonists reported in 2001 and their chemical structures

(1) (2) (3) (4) (5) (6) (7) (8) (9)

Name

Company

Chemical series

Development status

OPC-31260 OPC-41061 (Tolvaptan) SR121463 VPA-985 (Lixivaptan) WAY-140288 VP-343 and VP-339 VP-365 FR161282 None

Otsuka Otsuka Sanofi-Synthélabo Wyeth-Ayerst Wyeth-Ayerst Wakamoto Wakamoto Fujisawa Ortho-McNeil

Benzazepine derivative Benzazepine derivative N-Arylsulfonyl-oxindole derivative Benzodiazepine derivative Benzodiazepine derivative Quinoxaline derivative Benzodiazepine derivative Benzodiazepine derivative Benzothiazepine derivatives

Phase II Phase II Phase II Phase II Preclinical Preclinical Preclinical Preclinical Preclinical

H3C

N

CH3

OH

H

O

O

N O

CH3 CH2 O

N

N H

N

N

N

N O SO2

O NH

O

VP-343

HNOC

O

SR121463

OH

H3C

N

Cl

NH

NH

OCH 3

OPC-31260 H3C

O

O

VP-339

H 3C

OH

O

N

H3 C N

H3C

S

N

N

N

O

O

N

N

N

N

O

O

H3 C

NH

NH

CH3

O

O

O

F

has been intensively studied in pharmacological and clinical trials as a prototype drug (Serradeil-Le Gal et al., 1993). From several proof of concept studies performed with SR49059 — highly speculative for some of them — a number of clinical indications for AVP V1a receptor antagonists have clearly emerged. Since AVP exerts a powerful local vasoconstriction effect in several crucial vascular beds (renal, gastric, coronary. . . ), SR49059 effects were assessed in patients suffering from Raynaud’s disease. In a double-blind, placebo-controlled randomized cross-over study, at 300 mg p.o. once daily, SR49059 significantly antagonized (15%) cold-induced decrease in finger systolic blood pressure following a cold immersion test and accelerated temperature recovery (Hayoz et al., 2000). These preliminary results suggested an involvement of AVP in the abnormal vasoactive response of Raynaud’s patients

O

NH

NH

NH

WAY140288

H 3C

VPA-985

N

OC H3

O

Cl

O

OPC-41061

H

O

FR-161282 H3 C

O

O

(9)

together with a potential interest of V1a blockade in this pathology. Interesting results were also obtained with SR49059 in the treatment of dysmenorrhea. AVP plasma levels are thought to be increased in primary dysmenorrhea and this hormone is clearly involved in the development of pain by stimulating both vascular and uterine V1a receptors (Bossmar et al., 1997). In a double-blind, randomized cross-over Phase IIb study, SR49059 (100 and 300 mg) induced a dose-related positive effect in reducing pelvic pain during the first 24 h in primary dysmenorrhea, with a simultaneous decrease in the need for analgesic rescue (Brouard et al., 2000). Up to now, no convincing results are available with V1a receptor antagonists in hypertension, either in various animals models or in human, even if AVP seems to be involved in some forms of essential hypertension, in particular, in African–American

202 TABLE 3 Orally active, non-peptide AVP Va /V2 receptor antagonists reported in 2001 and their chemical structures

(1) (2) (3) (4) (5)

Name

Company

Chemical series

Development status

YM-087 (Conivaptan) YM-471 JVT-605 CL-385004 None

Yamanouchi Yamanouchi Japan Tobacco Wyeth-Ayerst Fujisawa

Benzazepine derivative Benzazepine derivative Thiazepine derivative Benzodiazepine derivative Benzazepine derivatives

Phase II Preclinical Preclinical Preclinical Preclinical

YM-471 HN

N

O

CH 3

N

CH 3 CH 3

N

N O

F

N

O

O(CH 2)3C

O

CH 3 N

N CH 3

NH

NH

O

O

N

O

O

CH 3

NH

N

N

N

S

F

CL-385004

F

NH

O

O N

JTV-605

YM-O87

O

OCH 3 N NH

(5)

O

TABLE 4 Main potential clinical indications for AVP V1a , V2 , dual V1a /V2 and V1b receptor antagonists V1a antagonists

V2 antagonists

Mixed V1a /V2 antagonists

V1b antagonists

Dysmenorrhea, preterm labor Raynaud’s disease Hypertension

Congestive heart failure SIADH Liver cirrhosis with ascites and water retention Hyponatremia Nephrotic syndrome Brain edema Glaucoma Hypertension Diabetic nephropathy Meniere’s disease

Congestive heart failure Hypertension Brain edema

Stress-related disorders, anxiety, depression ACTH-secreting tumors, Cushing’s syndrome HPA axis disorders

Congestive failure Brain edema Motion sickness Oncology (SCLC) CNS disorders

patients with elevated AVP plasma levels. An exploratory study with SR49059 (300 mg, single dose) in a situation of osmotic release of AVP (induced by a 5% hypertonic saline infusion) in black hypertensive patients failed to demonstrate a sustained blood pressure reduction (Thibonnier et al., 1999). It is

generally assumed that blockade of both V1a and V2 receptors needs to be achieved to reach a significant improvement in blood pressure alterations. A number of other clinical indications remain to be investigated, in particular CNS disorders with compounds able to cross the blood–brain barrier (Table 4).

203

Nonpeptide V2 receptor antagonists While only two AVP V1a receptor antagonists have been described, numerous selective, orally active AVP V2 receptor antagonists have been reported by several pharmaceutical companies. Ten lead compounds can be identified at various stages of investigation, with four molecules currently involved in clinical developments: OPC-31206, OPC-41061 (Tolvaptan), SR121463 and VPA-985 (Lixivaptan) (Table 2). By modifying the dihydroquinolinone chemical structure of their V1a receptor antagonist, OPC-21268, Otsuka reported in 1992 an orally active V2 compound, OPC-31260, and more recently a back-up/follow-up molecule, OPC-41061 (Tolvaptan) in the benzazepine chemical series (Yamamura et al., 1992, 1998). As shown in Table 2, the other V2 receptor antagonists reported by various pharmaceutical firms are all benzazepine derivatives, except SR121463, an oxindole derivative resulting from optimization and simplification of our V1a receptor antagonist, SR49059. In order to simplify the 1-phenylsufonyl indoline structure of SR49059 bearing three asymmetric carbons, we designed Narylsufonyl-oxindoles yielding highly specific V2 ligands among which SR121463 was chosen for further preclinical and clinical development (SerradeilLe Gal et al., 1996; Serradeil-Le Gal, 2001). Clinical indications for V2 receptor antagonists The generation of receptor-specific AVP V2 antagonists, so-called ‘aquaretics’, able to block the antidiuretic action of AVP in the collecting duct cells, and thus to specifically promote water excretion by preventing the insertion of AVP-specific water channels (aquaporin, AQP-2) into the luminal membrane, could be of high therapeutic value for the treatment of several water-retaining disorders such as SIADH, liver cirrhosis, certain stages of congestive heart failure and hypertension, nephrotic syndrome, renal failure. . . (Table 4). In most of these diseases an abnormal increase of circulating AVP plasma level, activating renal V2 receptors, seems to be the key event in water retention and subsequent hypotonic hyponatremia (Goldsmith et al., 1989; Gavras, 1991; Sorensen et al., 1995). Thus, for these pathologies, there is great clinical interest in the development

of potent V2 receptor antagonists to provide specific water diuretic/aquaretic compounds devoid of the well-known side effects of classical diuretic or saliuretic agents on the urine Na+ and/or K+ loss. According to the above rationale, several clinical trials are reported with OPC compounds and VPA-985 in CHF, cirrhosis with ascites, SIADH with subsequent hyponatremia and in congestive heart failure (Thibonnier, 1999; Paranjape and Thibonnier, 2001). Whatever the compound used, urine volume was increased, urine osmolality decreased and a normalization of serum Na+ observed. Results of repeated chronic treatments in these pathologies are eagerly awaited, but are available in some animal models. Of note, SR121463 demonstrated benefit in a model of cirrhosis (CCl4 -induced) in rats with ascites, water retention and impaired renal function after chronic treatment. Ten-day repeated oral administration of SR121463 (0.5 mg/kg) normalized serum Na+ and totally corrected hyponatremia. SR121463 restored normal urine excretion, urine osmolality and renal function since after a water overload, cirrhotic rats excreted similar urine volume as control noncirrhotic rats (Jimenez et al., 2000). In addition, due to the extrarenal localization of AVP V2 receptors (brain, endothelial, lung. . . ), other therapeutic areas deserve to be explored. For example, V2 receptor blockade could be of interest in brain edema or more surprisingly in glaucoma. As recently shown, in a rabbit model of ocular hypertension (α-chymotrypsin-induced), SR121463, after single or repeated (10 days at 1%) instillation, markedly decreased intraocular pressure with similar efficacy to the currently used α (clonidine) or β-adrenergic (timolol) treatments, demonstrating the potential benefits of V2 receptor antagonists in decreasing intraocular pressure via a mechanism of action that remains to be elucidated. Moreover, it suggested the presence of ocular V2 receptors (Lacheretz et al., 2000). Dual nonpeptide V1a , V2 receptor antagonists The class of dual V1a and V2 receptor antagonists has been extended and includes now several lead compounds reported in Table 3, but only one molecule YM-087 (Conivaptan) has been tested in humans; clinical trials are reported mainly in CHF (Norman et al., 2000; Udelson et al., 2000). Of note, all these

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compounds are benzazepine derivatives obtained by modifying the chemical structure of the first AVP receptor antagonists (OPC-2128, OPC-31260). According to the compound, the affinity and activity ratio at V1a and V2 receptors is highly variable, generating various pharmacological profiles for these drugs. Interestingly, this V1a /V2 ratio seems a key factor when considering therapeutic purposes. Dual V1a and V2 receptor blockade is expected to modify both systemic hemodynamic and renal parameters. This strategy could be of interest in developing antihypertensive agents. Pure V1a receptor antagonists are reported to be inactive per se in various animal and human models of hypertension (even with increased AVP plasma levels). It is assumed that blockade of both V1a and V2 receptors will achieve a decrease in blood pressure by modifying both peripheral resistances and circulating blood volume. Similarly, dual V1a /V2 blockade could improve hemodynamic and fluid status in CHF. Finally, treatment of brain edema with this class of drugs needs also to be explored, based on the rationale that pure V1a and pure V2 receptor antagonists have both shown a benefit in the development of this pathology by decreasing brain water content and restoring brain Na+ content with a decrease in neurogenic inflammation. An involvement of peripheral and probably direct effect on brain vessel permeability and choroid plexus could be speculated even if the intrinsic mechanism of action is not known (Bemana et al., 1997; Laszlo et al., 1999). V1b receptor antagonists V1b receptor ligands The recently cloned V1b receptor, mainly found in the adenohypophysis, is involved in the stimulating effect of AVP on ACTH secretion (De Keyzer et al., 1994; Sugimoto et al., 1994). AVP is a direct ACTH secretagogue but also synergizes corticotropin-releasing factor (CRF)-induced ACTH release in the pituitary (Gillies et al., 1982). This receptor has a wide distribution in various tissues such as the brain, adrenals, kidney and pancreas. To date, due to the lack of selective V1b receptor ligands (agonists/antagonists) and to the absence of orally active V1b receptor antagonists, the V1b receptor is still poorly characterized and the precise role of AVP

via central and peripheral V1b receptors remains to be elucidated. Interestingly, to explore the functions of this receptor, a knockout mouse has recently been generated (Lolait et al., 2000). In contrast to the numerous potent and selective, peptide and nonpeptide V1a and V2 receptor ligands, only a few non-selective V1b peptides are available: [D-3(pyridyl)Ala2 ]AVP, a specific V1b agonist in rats turned out to be a V1a , V1b ligand at human AVP receptors; dDAVP displays both agonist V2 /V1b properties and a recent series of dDAVP analogues modified at position 2 yielded full V1b /partial V1a agonists (Derick et al., 2000). Finally, the reference peptide antagonist, [deaminopenicillamine-O-Me-Tyr,Arg]AVP, (dPen), is a dual V1b /V1a ligand. Recently, we have developed the first selective, nonpeptide and orally active V1b receptor antagonist to be described, SSR149415 (Serradeil-Le Gal et al., 2002 (Table 5)). It results from chemical optimization in the field of the indoline/oxoindole chemical series which has previously yielded nonpeptide molecules highly selective for the V1a (SR49059, Tables 1 and 6) and the for the V2 (SR121463, Tables 2 and 6) receptors. Pharmacological profile of SSR149415 As shown in Table 6, SSR149415 displays nanomolar affinity for human V1b receptors. It exhibits a TABLE 5 Orally active nonpeptide AVP V1b receptor antagonists reported in 2002 Name

Company

Chemical series

Development status

SSR149415

SanofiSynthélabo

N-Arylsulfonyloxindole derivative

Preclinical

OH OCH3 Cl

N N N

CH3

O O

SO2

OCH3 C H3O

CH3

205 TABLE 6 Comparative affinities of the nonpeptide compounds, SR49059, SR121463 and SSR149415 for human AVP (V1a , V1b , V2 ) and OT receptors K i (nM)

SR49059 SR121463 SSR149415 AVP OT

h-V1a

h-V1b

h-V2

h-OT

6.3 ± 0.6 460 ± 120 91 ± 23 1.7 64

220 ± 30 >10,000 1.5 ± 0.8 1.1 1782

275 ± 50 4.1 ± 0.8 1412 ± 314 1.1 167

320 ± 168 1213 ± 383 174 ± 35 16 0.9

Affinities of the natural hormones, AVP and OT, as references. Inhibition constants (K i ) were determined from competition experiments calculated according to the Cheng and Prussoff equation. Values are the mean ± SEM of at least three determinations.

%

highly selective profile versus V1a , V2 and OT human receptors and has no measurable affinity for a number of other receptors (n = 100). As illustrated in Fig. 1, SSR149415 dose-dependently antagonized [3 H]AVP binding to human V1b receptors with an affinity for human V1b receptors close to that of the natural hormone, AVP (K i values of 1.54 ± 0.82 and 0.80 ± 0.25 nM, respectively). SSR149415 exhibited much higher affinity than the nonselective reference

-10

-9

-8

-7

-6

Fig. 1. Inhibition of [3H]AVP specific binding to human V1b receptors by SSR149415 () and reference peptide compounds: AVP (•); dDAVP, [desamino-[ D-Arg]vasopressin] ( ); dPen, [desamino-[ D-Arg]vasopressin] (◦) and dPal, [(deamino-Cys, D3-(Pyridyl)-Ala2 -Arg8 )-vasopressin] (). Binding assays were performed for 45 min at 20°C in the presence of 30 μg/assay of CHO membranes expressing the human V1b receptors. Results represent data from a typical experiment performed in duplicate, which was repeated three times without noticeable differences.

agonist (dDAVP, [D-3(pyridyl)Ala2 ]AVP) and antagonist (dPen) V1b peptides (K i values of 20 ± 8, 12 ± 5 and 21 ± 6 nM, respectively). K i values obtained for these peptides are consistent with affinities previously reported for the human V1b receptor. In saturation binding experiments followed by Scatchard analysis, SSR149415 inhibited [3 H]AVP binding in a competitive manner with a K i value of 2.51 ± 0.45 nM (Serradeil-Le Gal et al., 2002). Earlier cellular events upstream of ACTH release, provoked by occupancy of corticotroph V1b receptors by AVP, include the activation of phospholipase C, protein kinase C and the mobilization of intracellular free Ca2+ mainly via Gq/11 G-protein recruitment. In CHO cells transfected with the human V1b receptor other intracellular pathways have also been described (e.g. cAMP production, stimulation of DNA synthesis and cell proliferation), clearly depending on the level of V1b receptor expression (Thibonnier et al., 1997). In CHO cells expressing the human V1b receptors AVP-induced cell proliferation with an EC50 value of 0.14 ± 0.12 nM (n = 4) as measured by a colorimetric method with the tetrazolium compound (MTS) according to (Thibonnier et al., 1998). In this latter model, SSR149415 (Fig. 2) dose-dependently antagonized stimulation of cell proliferation by AVP (3 nM) with a K i value of 0.43 ± 0.41 nM consistent with the SSR149415 affinity found in binding studies using the same cellular preparation. In vivo, pharmacology performed measuring ACTH secretion induced by various stimulants, such as hormones and physical stress, confirmed the full antagonist profile of SSR149415. In all these situations, SSR149415 antagonized ACTH secretion, which constitutes a critical response of the organism to stress in emotional situations (Aguilera and Rabadan-Diehl, 2000). It is important to note that SSR149415 demonstrated high potency by the oral route on the potentiation of the effect on CRF by AVP, a mechanism described as typically V1b mediated. Significant inhibition of ACTH secretion was observed from a dose of 3 mg/kg p.o., total blockade occurred at 10 mg/kg p.o., an effect that lasted for more than 4 h (Fig. 3). Conversely, the selective, orally active V1a receptor antagonist, SR49059, was unable to inhibit AVP plus CRFinduced ACTH secretion, demonstrating a specific V1b -mediated effect. Various physical stresses are

206

%

(A)

-10

-9

-8

-7

-6

-5

Fig. 2. Inhibition by SSR149415 of AVP-induced proliferation of CHO cells transfected with the human V1b receptor. CHO cells were grown for 48 h in 96-well plates (5000 cells/well). Cell proliferation was measured using the CellTiter 96 cell proliferation assay from Promega (Madison, WI). Cells were washed with 200 μl PBS and treated with AVP (3 nM) and increasing concentrations of SSR149415. After 18 h of incubation (37°C, 5% CO2 , 80% humidity), 20 μl dye solution was added to each well. The plate was incubated for 4 h and absorbance was recorded at 492 nm wavelength. Results represent data from a typical experiment which was repeated four times without noticeable change.

able to induce ACTH secretion. The stress-induced release of ACTH is believed to involve the activation of several humoral and neural pathways, including that mediated by AVP (Linton et al., 1985). As shown in Fig. 4, in rats submitted to an immobilization period of 15 min, there was a significant increase (more than 5-fold) in plasma ACTH levels. Pretreatment with SSR149415 (3–10 mg/kg i.p.) 30 min before the restraint stress period, caused dosedependent inhibition of plasma ACTH elevation in comparison with stressed animals treated with the corresponding vehicle. The regulation of ACTH secretion and consequently of the HPA axis are largely mediated by AVP and V1b receptors, and SSR149415 offers a new tool for controlling emotional or physical stress. Indeed, several neuroendocrine studies strongly suggest that dysregulation of the HPA system plays a causal role in the development and the course of diseases such as generalized anxiety, depression and addiction (Holsboer, 1999). In conclusion, SSR149415 is a potent, selective and orally active V1b receptor antagonist. Whatever the model, SSR149415 is devoid of any V1b agonist

(B)

Fig. 3. Effect of oral SSR149415 on the potentiation of exogenous CRF by AVP on ACTH secretion in conscious rats. Dose–effect (A) and time-course (B) studies. (A) SSR149415 (1–10 mg/kg) was administered by gavage 2 h prior to CRF (0.1 μg/kg i.v.) and AVP (0.03 μg/kg i.v.) injection. Inset: effect of a selective orally active V1a receptor antagonist, SR49059 (1–10 mg/kg) in this model. (B) The time-course effect of SSR149415 was studied at 10 mg/kg p.o. on plasma ACTH secretion (n = 5–10). Values are expressed as the percentage of ACTH secretion versus the CRF plus AVP control. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s test or using the nonparametric Kruskal–Wallis test. (* P < 0.05; ** P < 0.01 versus control).

effect when tested alone. It is a unique tool for exploring the localization and the role of V1b receptors. This class of drugs exhibits a promising therapeutic profile for the treatment of stress-related disorders, anxiety and depression. However, due to the ubiquitous localization of the V1b receptor, several other

207

Fig. 4. Effect of SSR149415 on restraint stress-induced ACTH secretion in conscious rats. Rats (8–10 per group) received intraperitoneal injection of the vehicle (2 ml/kg 5% DMSO, 5% cremophor, 90% saline) or SSR149415 (3 and 10 mg/kg). Thirty minutes after the injection, rats were placed in plexiglass restrainers for 15 min. At the end of the immobilization period, they were sacrificed by decapitation and plasma ACTH levels were measured by RIA. Data are mean ± SEM. Statistical significance was assessed by an ANOVA on transformed logarithmic data followed by Dunett’s test (** P < 0.01 vs. control – unrestraint animals; ££ P < 0.01 vs. stress control – restraint animals).

therapeutic indications need to be investigated (Table 4). Conclusion AVP mediates a wide number of biological effects and may be involved in several pathological states. Selective blockade of the different AVP receptors, therefore, could offer new clinical perspectives for treating several diseases (Table 4). In 2001, four classes of orally active AVP receptor nonpeptide antagonists (V1a , V2 , V1a + V2 , and V1b ) were available, constituting the ‘Vaptan family’. In the past decade, various, selective, nonpeptide and orally active AVP V1a (OPC-21268, SR49059 (Relcovaptan)), V2 (OPC-31260, OPC-41061 (Tolvaptan), VPA985 (Lixivaptan), SR121463, VP-343, FR-161282, WAY-140288) and mixed V1a /V2 (YM-087 (Conivaptan), JTV-605, CL-385004) receptor antagonists have been intensively studied in various animal models and have reached, for some of them, Phase IIb clinical trials. It should be noted, however, that no compounds are on the market, many clinical trials and proof of concept studies are still ongoing and the results of long-term treatments are eagerly awaited to evaluate the benefit and the safety of this new class of drugs.

A wide variety of therapeutic indications could be targeted with these compounds: SR49059, a potent and selective V1a receptor antagonist may be of interest in the treatment of dysmenorrhea and in Raynaud’s disease while V2 receptor antagonists were of benefit in several water-retaining diseases (CHF, SIADH and hepatic cirrhosis) by inducing powerful aquaretic effects without modification of Na+ and/or K+ excretion, at variance with classical diuretics. They improved urine excretion, urine osmolality and renal function, and subsequently, normalized serum Na+ (partially or totally) and corrected hyponatremia. Extrarenal localization of V2 receptors has also been evidenced. Other therapeutic domains with V2 receptor antagonists remain to be explored, notably in glaucoma where SR121463, a selective V2 compound, decreased intraocular pressure by local instillations, as do the reference drugs currently used (timolol and clonidine). Finally, we have developed the first selective, orally active V1b receptor antagonist. This compound constitutes an invaluable tool for exploring this poorly characterized receptor and the precise role of AVP via central and peripheral V1b receptors. SSR149415, a representative member of this class, has potential for the treatment of anxiety, depression and stressrelated disorders. In addition, in view of the recently

208

described tissue localization of the V1b receptor protein and mRNA (brain, pancreas, adrenals. . . ), still other potential uses may be possible. Abbreviations ACTH Ang II AVP CHF CRF dDAVP dPen

adrenocorticotropin angiotensin II arginine vasopressin congestive heart failure corticotropin releasing factor desamino-[D-Arg]vasopressin [deaminopenicillamine-O-MeTyr,Arg]vasopressin HTS high throughput screening OT oxytocin SIADH syndrome of inappropriate antidiuretic hormone secretion VACM-1 receptor vasopressin-activated Ca2+ mobilizing receptor

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 16

Rat vasopressin mRNA: a model system to characterize cis-acting elements and trans-acting factors involved in dendritic mRNA sorting Evita Mohr ∗ , Iris Kächele, Carola Mullin and Dietmar Richter Universität Hamburg, Institut für Zellbiochemie und Klinische Neurobiologie, Martinistrasse 52, 20246 Hamburg, Germany

Abstract: The genes encoding the vasopressin (VP) and oxytocin (OT) precursors are expressed in magnocellular neurons of the hypothalamo-neurohypophyseal system. The neuropeptides have a dual function: (1) they are secreted from the nerve terminals into the systemic circulation to act as hormones on various peripheral target organs; and (2) VP and OT are also released from the dendrites into the central nervous system where they presumably play a role as either neurotransmitters or as modulators of the classical transmitters. Substantial amounts of VP and OT mRNAs are sorted to both axons and dendrites. Since the latter are equipped with components of the translational machinery, the peptide hormone precursors are likely to be locally synthesized in dendrites of magnocellular neurons. Evidence for axonal precursor synthesis, on the other hand, has not been obtained. Subcellular mRNA localization is a complex pathway. It is determined by sequences (cis-acting elements) within the RNA and proteins (trans-acting factors) which interact with these elements in order to guide the molecules to their ultimate destination. We have investigated the mechanisms involved in mRNA targeting in neurons by using VP mRNA as a model system. Recombinant eukaryotic expression vectors harboring the VP cDNA have been microinjected into the cell nuclei of cultured superior cervical ganglion (SCG) neurons. The subcellular distribution of the vector-expressed mRNAs was determined by non-radioactive in situ hybridization techniques. This revealed transport of VP mRNA to the dendrites, but not to the axonal compartment of SCG neurons. A complex dendritic localizer sequence (DLS) that spans part of the coding region as well as the 3 -untranslated region was identified by microinjecting constructs encoding partial sequences of the VP mRNA. In order to characterize trans-acting factors interacting with this element, protein/RNA binding experiments with radiolabeled in vitro synthesized VP RNA probes and proteins extracted from rat brain have been carried out. A protein specifically interacts with the DLS of the VP mRNA but not with sequences that obviously lack a role in subcellular RNA transport. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions include functions such as translational silencing. The translational state of mRNAs subject to dendritic sorting is most likely influenced by external stimuli. Consequently, PABP could represent one of several components necessary to regulate local synthesis of the VP precursor and possibly of other proteins. Keywords: cis-acting element; Oxytocin; Poly(A)-binding protein; RNA-binding protein; RNA localization; trans-acting factor; Vasopressin; UV-crosslinking

∗ Correspondence to: E. Mohr, Institut für Zellbiochemie und klinische Neurobiologie, Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany. Tel.: +49-40-42803-4553; Fax: +49-40-42803-4541; E-mail: [email protected]

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Introduction mRNA transport to cytoplasmic regions outside the cell somata is observed in various nerve cell types of the central and peripheral nervous system. Most often, defined transcripts are specifically delivered to dendrites, and the functional significance of this transport process appears to be evident: Dendrites are equipped with ribosomes and many additional components essential for translation and are, thus, competent for local protein synthesis (for review see Mohr, 1999; Tiedge et al., 1999; Steward and Worley, 2001). In fact, there is an ever-growing body of evidence indicating that on-site translation in dendrites is necessary for establishing at least particular forms of synaptic plasticity (for review see Schuman, 1999; Steward and Schuman, 2001). Hence, mRNA localization to the dendrites of the nerve cells is presumably important because synthesis of proteins involved in modulating synapse functions may be regulated in terms of both space and time. As far as mammalian nerve cells are concerned, definite proof for local protein biosynthesis in axons with the exception of the unmyelinated initial segment is still lacking. Notwithstanding, in vivo, specific delivery of distinct mRNA species to axons of nerve cells, for example primary sensory neurons projecting to the olfactory bulb and hypothalamic magnocellular neurons, has been observed (for review see Mohr and Richter, 2000), but their functional role has remained uncertain. In even rarer cases, the same mRNA species is localized to the axon as well as to dendrites, examples being the VP mRNA and transcripts encoding the Purkinje cell-specific L7 protein (for review see Mohr, 1999). By no means is subcellular mRNA localization restricted to neurons. On the contrary, this phenomenon is apparently conserved during evolution and is observed in many cell types from organisms as diverse as yeast and human. It is perhaps not surprising, however, that work on RNA sorting in postmitotic nerve cells has remained more or less descriptive for many years. Early on, the lead in this field was taken by investigations aimed at characterizing the RNA localization mechanisms in non-neuronal cells. Particularly well studied examples include developmental systems such as Xenopus oocytes, Drosophila oocytes and early embryos, but also dif-

ferentiated cell types in various organisms from yeast to mammalian species (for review see Bashirullah et al., 1998; Barbarese et al., 1999; Gonzalez et al., 1999; King et al., 1999; Lipshitz and Smibert, 2000). These studies demonstrated that RNA localization is remarkably complex and is determined by a variety of molecular entities: (1) sequence elements or motifs within the RNA molecule (cis-acting elements) that may form secondary, tertiary and, at least in some cases, quarternary structures; (2) proteins (trans-acting factors) as mediators of RNA sorting via a direct or indirect interaction with the mRNA to be transported; and (3) mechanisms of translational control including silencing and subsequent activation (or derepression). In addition, cytoskeletal elements provide the transport tracks and anchor sites of the ribonucleoproteins (for review see Bashirullah et al., 1998; Barbarese et al., 1999; Gonzalez et al., 1999; King et al., 1999; Lipshitz and Smibert, 2000). It may be assumed that mRNA localization in nerve cells takes place in a similar manner. This is underscored by the recent identification of cisacting elements, termed dendritic targeting element or dendritic localizer sequence, necessary and sufficient for dendritic targeting of mRNAs encoding the microtubule-associated protein 2 (MAP2; Blichenberg et al., 1999), the VP precursor (Prakash et al., 1997) and the α-subunit of Ca2+ /calmodulindependent protein kinase II (Mori et al., 2000; Blichenberg et al., 2001), and of the non-coding BC1 RNA (Muslimov et al., 1997). Furthermore, transacting factors have been characterized that interact, at least in vitro, with the cis-acting elements within the MAP (Rehbein et al., 2000; Monshausen et al., 2001) and the VP (Mohr et al., 2001a,b) mRNAs. In the present review, we will summarize current knowledge of mechanisms involved in VP mRNA sorting to the neurites of nerve cells in vivo and in primary cultured sympathetic neurons microinjected with eukaryotic expression vectors. In hypothalamic magnocellular neurons, VP mRNA is delivered to axons and dendrites Two different populations of magnocellular neurons (MCN) of the hypothalamic paraventricular and supraoptic nuclei express the genes encoding the VP and the structurally closely related OT precursors.

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MCNs are remarkable since both VP and OT are released from their dendrites into the central nervous system (Morris et al., 1998) in addition to being secreted into the periphery from axon terminals that project to the posterior pituitary. Apparently, VP and OT have multiple functions. Firstly, they influence in a well characterized manner the physiological state of a variety of peripheral target organs (for review see Birnbaumer, 2000; Gimpl and Fahrenholz, 2001). Secondly, they act as neurotransmitters or as modulators of the classical transmitters in the brain even though these functions are less well understood (for review see Raggenbass, 2001). Early on, we and others have noted that both VP and OT mRNAs are sorted to the axon and to the dendrites of hypothalamic MCNs. Yet, the axonal and the dendritic transcripts exhibit different characteristics implying that they are functionally not equivalent (for review see Mohr, 1999; Mohr and Richter, 2000). Interestingly, within MCNs, VP and OT transcripts exhibit a polymorphism concerning the length of their poly(A) tails: mRNAs residing in the axonal domain have a considerably shorter poly(A) tail than those located in the cell somata and in the dendrites (which are identically sized). The functional significance of this poly(A) tail shortening that must occur prior to axonal targeting of the mRNAs is not understood to date. Moreover, axonal transcripts are invariant with respect to the length of the poly(A) tail. This is a surprising feature of the axonal mRNAs because in the cell somata, the poly(A) tails of VP and OT mRNAs can be subject to ample elongation, for instance during osmotic stimulation. We have shown that mRNA transport to the axon most likely takes place subsequent to translation, but evidence for local axonal translation of both mRNA species has not been obtained since they are not associated with ribosomes (Mohr et al., 1995). So their function continues to remain unknown. The dendritic VP and OT mRNAs, in contrast, are localized to dendrites prior to translation (Mohr et al., 1995). Dendrites differ from axons both morphologically and functionally. Immunohistochemical studies at the ultrastructural level have indicated that peptide hormone precursor synthesis might occur in dendrites, at least under certain physiological circumstances. Careful analyses by Morris et al. (1998) have shown that the dendrites of MCNs contain most of the components required

for protein synthesis such as ribosomes, polysomes, rough endoplasmic reticulum (ER), and tRNA. A currently unresolved problem is the apparent lack of Golgi-like structures. Golgi marker proteins are only detectable in parts in close apposition to the cell body. What has been noticed, however, are morphological structures, tubular/vesicular organelles with associated ribosomes, that might possibly represent the Golgi apparatus-equivalent of dendrites (Morris et al., 1998). Similar observations have been reported using other nerve cell types (Tiedge and Brosius, 1996; Torre and Steward, 1996). Even though it is likely that the majority of neurosecretory vesicles originate from the cell soma it remains an appealing assumption, given the translational capacity of dendrites, that VP (and OT) are synthesized on-site in the dendrites of hypothalamic neurons. Precisely localized precursor synthesis might provide the mutual opportunity to modulate and/or fine tune defined synaptic connections within the paraventricular and supraoptic nuclei. Molecular determinants of the mRNA localization pathway Localization of mRNAs and non-coding transcripts to distinct cytoplasmic regions is conserved during evolution and is observed in organisms as diverse as yeast and mammals. By means of mRNA localization synthesis of particular proteins may be very precisely restricted to subcellular microdomains. Initially, this phenomenon has been described in developmental systems. In Drosophila oocytes and early embryos, for example, embryonic development is specified by the asymmetrical distribution of cell fate determinants. This segregation is achieved, at least in part, by localization of the respective mRNAs (for review see Bashirullah et al., 1998). Meanwhile, it is well known that RNA compartmentalization takes also place in differentiated cells, including neurons, to allow for the establishment and maintenance of cell polarity (for review see Bashirullah et al., 1998; Hazelrigg, 1998; Mohr, 1999; Carson et al., 2001). The molecular entities specifying the entire process of RNA localization appear to be remarkably complex. First, the mRNA (or the non-coding transcript) destined for transport must be selected from a large pool of RNAs. RNA localization may even begin,

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at least in some cases, in the cell nucleus (Schnapp, 1999; Long et al., 2001). Distinct sequences of the RNA molecule serve as targets for RNA-binding proteins, the trans-acting factors. These mediate, together with additional proteins that might associate the complex, transport of the RNAs to a defined cytoplasmic location along either microfilaments or microtubules (for review see Jansen, 1999). Anchor proteins that have very recently begun to be characterized (Zhao et al., 2001) are presumably required to attach the RNAs upon arrival at their ultimate destinations. Furthermore, the RNA localization process includes mechanisms of translational control. For instance, in Drosophila oocytes and early embryos and in Xenopus oocytes translation of localized mR-

NAs is repressed as long as they are in an incorrect subcellular environment. Only at their proper destination, this translational block is removed and protein synthesis can commence (Castagnetti et al., 2000; Wilhelm et al., 2000). Unfortunately, current knowledge as to whether or not translational control plays a role in mRNA localization in nerve cells is still scarce. Yet, there is accumulating evidence to suggest that protein synthesis within dendrites is triggered by synaptic activity implying mechanisms of translational silencing and activation of mRNAs localized to the dendritic compartment (for review see Wells et al., 2000; Steward and Schuman, 2001). Based on the available information we propose a — in many aspects still hypothetical — model (Fig. 1)

Fig. 1. Hypothetical scheme of VP mRNA transport mechanisms to axons and dendrites in hypothalamic magnocellular neurons. Two different pathways exist delivering VP mRNA molecules either to the dendrites (pathway 1) or to the axonal compartment (pathway 2). Pathway 1: a current concept proposes that mRNAs may be selected for transport in the cell nucleus. A nuclear RNA-binding protein binds to a specific sequence or structural motif and accompanies the mRNA to the cytoplasm. There, additional RNA-binding proteins join the complex and fix the cargo to cytoskeletal elements (not shown in this scheme) along which mRNA targeting to dendrites occurs. During transport, mRNAs are presumed to be translationally silenced. After delivery at the ultimate destination, the ribonucleoprotein complex is reorganized in such a way as to anchor the mRNA by still ill-defined anchor proteins. Protein synthesis is initiated by unknown mechanisms. That event is likely to be triggered by synaptic activation. The recruitment of the translational machinery presumably requires additional proteins not depicted in this scheme. Pathway 2: after entering the cytoplasm, VP mRNA is first translated at the rough endoplasmic reticulum. Gradually, poly(A) tail shortening takes place, until it exhibits a length that might no longer support an efficient precursor synthesis. Then, VP mRNA is directed toward the axon, presumably along microtubules. Axonal transport may also be mediated by RNA/protein interactions. Evidence obtained by electron spectroscopic imaging techniques suggests that the axonal transcripts are indeed organized into ribonucleoprotein particles (Mohr et al., 1993). The axonal transcripts are not translated, their functional significance has remained elusive.

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for axonal and dendritic sorting of VP (and possibly OT) mRNA. In hypothalamic MCNs obviously two different mRNA localization pathways exist. VP mRNA destined to be localized to the dendrites (pathway 1) may be marked for transport in the cell nucleus. If true, a nuclear RNA-binding protein would interact with a specific localizer sequences and accompany the mRNA to the cytoplasm. There, additional cytoplasmic proteins join the complex and fix it to the cytoskeleton, most likely to microtubules (Mohr et al., 2001b). After delivery of VP mRNA molecules at their ultimate destination(s), the ribonucleoprotein complexes are presumably reorganized in order to allow for anchoring and active translation of the hitherto translationally dormant VP mRNA. This may occur only upon certain physiological circumstances accompanied by synaptic activation. Pathway 2 begins with nuclear export and translation in the cell soma of VP mRNA. Gradually, poly(A) tail shortening takes place until it gains a length which may no longer support an efficient precursor synthesis. At that time, VP mRNA is directed toward the axonal compartment. This localization step might be mediated by proteins as well. Earlier data have suggested the involvement of microtubules in axonal VP mRNA transport in vivo (Levy et al., 1990). The functional role of the axonal transcripts is, however, virtually unknown. Identification of cis-acting elements within VP mRNA We have employed a well established and extensively characterized primary nerve cell culture system, namely SCG neurons, to delineate cis-acting sequences that mediate VP mRNA sorting to the nerve cell processes (for review see Mohr, 1999). Eukaryotic expression vectors schematically outlined in Fig. 2A have been introduced into these cells (which lack endogenous VP gene expression) by computer-assisted nuclear microinjections. These initial experiments, summarized in Fig. 2B–D, indicate that either a sequence motif or a secondary structure of the VP mRNA must contain information essential for dendritic mRNA sorting: when injections were done with a construct driving the expression of sense RNA, substantial amounts of VP transcripts were detectable in dendrites in addition to

the cell soma (Fig. 2B). VP anti-sense RNA, in contrast, remained confined to the cell bodies (Fig. 2C). Vector-expressed α-tubulin mRNA (Fig. 2D), like its endogenously transcribed counterpart, is not localized to dendrites further underscoring specificity of dendritic VP mRNA sorting. Parenthetically, sorting to the axonal domain was not observed using this detection protocol. Thus, the determinants specifying axonal mRNA transport might be more cell-specific than those required for dendritic mRNA localization. Yet, the possibility still remains that more sensitive RNA detection procedures would reveal transport of VP mRNA to the axonal compartment of SCG neurons as well. Our earlier data (see above) have suggested VP mRNA sorting to the dendrites of MCNs takes place well before translation commences. Thus, ongoing translation should not be required for the sorting process. If true, VP mRNA should continue to be delivered to dendrites while translation is blocked. As shown in Fig. 3, culturing microinjected SCG neurons in the presence of cycloheximide dramatically reduced translation of the VP mRNA but had no recognizable effect on its transport to the dendrites, a result in line with data reported by others (Kleiman et al., 1993). Subsequent analyses specified the last 395 nucleotides of the VP mRNA as being essential for an efficient dendritic mRNA transport, because this sequence, referred to as DLS, was able to confer dendritic localization to the non-localized α-tubulin mRNA used as a reporter transcript (Mohr, 1999). Notably, the DLS is composed of coding sequences in addition to the complete 3 -untranslated region (UTR). Initially, this was regarded as an unusual feature, because previously characterized RNA localization elements were always confined to the 3 -UTR. Meanwhile, however, several localized mRNAs, in Drosophila and in yeast, have been shown to accommodate targeting information within the proteinencoding moiety of the mRNA molecule (for review see Gonzalez et al., 1999; Mohr and Richter, 2001). More recently, we have been able to show that the DLS contains several subelements, weak localizer elements on their own, only the concerted action of which mediates an efficient dendritic sorting of reporter mRNA molecules comparable to that achieved by the VP mRNA alone (Mohr, 1999).

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Fig. 2. VP mRNA is sorted to the dendrites of primary cultured SCG neurons. (A) Schematic view of eukaryotic expression vectors microinjected into the cell nuclei of in vitro cultured SCG neurons. The expression of any inserted cDNA is driven by the cytomegalovirus (CMV) promoter. A short sequence of the bacterial β-galactosidase (β-gal) gene was included such that it forms part of the 5 -UTR of the resulting transcripts. This allows to discriminate mRNAs that are endogenously expressed in SCG neurons from those derived by transcription of the expression vector by performing in situ hybridizations with β-gal anti-sense oligodeoxyribonucleotides. Addition of a poly(A) tail is mediated by the bovine growth hormone (BGH) gene poly(A) addition sequence. (B–D) In situ hybridization analyses of cells microinjected with three different expression vector constructs. (B) Injection of a construct containing the rat VP cDNA inserted in sense orientation leads to transport of the mRNA into proximal and distal parts of the dendrites (arrows). Labeling of the axon has not been observed. (C) A cell is shown that expresses VP anti-sense transcripts. In this case, sorting out of the cell somata does not occur (arrowheads). (D) The vector-derived mRNA encoding α-tubulin is confined to the cell somata (arrowheads). Microinjected cells have been processed for in situ hybridization approximately 18 h following injections. For experimental details see (Prakash et al., 1997). From Mohr et al., 2001b. Reproduced with permission.

Mutations within the DLS impair its dendritic targeting capacity The DLS contains two motifs rich in pyrimidine nucleotides termed E1 and E2 (Fig. 4A). Short repetitive motifs are involved in the subcellular transport of some mRNAs in non-neuronal cells (Mowry and Cote, 1999). In order to determine if E1 and/or E2 have an impact on VP mRNA sorting to the nerve cell processes the elements have been deleted from the DLS either individually or together. The mutagenized DLSs were inserted as part of the 3 -UTR of α-tubulin cDNA cloned into a eukaryotic expression vector which is schematically depicted in Fig. 4B. In line with earlier observations (Mohr, 1999) the majority of SCG neurons microinjected with the construct containing the wild-type DLS (construct 1: pCMV β-gal α-tub/VP-DLS) harbor the chimeric

mRNA in the dendritic domain, predominantly in proximal and distal dendritic segments (Fig. 5A,B and Fig. 6). Deletion of element E1 (construct 2: pCMV β-gal α-tub/VP-DLSE1) reduced dendritic targeting of the recombinant mRNA substantially even though it was not completely abolished. In most cells, however, the mRNA was confined to the cell somata (Fig. 5C,D and Fig. 6). Only residual dendritic targeting was observed in the remainder of cells with localization of chimeric transcripts primarily in basal and proximal dendritic segments. Upon deletion of element E2 (construct 3: pCMV β-gal α-tub/VP-DLSE2) a somewhat weaker but still remarkable reduction of the dendritic localizer capacity was noted (Fig. 5E,F and Fig. 6). Concomitant deletion of both elements (construct 4: pCMV βgal α-tub/VP-DLSE1 + E2) had no further effect on the subcellular RNA distribution pattern which

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Fig. 3. Dendritic VP mRNA transport occurs independently of ongoing translation. In vitro cultured SCG neurons were injected with a eukaryotic expression vector (see Fig. 2A) containing the rat VP cDNA in sense orientation. Following overnight incubation, cells were subjected to in situ hybridization with a digoxigenin-labeled VP-specific anti-sense riboprobe. Probe detection was done immunocytochemically by sequentially adding a sheep anti-digoxigenin antibody and a biotinylated donkey anti-sheep antibody, respectively. The hybridization signal was finally amplified using the Renaissance TSA-indirect system (NEN) with Neutravidin-conjugated Alexa 488 (Molecular Probes) as a fluorochrome. Concomitantly, the neurophysin II (NP II) moiety of the VP precursor was visualized using a rabbit antineurophysin II-antiserum followed by Cy3-conjugated donkey anti-rabbit immunoglobulin. Micrographs were taken using the appropriate filter sets to allow for discrimination between the two fluorochromes. (A–C) Control SCG neuron cultured in the absence of cycloheximide; (D–F) SCG neuron cultured in the presence of 40 μM cycloheximide for the entire period of the experiment. The drug was added to the culture media 1 h prior to the injection. (A,D) micrographs showing the distribution of VP mRNA; (B,E) micrographs showing the distribution of neurophysin II; (C,F) phase contrast micrographs. ICC, immunocytochemistry; ISH, in situ hybridization. Scale bar: 50 μm.

Fig. 4. Deletion of elements rich in pyrimidine nucleotides within the dendritic localizer sequence of the VP mRNA (VP-DLS) reduces its capacity to confer dendritic localization to the non-localized α-tubulin (α-tub) RNA. (A) Schematic representation of the 395-bp VP-DLS (nucleotide residues 190–584) which contains two highly homologous elements, E1 and E2, enriched in pyrimidine nucleotides. These elements were deleted either alone or together. (B) The wild-type and the mutagenized DNA fragments, respectively, were individually ligated in sense orientation to the 3 -end of the rat α-tubulin cDNA cloned into a eukaryotic expression vector. Expression of the chimeric mRNAs is driven by the cytomegalovirus (CMV) promoter. In addition, the vector contains a 50-bp fragment derived from the bacterial β-galactosidase (β-gal) gene. This sequence forms part of the 5 -untranslated region of vector-expressed transcripts. It is not relevant for the data presented here (for experimental details see Prakash et al., 1997). BGH, bovine growth hormone.

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resembled that seen with pCMV β-gal α-tub/VPDLSE1 (Fig. 5G,H and Fig. 6). Obviously, deletions within the DLS are no longer compatible with a coordinated interplay of the presumably complex and multifactorial transport machinery overall resulting in a reduced dendritic transport. These results are not surprising. Cis-acting mRNA transport signals are rather complex usually consisting of several hundreds of nucleotides (reviewed in Bashirullah et al., 1998). As outlined above, the same holds true for the DLS of the VP mRNA. The DLS might contain several conserved sequence elements individually recognized by proteins of the localizing machinery. Alternatively, the interaction of trans-acting factors could primarily depend on the secondary or tertiary structure adopted by the RNA molecule. The DLS contains one copy each of the motifs E1 and E2 in its proximal and its distal half, respectively. On their own, both of these DLS-subsegments are able to direct the non-localized α-tubulin mRNA to dendrites, but only to regions proximal to the cell body (Prakash et al., 1997). According to our definition, these segments would represent weak localizer elements. Interestingly, short reiterated pyrimidine nucleotide-rich elements play a critical role in sorting Vg1 mRNA to the vegetal hemisphere in Xenopus oocytes. Upon deletion, Vg1 mRNA localization is either abolished or impaired, depending on the motif under investigation (Mowry and Cote, 1999). Deletion of E1 and E2 within the DLS likewise has a severe impact on dendritic mRNA transport, but a residual activity could still be observed. Consequently, E1 and E2 are not the sole dendritic targeting elements. Besides E1 and E2, other primary sequences might exist which harbor the residual dendritic localization capacity displayed by the mutagenized sequences. Alternatively, by deleting

these elements the DLS secondary or tertiary structure could have been altered such as to disturb the interplay with the transport machinery considerably since higher order structure of an RNA molecule is determined by its primary sequence (SenGupta et al., 1996). The data presented here do not allow to discriminate between these alternatives but they clearly show that the DLS of the VP mRNA is organized in a complex way as are the targeting elements of localized transcripts in diverse organisms. Rat poly(A)-binding protein interacts specifically with the DLS of the VP mRNA In non-neuronal systems, and probably in nerve cells as well, proteins play a pivotal role in any of the multiple events of mRNA localization (Fig. 1). By using UV-crosslinking assays with rat brain cytosolic protein extracts and radiolabeled RNA probes we have recently characterized a protein that interacts specifically with the DLS of the VP mRNA but not with its 5 -end that lacks a role in dendritic VP mRNA targeting (Mohr et al., 2001a). The identity of this protein, initially termed VP-RBP (VP mRNA-binding protein), was revealed by biochemical purification (Mohr et al., 2001b) showing that it is the well known and multifunctional PABP. As demonstrated in Fig. 7A, PABP, while specifically interacting with VP mRNA, fails to bind to a variety of other transcripts including the dendritic targeting element of the MAP2 transcript and α-tubulin mRNA. Since PABP is a ubiquitous and an extremely abundant protein (Görlach et al., 1994) it is very surprising to find the highest binding activity to the VP mRNA in brain extracts whereas peripheral tissues and non-neuronal cell lines contain very little if any binding activities (Fig. 7B). A possible

Fig. 5. Micrographs showing primary cultured SCG neurons microinjected into the cell nuclei with eukaryotic expression vector constructs containing the wild-type VP-DLS or mutagenized sequences thereof (schematically outlined in Fig. 4). (A,B) Microinjection of construct 1 (pCMV βgal αtub/VP-DLS) containing the wild-type VP-DLS. (C,D) Microinjection of construct 2 (pCMV βgal αtub/VP-DLSE1), lacking element E1. (E,F) Microinjection of construct 3 (pCMV βgal αtub/VP-DLSE2) lacking element E2. (G,H) Microinjection of construct 4 (pCMV βgal αtub/VP-DLSE1 + E2) lacking both element E1 and element E2. The subcellular distribution of the chimeric RNAs was determined by in situ hybridization with a digoxigenin-labeled in vitro synthesized VP anti-sense riboprobe. The wild-type VP-DLS mediates RNA transport to distal parts of the dendrites (arrows). In contrast, chimeric RNAs containing either of the mutagenized VP sequences are detectable, in very low amounts, in basal and proximal dendritic segments (arrows) in the majority of the injected cells (for experimental details see Prakash et al., 1997). αtub, α-tubulin; βgal, β-galactosidase; DLS, dendritic localizer sequence; VP, vasopressin. Scale bar: 25 μm.

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Fig. 6. Evaluation of distribution patterns of chimeric transcripts in primary cultured SCG neurons microinjected with constructs schematically shown in Fig. 4. All expression vector constructs have been injected in at least 10 different experiments with embryonic cell cultures prepared from different pregnant females (800–1000 cells/construct). The relative number of cells injected with the four constructs that harbor the chimeric mRNAs in the cell body only, or in addition in basal, proximal and distal parts of the dendrites, schematically outlined in A, are graphically summarized in B.

explanation to this obvious discrepancy would be to assume that PABP’s binding specificity to VP mRNA is determined by additional parameters. For example, a covalent modification or the association with additional proteins to form a larger complex could alter PABP’s sequence selection properties. Apparently, this ‘specificity factor’, the nature of which remains to be determined, is brain specific. The literature on proteins that might play a role in mRNA transport to the neurites of nerve cells is still rather scarce. Recently, however, trans-acting factors have been characterized that interact with the

dendritic targeting element of the MAP2 mRNA, an mRNA species localized to the dendrites of various nerve cell types (for review see Kindler et al., 2001). Two proteins, termed MARTA1 (Rehbein et al., 2002) and MARTA2 (the identity is not yet known), appear to associate selectively with MAP2 transcripts because they neither bind to the rat VP mRNA nor to other transcripts known to be sorted to dendrites (Rehbein et al., 2000). The third protein found to interact with MAP2 mRNA is the rat Staufen protein (Monshausen et al., 2001). Even though Staufen binds to any RNA in vitro, its

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species such as bicoid, oskar and prospero transcripts (for review see Bashirullah et al., 1998). Rat Staufen is expressed in many tissues, interestingly, however, in neurons it shows a somato-dendritic distribution suggesting that it might be involved in dendritic mRNA localization (for review see Kiebler and DesGrosseilliers, 2000). Taken together, the available information supports the view that the molecular determinants required for localizing different mRNA species to dendrites of nerve cells appear to be surprisingly specific for a given transcript. These findings are not completely unexpected, though, given the different temporal and spatial distribution patterns of mRNAs that have been observed in nerve cells (for review see Mohr, 1999). Proposed function of PABP in VP mRNA localization

Fig. 7. PABP binds specifically to the DLS of the VP mRNA. (A) Autoradiogram of UV-crosslinking analyses performed with 7.5 μg rat brain cytosolic protein extract and 5 fmol of the radiolabeled DLS riboprobe (lacking a poly(A) tail). Unlabeled competitor RNAs were added at a 100-fold molar excess. Lane 1, no competitor; lane 2, full size VP RNA; lane 3, dendritic targeting element of rat MAP2 mRNA; lane 4, full-size rat αtubulin RNA. The positions of molecular size marker proteins is indicated on the right. The arrow denotes the PABP/VP RNA complex. All competitor RNAs represent the sense strands; none of the transcripts possess poly(A) tails. (B) Autoradiogram of UV-crosslinking analyses performed with 7.5 μg each of various rat tissue/cell line cytosolic protein extracts and 5 fmol of the radiolabeled DLS riboprobe (lacking a poly(A) tail). Proteins were prepared from: lane 1, total brain; lane 2, hypothalamus; lane 3, heart; lane 4, lung; lane 5, spleen; lane 6, liver; lane 7, rat I cells; lane 8, PC 12 cells. The positions of molecular size marker proteins is indicated on the right. The arrow denotes the PABP/VP RNA complex. For experimental details see (Mohr et al., 2001a). comp., competitor; hypoth., hypothalamus; MAP2, microtubule-associated protein 2; PABP, poly(A)-binding protein; tot. brain, total brain; tub., tubulin; VP, vasopressin. From Mohr et al., 2001b. Reproduced with permission.

in vivo binding properties are remarkably specific. In Drosophila oocytes and neuroblasts, Staufen is involved in subcellular targeting of defined mRNA

PABP is an evolutionarily conserved RNA-binding protein. It consists of four RNA recognition motifs (RRM; each 80–100 amino acids in length) at the Nterminus of the protein and a C-terminal proline-rich auxiliary domain (for review see Burd and Dreyfuss, 1994). It exhibits high affinity binding to the poly(A) tail of mRNAs, mainly via RRMs 1 + 2, and this interaction is essential for translational efficiency. PABP enhances translation initiation by still unknown mechanisms via its interaction with initiation factors bound at the 5 -end of the mRNA (Preiss et al., 1998; Fig. 8A). Moreover, it is required for translation-dependent mRNA stabilization (Coller et al., 1998). In vitro binding assays performed with either individual RRMs or various combinations of RRMs revealed remarkable features. While single RRMs do not interact with poly(A) sequences at all, RRMs 1 + 2 bind with high affinity indistinguishable of that displayed by the full-size protein. In contrast, the affinity of RRMs 3 + 4 for poly(A) sequences is much lower. In fact, these RRMs bind more efficiently to poly(U) and poly(G) than to poly(A) sequences (Burd et al., 1991; Kühn and Pieler, 1996). Notably, PABP is essential for cell viability in yeast. Yet, critical function(s) are displayed by RRM4 together with part of the C-terminus rather than by RRMs 1 + 2 the removal of which still supported growth (Burd et al., 1991). Consequently, the RRMs of PABP are functionally diverse and its most impor-

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Fig. 8. Functional roles of PABP in translation. (A) PABP, bound in multiple copies to the poly(A) tail of mRNAs, plays a major role in translation by stimulating the initiation of protein biosynthesis. PABP interacts with eIF4G which forms, together with the cap-binding protein eIF4E and eIF4A, an ATP-dependent RNA helicase, a ternary complex called eIF4F. Thus, the 3 -end is brought in close proximity to the 5 -end of the mRNA. This is believed to facilitate joining of the ribosomal subunits that are liberated at the stop-codon of the mRNA. (B) PABP bound to the DLS of the VP mRNA (VP-DLS) could possibly play a role in its translational silencing. By interaction of PABP bound to the DLS with those PABP molecules bound to the poly(A) tail (either directly by homophilic interactions or via an additional protein factor) translational initiation could be inhibited, because this interaction would interfere with the interaction of the poly(A) tail-bound PABP with eIF4G at the 5 -end of the VP mRNA. AUG, translational start codon; cap, cap structure at the 5 -end of the mRNA; DLS, dendritic localizer sequence; eIF, eukaryotic initiation factor; PABP, poly(A)-binding protein; poly(A), poly-adenylate; VP, vasopressin.

tant feature is clearly not the high affinity binding to poly(A) sequences. Earlier estimates have suggested PABP to be present at a concentration exceeding binding sites on poly(A) tails of mRNAs approximately three-fold (Görlach et al., 1994). Since it is able to interact with non-poly(A) sequences, at least in vitro (Görlach et al., 1994; Kühn and Pieler, 1996), PABP presumably has multiple functions in mRNA metabolism. This view is supported by recently published data. For instance, PABP expression is regulated at the translational level (Wu and Bag, 1998; Hornstein et al., 1999). Translational silencing of PABP mRNA appears to be brought about by a specific interaction of PABP itself with sequences in the 5 -UTR (De Melo Neto et al., 1995; Bag and Wu, 1996). Moreover, PABP, in concert with additional proteins, is involved in controlling the translationdependent turnover of the c-fos mRNA (Grosset et al., 2000). Thus, it is conceivable that it may also be implicated in regulating the translational state of the VP mRNA. Ever-growing evidence supports the current view that mRNAs residing in dendrites are translationally silenced until external stimuli activate protein synthesis (for review see Marin et al., 1997; Schuman, 1999; Steward and Schuman, 2001). Downregulation of dendritcally localized VP mRNA translation by PABP could, for example, be brought

about by its binding the DLS. The DLS-bound PABP could, either directly or via additional factors, interact with PABP molecules on the poly(A) tail. As a net result, this could effectively interfere with translational stimulation because it would prevent the poly(A) tail-bound PABP interaction with translational initiation factors at the 5 -end of the mRNA (Fig. 8B). Beyond doubt, we are currently still missing many of the components necessary for dendritic (and axonal) VP mRNA trafficking. Presumably, PABP is only one member of a multi-factorial complex. Yet, by applying techniques such as the yeast two hybrid system with PABP as a bait and immunoprecipitation once an anti-rat PABP antiserum is available we should be able to identify and characterize additional constituents of the sorting machinery. This will pave the way to gain a deeper understanding of the RNA localization pathways in nerve cells. Abbreviations BGH CMV DLS ER MAP2

bovine growth hormone cytomegalovirus dendritic localizer sequence endoplasmic reticulum microtubule-associated protein 2

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MCN OT PABP RRM SCG UTR VP VP-RBP

magnocellular neuron oxytocin poly(A)-binding protein RNA recognition motif superior cervical ganglion untranslated region vasopressin VP RNA-binding protein

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Preiss, T., Muckenthaler, M. and Hentze, M.W. (1998) Poly(A)tail-promoted translation in yeast: implications for translational control. RNA, 4: 1321–1331. Raggenbass, M. (2001) Vasopressin- and oxytocin-induced activity in the central nervous system: electrophysiological studies using in-vitro systems. Prog. Neurobiol., 64: 307–326. Rehbein, M., Kindler, S., Horke, S. and Richter, D. (2000) Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs. Mol. Brain Res., 79: 192–201. Rehbein, M., Wege, K., Buck, F., Schweizer, M., Richter, D. and Kindler, S. (2002) Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs. J. Neurochem., in press. Schnapp, B. (1999) RNA localization: a glimpse of the machinery. Curr. Biol., 9: R725–R727. Schuman, E. (1999) mRNA trafficking and local protein synthesis at the synapse. Neuron, 23: 645–648. SenGupta, D.J., Zhang, B., Kraemer, B., Pochart, P., Fields, S. and Wickens, M. (1996) A three-hybrid system to detect RNA–protein interactions in vivo. Proc. Natl. Acad. Sci. USA, 93: 8496–8501. Steward, O. and Schuman, E.M. (2001) Protein synthesis at synaptic sites on dendrites. Annu. Rev. Neurosci., 24: 299– 325. Steward, O. and Worley, P. (2001) Localization of mRNAs at synaptic sites on dendrites. In: D. Richter (Ed.), Results and Problems in Cell Differentiation: Cell Polarity and Subcellular RNA Localization, Vol. 34. Springer-Verlag, Heidelberg, pp. 1–26. Tiedge, H. and Brosius, J. (1996) Translational machinery in dendrites of hippocampal neurons in culture. J. Neurosci., 15: 7171–7181. Tiedge, H., Bloom, F.E. and Richter, D. (1999) RNA, whither goest thou?. Science, 283: 186–187. Torre, E.R. and Steward, O. (1996) Protein synthesis within dendrites: glycosylation of newly synthesized proteins in dendrites of hippocampal neurons in culture. J. Neurosci., 16: 5967–5978. Wells, D.G., Richter, J.D. and Fallon, J.R. (2000) Molecular mechanisms for activity-regulated protein synthesis in the synapto-dendritic compartment. Curr. Opin. Neurobiol., 10: 132–137. Wilhelm, J.E., Vale, R.D. and Hedge, R.S. (2000) Coordinate control of translation and localization of Vg1 mRNA in Xenopus oocytes. Proc. Natl. Acad. Sci. USA, 97: 13132–13137. Wu, J. and Bag, J. (1998) Negative control of the poly(A)binding protein mRNA translation is mediated by the adeninerich region of its 5 -untranslated region. J. Biol. Chem., 273: 34535–34542. Zhao, W., Jiang, C., Kroll, T.T. and Huber, P.W. (2001) A proline-rich protein binds to the localization element of Xenopus Vg1 mRNA and to ligands involved in actin polymerization. EMBO J., 20: 2315–2325.

D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 17

Dendritic action potentials in magnocellular neurons Jaideep S. Bains * Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, Calgary AB, Canada

Abstract: In the central nervous system, information is traditionally thought to flow from synapses to dendrites to soma. Recent evidence, however, suggests that dendrites play more of an active role in signal processing than previously thought. This review will examine the evidence in support of dendritic spikes in magnocellular neurons. Additionally, it will shed light on a number of important roles these spikes may play in regulating the excitability of magnocellular neurons. Keywords: Oxytocin; Vasopressin; Voltage-gated channel; Calcium; Retrograde inhibition

Introduction A putative function for neuronal dendrites was first formulated by Ramon y Cajal who suggested they served to propagate impulses towards the soma. Over 50 years later this concept was formalized, stating in effect that dendrites served as cable-like structures that summed excitatory and inhibitory inputs and delivered the net sum to the soma in order to generate an action potential (Rall, 1959). Shortly after this time, the hypothesis was put forward that dendrites may also amplify or boost synaptic signals (Loronté de Nó and Coundouris, 2001). Although this was a major shift in how neuroscientists thought about the dendrite, the experimental evidence in support of this idea was still lacking. In 1961, Spencer and Kandel described an electrophysiological phenomenon in hippocampal pyramidal cells that they dubbed ‘fast pre-potentials’ (FPPs) (Spencer and Kandel, 1961). These discrete events, which preceded an action potential likely originated, according to the authors, in distant dendrites in areas they referred to as ‘trigger zones’. They surmised

∗ Correspondence to: J.S. Bains, Department of Physiology and Biophysics, Neuroscience Research Group, University of Calgary, Calgary AB, Canada.

these events were true spikes since they did not exhibit a graded response as is often observed with excitatory postsynaptic potentials, but rather were all-or-none, much like the classical Na+ action potentials. Since this seminal observation, our knowledge of dendrites has grown extensively (recent reviews see: Yuste and Tank, 1996; Magee et al., 1998; Segev and Rall, 1998; Johnston et al., 1999; Hausser et al., 2000; Segev and London, 2000), but it is safe to say that aside from the electrophysiology afficianados, relatively few neuroscientists truly grasp the fundamental role that dendrites play in dictating the way in which information is transduced in the central nervous system (CNS). In the magnocellular neurons, for example, we appreciate that the dendrites contain and indeed release the neuropeptides vasopressin (VP) and oxytocin (OT) (Pow and Morris, 1989) (for review see: Ludwig, 1998), and that these peptides have a profound effect on cellular activity (Poulain and Wakerley, 1982; Yamashita et al., 1987; Moos and Richard, 1989; Kombian et al., 1997, 2000; Moos et al., 1998; Hirasawa et al., 2001) yet we still do not fully appreciate how these interactions are initiated. The primary reason for this dearth of knowledge is that our understanding of how the nervous system works is clouded by our soma-centric perspective. Although the vast majority of the CNS is a tangle of dendrites and axons, neurophysiologists have

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focused primarily on the cell bodies. To a large degree this stems from the experimental inaccessibility of dendrites and axons. Although in some structures, the hippocampus and cerebellum in particular, a number of groups have succeeded in the heroic feat of obtaining ‘blind’ recordings from dendrites (Spencer and Kandel, 1961; Llinas and Nicholson, 1971; Llinas and Hess, 1976; Llinas and Sugimori, 1980). As a result of recent advances in microscopy, it is now possible to obtain electrical recordings directly from the dendrites of CNS neurons on a relatively consistent basis (Stuart et al., 1993). The information gained from these experiments has confirmed what many had intuitively thought: The dendrites are dynamic structures that are far more than a simple cable linking the synapse to the cell body. Virtually all of these data has been obtained from pyramidal cells in either the neocortex or hippocampus where the architecture of these cells makes both the dendrites and the cell body readily accessible for the experimenter. Unfortunately, this is not the case with the magnocellular neurons of the hypothalamus, where dendrites are interwoven with cell bodies and axons, making intradendritic recording considerably more challenging. This means that we are only starting to scratch the surface of truly understanding just how important the role of dendrites may be in the life of a magnocellular neuron. This review will focus on one particular aspect of dendritic function, the dendritic action potential. I will cover the brief history of dendritic spikes in magnocellular neurons and then explore the potential source of these spikes. Finally, I will discuss a number of putative roles for dendritic spikes that may be unique to magnocellular neurons. I will not dwell on a number of important functions mediated by dendritic spikes, such as boosting synaptic signals or providing a substrate for a Hebbian form of plasticity. These topics have been covered extensively by several excellent reviews (Magee et al., 1998; Hausser et al., 2000; Segev and London, 2000). I will instead focus on the potential contributions of dendritic spikes to three aspects that are important to the physiology of magnocellular neurons: burst firing, dendritic release of neuromodulators (VP, OT and nitric oxide (NO)) and resistance to excitotoxicity. The recent explosion of work that has brought the dendrite to the forefront of neuroscience owes

much of its gratitude to the improvements in microscope optics. The use of an upright microscope with differential interference contrast (DIC) optics in combination with a high numerical aperture, long working distance, water immersion objective, has made it possible to make direct recordings from these structures (Stuart et al., 1993). Based on these recordings, we can now say definitively that in addition to ligand-gated receptors, dendrites are also endowed with a rich repertoire of voltage-gated conductances. Much of this work has been extensively reviewed elsewhere and the reader is referred to several excellent recent reviews on various topics related to dendritic physiology (Magee et al., 1998; Johnston et al., 1999; Hausser et al., 2000; Segev and London, 2000). Without the benefit of dendritic recordings, but with the use of a little deductive reasoning, it can be argued that dendrites in magnocellular neurons also possess a number of voltage-gated conductances. The first observation that, in hindsight, hinted at the dynamic dendrite in magnocellular neurons was the presence of TTX-insensitive action potentials (Andrew and Dudek, 1983; Mason and Leng, 1984). These spikes were not due to activation of a novel, TTX-insensitive Na+ conductance, but rather were mediated by Ca2+ ions (Mason and Leng, 1984; Bourque and Renaud, 1985). Slower, long-lasting Ca2+ -mediated depolarizing potentials had been described in fetal mouse hypothalamic neurons (Legendre et al., 1982), but their relationship to these brief, transient events was unclear. (In hindsight, it seems these events, which were observed when K+ conductances were blocked may have been prolonged dendritic depolarizations (see below).) Ca2+ spikes had been described previously in the dendrites of cerebellar Purkinje cells (Llinas and Hess, 1976) and a similar site of initiation was proposed for these events in magnocellular neurons. This was based on the observation that a dual spike waveform was present when extracellular recordings were obtained from magnocellular neurons, but not when intracellular recordings were made exclusively from the soma (Mason and Leng, 1984). Additionally, it was reported that magnocellular neurons exhibited non-synaptic depolarizing potentials (NSDPs) which were small spike-like events that were sensitive to extracellular Ca2+ (Bourque et al., 1986). The final piece of ev-

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idence supporting a dendritic site of initiation was the demonstration that intense activation of NMDA receptors, which are located on the dendrites of magnocellular neurons, resulted in the activation of TTXinsensitive, Ca2+ -mediated spikes (Bains and Ferguson, 1999). This provided the first direct evidence that transduction of a synaptically generated EPSP to the soma may not just be a simple case of propagating an ever-decaying signal down a long, narrow tube. If we cannot record from the dendrites, how do we really know the spikes are dendritic in origin? Although the work cited above is suggestive of a dendritic origin for the Ca2+ spikes, a more rigorous set of criteria needed to be met before a somatic

site of initiation could be ruled out completely. We decided to implement a number of criteria that would allow us to safely conclude that the events we observed following NMDA receptor activation were indeed dendritic. First, in response to NMDA receptor activation, we observed rapid, inward current spikes in magnocellular neurons that were voltage clamped (Fig. 1a). Although under these conditions, the membrane potential of the cell should, theoretically, be ‘clamped’, this is rarely the case with neurons (Bains and Ferguson, 1999). Because of a cell’s dendritic tree, it is difficult to effectively clamp the voltage in the distal dendrites. Therefore, synaptic input, if sufficiently depolarizing, should activate voltage-gated channels located in these regions. Since the voltage in the soma of the neuron under examination should be clamped at the holding potential, it would be dif-

Fig. 1. Criteria for dendritic spikes. (A) NMDA agonist (1 μM, arrow) in TTX (2 μM) elicits slow inward currents accompanied by fast transients at the onset of response. These are likely unclamped Ca2+ conductances generated in the distal region of the dendrites. (B) Voltage responses to somatic depolarizing current pulses (10–50 pA) in TTX (2 μM). Large amplitude depolarization elicits a sharp spike. (C) Threshold for Ca2+ spike activation varies with stimulus. Top traces are responses of a cell to NMDA agonist application (arrow) and +10 pA depolarizing pulse. The dotted line represents spike activation threshold. Note the invariant threshold for somatic Na+ spikes. Lower traces depict the same experiment in the presence of TTX. Note the marked difference in spike thresholds between current pulses (soma) and NMDA receptor activation.

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ficult to activate voltage-gated conductances near the recording site. Therefore, these current spikes likely indicate the activation of voltage-gated conductances in a distal region of the dendrite that was not adequately clamped (Bains and Ferguson, 1999). Similar observations were made in neocortical pyramidal cells where unclamped current spikes were observed in response to focal application of glutamate to distal segments of the apical dendrite (Schwindt and Crill, 1997, 1998). Subsequent recordings from the dendrites of these cells have confirmed the presence of dendritic Ca2+ spikes (Schiller et al., 1997, 2000). Second, the high threshold for activation following the injection of depolarizing current into the soma (Bourque and Renaud, 1985; Bains and Ferguson, 1999) suggests that the location of the spike initiation zone is some distance from the soma (Fig. 1). A pulse injected into the soma will be subjected to electrotonic filtering, so lower amplitude pulses which are sufficient to activate axonal Na+ spikes will not be sufficient to depolarize the dendrites and initiate dendritic spikes. (The smaller magnitude pulse required for Na+ spikes may also be due to a lower threshold for activation for Na+ channels as well as a requirement that fewer Na+ channels need to be recruited before a spike is initiated. This issue remains unresolved.) It is also possible that the high threshold is indicative of the opening of L-type, or high-threshold somatic Ca2+ channels, that open at potentials more depolarized than −40 mV. However, recordings from dissociated magnocellular neurons indicate the activation threshold of these currents is approximately −50 mV (Fisher and Bourque, 1995). These channels also exhibit little if any inactivation (Fisher and Bourque, 1995) indicating that a depolarizing event due to activation of these channels should be virtually the same duration as the depolarizing current pulse. This, however, is not the case. The spikes recorded in TTX were brief, transient events (Andrew and Dudek, 1983; Bourque and Renaud, 1985; Bourque et al., 1986; Bains and Ferguson, 1999), consistent with a channel that opens and inactivates quickly, much like a T-type, or lowthreshold Ca2+ channel (Fisher and Bourque, 1995). There are reports of dense distributions of T-type Ca2+ channels in dendrites. These may even be clustered at branch points, forming ‘hotspots’ to boost synaptic signaling. This would be analogous to and

in fact a confirmation of the ‘trigger zones’ proposed by Spencer and Kandel (1961). Finally, the putative dendritic spikes in magnocellular neurons exhibit a varying threshold for activation. This is in contrast to somatic Na+ spikes which exhibit an invariant threshold regardless of whether they are activated by current injection into the soma, or NMDA receptor activation at the dendrites (Fig. 1c). Again, similar observations have been made in neocortical pyramidal cells where dendritic spikes were evoked by application of glutamate directly to the apical dendrites of these cells (Schwindt and Crill, 1997, 1998). The presence of these dendritic spikes has been confirmed with direct dendritic recordings (Schiller et al., 2000). Is there more to a dendritic spike than just Ca2+ flux through voltage-sensitive calcium channels (VSCCs)? The following observations indicate that activation of VSCCs is necessary for dendritic spikes: (1) They are activated by depolarizing current pulses (Bourque and Renaud, 1985; Bains and Ferguson, 1999). This suggests a mechanism that is activated by a change in membrane voltage. (2) They are abolished by CdCl2 , a blocker of Ca2+ channels. In spite of this evidence, we should not be overly hasty in ruling out an additional, perhaps complementary mechanism — the direct influx of Ca2+ through the pore of the NMDA receptor/channel complex itself (Schiller et al., 2000). We have reasoned that activation of NMDA receptors resulted in a local depolarization that was sensed by VSCCs located close to receptors and that this depolarization was sufficient to open VSCCs resulting in a rapid influx of Ca2+ (Bains and Ferguson, 1999). This is entirely consistent with work in the neocortex (Markram and Sakmann, 1994; Schiller et al., 1997), cerebellum (Llinas and Hess, 1976) and hippocampus (Golding et al., 1999). Furthermore, direct dendritic recordings from neocortical neurons suggest that backpropagating action potentials from the soma readily activate dendritic calcium channels resulting in the generation of fast spikes (Stuart and Spruston, 1998; Larkum et al., 1999, 2001). In spite of this evidence, the recent work by Schiller et al. demonstrating that a component of the dendritic spike may result from

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the flux of Ca2+ ions directly through the NMDA receptor itself (Schiller et al., 2000) cannot be completely ruled out. They elegantly demonstrate, using two-photon laser microscopy that the co-activation of neighboring synaptic inputs onto cortical pyramidal cells results in local, transient membrane depolarizations that are mediated to a large degree by charge movement through the NMDA receptors and may not require activation of VSCCs. A resolution of this issue in magnocellular neurons will require either a similar imaging of, or direct electrophysiological recordings from the dendrites of these cells. What influences the propagation of dendritic spikes? Although Ca2+ spikes can be generated in the dendrites of magnocellular neurons, there is still no guarantee they will reach the soma, and more importantly, that they will alter the firing behavior of the cell. In fact, it is rare that these spikes are even observed during somatic recordings in the absence of TTX. This may be largely due to the fact that a Ca2+ spike would lead to a rapid Na+ spike which would obscure the initial depolarizing event. There is, however, also evidence that the dendrites may be capable of ‘absorbing’ the dendritic spike so that it never reaches the soma. This may result either from the abundant collection of K+ conductances present on dendrites (Hoffman et al., 1997), or alternatively from a limitation imposed by the actual geometry of the dendritic tree (Vetter et al., 2001). First let us examine the influence of other dendritic voltage-gated conductances on dendritic spike propagation. In addition to Ca2+ channels, the dendrites of neurons also possess a rich diversity of K+ (Hoffman et al., 1997) and Na+ (Colbert et al., 1997; Jung et al., 1997; Golding and Spruston, 1998). It is the interaction of these channels, their distribution along the dendrite as well as the recent synaptic history of the cell that will dictate whether a dendritic spike invades the soma. A number of different K+ channels have been located on the dendrites of neurons. They can be classified, broadly, into two categories: K+ channels that buffer synaptic signals (IA and ID families) (Hoffman et al., 1997; Magee and Carruth, 1999; Migliore et al., 1999) and K+ channels that boost synaptic signals (Ih ) (Magee,

1998, 1999). For now, I will focus on the first group of channels that may effectively shunt the dendritic Ca2+ spike before it reaches the soma. In hippocampal CA1 pyramidal cells, a gradient of K+ channel distribution has been established on the dendrites, with a linear increase in IA channel density with respect to distance from the soma (Hoffman et al., 1997). This relationship is subtype specific since the delayed rectifier shows no such gradient, while the calcium activated K+ channel is present almost exclusively in the soma (Poolos and Johnston, 1999). Although the distributions of the respective K+ channels have not been mapped for magnocellular neurons, we can, for the sake of the current discussion, assume they are similar to those observed in other areas of the brain. Altering the relative contributions of these conductances to the propagation of synaptic signals can have profound effects on dendritic spiking. Pharmacological inhibition of IA can lead to an increase in dendritic spiking and a summation of consecutive spikes. The net result is a nearly constant depolarization in the dendrites, which translates into a continuous mode of firing in the soma (Hoffman et al., 1997). These observations, however, may overestimate the role of IA in regulating dendritic excitability since blocking IA with 5 mM 4-aminopyridine (4-AP) will also block another K+ current known as the delay current, ID . This current activates rapidly, like an IA , but it inactivates at a much slower rate, and perhaps of greater significance for dendritic signaling, it remains inactive for tens of seconds (Storm, 1988). It also exhibits a greater sensitivity to 4-AP (micromolar doses) (Storm, 1988) and is also sensitive to α-dendrotoxin (Rudy, 1988). Selective inhibition of ID in the dendrites of hippocampal CA1 pyramidal neurons results in the unmasking of clustered doublets of spikes in response to somatic current injection (Golding et al., 1999). In magnocellular neurons, this manipulation results in the generation of large, plateau-like depolarizations in response to NMDA receptor activation (Bains and Ferguson, 1998). This likely results from a summation of Ca2+ spikes in the dendrites leading to a depolarization that is propagated to the soma (Fig. 2). These findings suggest that under most physiological conditions, dendritic K+ effectively serve to uncouple the dendrites from the soma during intense

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Fig. 2. Unmasking of dendritic spikes by K+ channel blockade. (A) Magnocellular neurons respond to NMDA agonist with a burst of action potentials that ride a depolarizing envelope. Following blockade of ID with 100 μM 4-AP, a similar concentration of agonist results in a plateau like depolarization. (B) The onset of the response is characterized by a broadening of spikes resulting in an eventual sustained depolarization. (C) A schematic of the presumptive interaction between calcium ions, potassium ions and dendritic spike propagation. (D) A schematic depicting the possible role of ANG in regulating potassium channels and thus influencing the propagation of dendritic spikes.

synaptic activity. This uncoupling is not observed for single EPSPs that are subthreshold for these currents. A larger depolarization such as a Ca2+ spike, however, would activate these currents and be shunted precluding it from reaching the soma. This offers a possible explanation for the observation of multipeaked waveforms in extracellular recordings (Mason and Leng, 1984). They represent a spike that is a compilation of both dendritic and somatic activity. Under certain conditions, however, dendritic spikes may invade the soma and lead to ‘intentional’ bursts. For example, if the dendrites are primed by a second transmitter that will depolarize the dendrites or inactivate K+ currents, this may provide a break in the shunt between dendrite and soma allowing dendritic depolarizations to reach the soma. The ID , in particular has a very slow recovery from inactiva-

tion. Therefore, synaptic signals that are temporally associated with the inactivation of ID can result in a relatively long (tens of seconds) depolarization. This would result in an uninterrupted run of action potentials in the soma that would be terminated only when a sufficient number of ID channels recover from inactivation to once again dampen dendritic excitability. Since this recovery is time and voltage dependent, this could occur in one of two ways: either there is a cessation of excitatory synaptic input to the dendrites for a sufficiently long time to allow recovery, or there is an increase in inhibitory synaptic input resulting in transient hyperpolarizations in the dendrites that relieve the inactivation. This pattern of sustained phasic activity has been observed in OT neurons (Poulain and Wakerley, 1982) and as yet a complete mechanism to explain this phenomenon

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has not been put forward (but see: Jourdain et al., 1998). Retrograde inhibition of magnocellular neurons Magnocellular neurons can also release chemicals locally to modulate their own activity. The direct effects of exogenous OT and VP on the firing of magnocellular neurons have long been known (Poulain and Wakerley, 1982; Yamashita et al., 1987; Moos and Richard, 1989). There is now ample evidence that these peptides are released from the dendrites under certain physiological conditions (Ludwig, 1995, 1998). Magnocellular neurons can also synthesize and release the labile gas, nitric oxide. Blockade of NO synthesis potentiates excitatory neurotransmitter actions in PVN (Bains and Ferguson, 1994), and augments the release of both corticotrophinreleasing factor (Rivier and Shen, 1994) and VP (Yasin et al., 1993). Furthermore, competitive inhibition of NOS by NT -nitro L-arginine methyl ester (L-NAME) potentiates the membrane depolarization of magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) in response to NMDA (Cui et al., 1994). Although incredibly different in physical properties, NO and the neuropeptides share common features when it comes to regulating neurohypophyseal function. According to recent studies in slice preparations, each seems to have a net inhibitory effect on the activity of these cells. NO does so by increasing GABAergic drive to the magnos (Bains and Ferguson, 1997) while OT (Kombian et al., 1997; Hirasawa et al., 2001) and VP (Kombian et al., 2000) act at presynaptic excitatory terminals to decrease the release of glutamate. This latter effect, although robust under slice conditions is at odds with other work demonstrating an increase in the activity of magnos in response to exogenous OT (Yamashita et al., 1987; Moos and Richard, 1989; Jourdain et al., 1998). Another question that remains unresolved concerns the mechanism responsible for the release of these messengers. OT can be released in response to back-propagating action potentials in the slice (Kombian et al., 1997). Although this may occur in vivo, it is likely not the stimulus that causes OT release from the dendrites since this release is insensitive to TTX (Ludwig, 1998). The release of both OT and VP, however, is sensitive to extracellular Ca2+

(Ludwig, 1998). Unlike classical neurotransmitters or neuropeptides, NO does not depend on the activation of Ca2+ sensitive machinery and exocytosis of vesicles. Since it is a gas, it can readily diffuse across the membrane. The Ca2+ -dependence of NO is one step earlier in the pathway — during production. The enzyme responsible for NO production, nitric oxide synthase (NOS), is a Ca2+ /calmodulin dependent protein which can be activated by Ca2+ influx that is associated with the activation of NMDA receptors (Garthwaite et al., 1988; Garthwaite, 1991). It can be activated by an increase in intracellular Ca2+ and its activity has been linked to increase in excitatory synaptic activity. Thus, a strong Ca2+ signal, such as a dendritic spike may be utilized as a local signal by a neuron to release vesicles of VP and OT or to manufacture NO which can then diffuse into the surrounding neuropil. The two signals may act in concert to decrease excessive excitation in specific compartments of the dendritic tree (Fig. 3).

Fig. 3. Dendritic spikes and release of retrograde messengers. The schematic depicts the potential role of dendritic spikes in regulating the release of the retrograde messengers. The influx of Ca2+ may be sufficient to cause the exocytotic release of OT or VP which can inhibit the release of glutamate. In addition, Ca2+ can drive the conversion of L-ARG to NO. Once synthesized, NO can diffuse across the membrane to potentiate the release of GABA on to the magnocellular neuron.

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Dendritic spikes: the darker side As mentioned above, altering the relative contributions of K+ conductances can have profound effects on dendritic spiking. In magnocellular neurons, blocking ID results in a plateau-like depolarization following activation of NMDA receptors (Bains and Ferguson, 1998). This somatic response is likely a symptom of a sustained depolarization in the dendrites of these cells. These plateau depolarizations, however, are not observed when K+ channels are fully functional, suggesting that under most conditions, K+ channels in the dendrites can effectively restrict large depolarization to very selective compartments. When ID is blocked, however, we observe a profound broadening of spikes and appearance of doublets and triplets following activation of NMDA receptors which precedes a sustained depolarization (Fig. 2). This electrophysiological response can be strongly correlated with an increase in susceptibility of magnocellular neurons to the excitotoxic actions of glutamate (Bains et al., 2001). Does this suggest that dendritic spikes may be deleterious to the cell under certain conditions? This is certainly possible during periods of intense glutamatergic excitation. The interplay between Ca2+ spikes and dendritic K+ channels also raises a second interesting possibility: the rise in Ca during a dendritic spike is sufficient to activate a K+ -channel interacting proteins that can increase the conductance and inactivation time constant of Kv 4.2 in a Ca2+ -dependent manner (An et al., 2000). These proteins may be activated in response to the surge in Ca2+ that accompanies synaptic activation, specifically, but not exclusively when the NMDA receptor is involved (An et al., 2000). This would result in an increase in the conductance of K+ channels and provide a safety-valve that would protect the cell by preventing the passage of large depolarizations from the dendrites to the soma (Golding et al., 1999) following intense synaptic activity. Long-term effects of dendritic spikes Any process that requires a rapid Ca2+ flux for activation is a potential target for dendritic Ca2+ spikes. Many of the processes implicated in altering synaptic strength, i.e. changes in expression of post-synaptic

receptors, fall into this category. In magnocellular neurons, there are additional morphological changes that occur that may be sensitive to the effects of dendritic spikes. For example, there is considerable evidence that the dendrites of OT neurons may undergo pruning during lactation (Stern and Armstrong, 1998). Might this pruning be initiated by a dramatic change in synaptic activity? The synapses that are strongly activated may be strengthened, while those that exhibit a decrease in activity are pruned off. This may be a way for the neuron to increase its signal-tonoise ratio and allow it to focus more efficiently on the task at hand. Alternatively, perhaps the pruning acts to facilitate the propagation of dendritic spikes to the soma, thereby increasing the probability that any given synaptic potential will generate a somatic action potential. Summary Although our understanding of dendritic physiology has advanced greatly in the last decade, we are only beginning to scratch the surface of what these new findings mean for the release of OT and VP. While making the link between active dendritic conductances and the output of magnocellular neurons has been hindered by our inability to record faithfully from dendrites, we have still been able to make some critical advances. Most notably, we know that dendritic spikes can be activated by intense synaptic activity, perhaps mediated by NMDA receptors. We also know that the powerful dendritic K+ channels must be suppressed in order for dendritic spikes to be faithfully translated into somatic action potentials. Furthermore, if coupled with the episodic release of glutamate dendritic spikes the inactivation of K+ currents may be the substrate for burst initiation in the soma. Finally, dendritic spikes seem like the most likely candidate mechanism utilized by magnocellular neurons to release chemical messengers from their dendrites and communicate in a retrograde fashion. It is unclear whether dendritic spikes act as local signals that are restricted to selective compartments and then quickly terminated by powerful K+ conductances, or whether they increase the probability that synaptic signals that may otherwise be lost in the background are now heard loud and clear.

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 18

Modulation of synaptic transmission by oxytocin and vasopressin in the supraoptic nucleus S.B. Kombian 1,∗ , M. Hirasawa 2, D. Mouginot 3 and Q.J. Pittman 2 2

1 Faculty of Pharmacy, Kuwait University, Kuwait Neuroscience Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada 3 CHUL Research Center, Neuroscience Unit, Laval University, Sainte-Foy, QC, Canada

Abstract: It is now generally accepted that magnocellular neurons of the supraoptic and paraventricular nuclei release the neuropeptides oxytocin and vasopressin from their dendrites. Peptide release from their axon terminals in the posterior pituitary and dendrites differ in dynamics suggesting that they may be independently regulated. The dendritic release of peptide within the supraoptic nucleus (SON) is an important part of its physiological function since the local peptides can regulate the electrical activity of magnocellular neurons (MCNs) which possess receptors for these peptides. This direct postsynaptic action would affect the output of peptide in the neurohypophysis. Another way that these peptides can regulate MCN activity would be to modulate afferent inputs unto themselves. Although the influence of afferent inputs (inhibitory and excitatory) on SON magnocellular neuron physiology has been extensively described in the last decade, a role for these locally released peptides on synaptic physiology of this nucleus has been difficult to show until recently, partly because of the difficulty of performing stable synaptic recordings from these cells in suitable preparations that permit extensive examination. We recently showed that under appropriate conditions, oxytocin acts as a retrograde transmitter in the SON. Oxytocin, released from the dendrites of MCNs, decreased evoked excitatory synaptic transmission by inhibiting glutamate release from the presynaptic terminals. It modulated voltage-dependent calcium channels, mainly N-type and to a lesser extent P/Q-type channels, located on glutamatergic terminals. Although evidence is less conclusive, it is possible that vasopressin has similar actions to reduce excitatory transmission. This synaptic depressant effect of oxytocin and/or vasopressin, released from dendrites, would ensure that MCNs regulate afferent input unto themselves using their own firing rate as a gauge. Alternatively, it may only be a subset of afferent terminals that are sensitive to these peptides, thereby providing a means for the MCNs to selectively filter their afferent inputs. Indeed its specificity is partly proven by our observation that oxytocin does not affect spontaneous glutamate release, or GABA release from inhibitory terminals (Brussaard et al., 1996). Thus, the dendrites of MCNs of the supraoptic nucleus serve a dual role as both recipients of afferent input and regulators of the magnitude of afferent input, allowing them to directly participate in the shaping of their output. This adds to a rapidly growing body of evidence in support of the concept of a two-way communication between presynaptic terminals and postsynaptic dendrites, and shows the potential of this nucleus as a model to study such form of synaptic transmission. Keywords: Supraoptic nucleus; Vasopressin; Oxytocin; Excitatory postsynaptic current; Retrograde transmission; Somatodendritic release; Voltage-activated calcium channel

∗ Correspondence to: S.B. Kombian, Faculty of Pharmacy, Kuwait University, Kuwait. Fax: +965-534-2807; E-mail: [email protected]

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Introduction A long held and classic concept of neurotransmission is that dendrites of neurons only serve as recipients of synaptic inputs where they function only to integrate and relay information. Inherent in this concept is the unidirectional transfer of information from the presynaptic neuron to the postsynaptic neuron. This concept is now giving way to a more prominent role for these anatomical structures such that, in addition to receiving, integrating and relaying afferent information, they may also regulate afferent inputs unto themselves thus, helping to model the input and hence their own output (Conde, 1992; Pucak and Grace, 1994). This evolving concept implies a bidirectional mode of communication between neurons whereby information can flow from the postsynaptic neuron back to the presynaptic neuron terminal and vice versa. Thus, anterograde and retrograde synaptic transmission can occur at the synapse concurrently. In keeping with this additional role, dendrites have recently been shown to elaborate, store and release transmitters that act as retrograde messengers to affect afferent input. Conventional transmitters such as dopamine, glutamate and GABA (Cheramy et al., 1981; Klitenick et al., 1992; Glitsch et al., 1996; Zilberter et al., 1999) as well as endogenous opioids and cannabinoids (Drake et al., 1994; Kreitzer and Regehr, 2001) have all been shown to carry out such a function in several brain regions. As well, the neuropeptides oxytocin (OXT) and vasopressin (VP) have recently been shown to engage in retrograde neurotransmission where they influence afferent input onto themselves (Kombian et al., 1997; Hirasawa et al., 2001). The SON and the paraventricular nucleus (PVN) of the hypothalamus are the main sources of circulating OXT and VP in mammals (Bargmann and Scharrer, 1951). Most of this review will deal with the SON, although most of the findings here may well apply to the PVN. The SON has been shown by neurochemical, immunocytochemical and electron microscopic studies to possess all the prerequisites for engaging in retrograde neurotransmission. Immunocytochemical studies show that the soma and dendrites of magnocellular neurons have vesicles that contain the peptides (Castel and Morris, 1988). Release profiles have also been demonstrated in the

somatodendritic regions of the MCNs using electron microscopic studies (Morris and Pow, 1993). Finally, microdialysis studies reveal the presence of extracellular OXT and VP locally in this nucleus (Di Scala-Guenot et al., 1987; Ludwig and Landgraf, 1992; Neumann et al., 1993, 1996). This local release was calcium-dependent and tetrodotoxinresistant (Di Scala-Guenot et al., 1987; Ludwig and Landgraf, 1992). All the above evidence suggest that the somatodendritic region of the magnocellular neuron is equipped with the necessary components that enable them to engage in peptide release using processes similar to those that have been described at the axon terminal (Pow and Morris, 1989). As axon collaterals are very rare in the SON (Mason et al., 1984; Pow and Morris, 1988) and peptidergic afferents from other thalamic regions are scarce (Conrad and Pfaff, 1976; Saphier and Feldman, 1987), most of the local peptide must therefore arise from somatodendritic release. Not only are these peptides present in the SON and/or PVN, their receptors have been demonstrated in these regions (Brinton et al., 1984a,b; Van Leeuwen et al., 1985) and functional roles for these local, somatodendritically released peptides have been reported (Lambert et al., 1993; Neumann et al., 1996). The current issue to resolve then is: How do these peptides produce these effects? Action of OXT/VP on magnocellular neurons OXT or VP have both been shown to cause direct effects on magnocellular neurons (MCNs; the same cells that manufacture, store and secrete them) by acting on autoreceptors located on these cells, both in vivo and in vitro (Freund-Mercier and Richard, 1981; Yamashita et al., 1987). These peptides cause an increase in intracellular calcium (Lambert et al., 1994; Dayanithi et al., 1996) that may contribute to the excitation of MCNs and trigger more local and systemic peptide release. In addition to these direct effects of the peptides on postsynaptic cell, OXT has also been reported to decrease extrinsic synaptic influences on these cells by postsynaptically modulating inhibitory GABAA receptor-mediated postsynaptic currents (Brussaard et al., 1996). In this study, OXT was found to decrease spontaneous and evoked IPSC amplitude but not the frequency of occurrence of spontaneous IPSCs. These effects of

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OXT could be mimicked by intracellular manipulation of upstream signal transduction systems such as activating or inhibiting G-proteins and mobilization of intracellular calcium in the MCN. Retrograde action of OXT/VP Although the MCNs of the hypothalamus possess several intrinsic conductances that are important to the function of SON and PVN, extrinsic synaptic inputs are very important as well (Renaud and Bourque, 1991; Armstrong, 1995). The SON receives both excitatory and inhibitory synaptic inputs from several regions including the circumventricular organs (Tribollet et al., 1985; Chaudhry et al., 1989; Leng et al., 1989; Honda et al., 1990; McKinley et al., 1992; Richard and Bourque, 1995). Both these excitatory and inhibitory inputs have recently been intensely studied as they turn to affect the excitability of the MCNs and may be involved in the generation of appropriate firing patterns to optimize peptide release in the periphery as well as locally within the nucleus. The role of these locally released peptides (OXT and VP) on these inputs is the main thrust of this review. Using an in vitro hypothalamic slice preparation containing the SON and the nystatin-patch whole-cell recording technique, we examined the role of these peptides on evoked and spontaneous synaptic events in the SON. Bath (exogenous) application of OXT, its agonist or VP all caused concentration dependent decreases in evoked glutamate-mediated excitatory postsynaptic currents (Fig. 1; Kombian et al., 1997) These synaptic depressant effects were blocked by Manning compound (MC), a non-selective OXT/VP receptor antagonist (Kruszynski et al., 1980) as well as by a selective OXT receptor antagonist. Although OXT and VP produced these effects when they were exogenously applied, a more crucial question was whether endogenous (local, dendritically released) peptides produced these same effects. To answer this question, we exploited the fact that OXT and VP, after being released, are rapidly degraded by local aminopeptidase enzymes (Burbach and Lebouille, 1983; Claybaugh and Uyehara, 1993). Therefore, we used an aminopeptidase inhibitor, amastatin and showed that this compound produced a synaptic depression similar to that produced by exogenous

peptides. This effect was specific for the aminopeptidase inhibitor as an inhibitor of endopeptidase (an enzyme present in the SON but not involved in the metabolism of OXT/VP), phosphoramidon, did not have an effect on evoked responses. Furthermore, the amastatin-induced synaptic depression was blocked by the peptide antagonist (MC) suggesting that amastatin caused synaptic depression indirectly by enhancing extracellular levels of OXT and/or VP. Thus, endogenous dendritically released peptides, when not rapidly destroyed by degradative enzymes, can decrease afferent excitation unto MCNs. The presence of these aminopeptidases in the SON suggest that there is, routinely, no tonic action of OXT and/or VP in this nucleus. This may be necessary to ensure that peptide receptors do not desensitize due to prolonged contact with agonist. The peptides may therefore produce an effect in the SON only when the enzymes are overwhelmed following massive peptide release. This may happen when MCN activity (action potential firing) is intense following large or extended peripheral demand resulting in huge/massive terminal and dendritic release. We attempted to increase intra-SON peptide level by using high-frequency afferent stimulation (HFS) that has been reported to optimize peptide release (Bicknell, 1988). HFS of afferents at 100 Hz for 1 s was followed by synaptic depression that lasted almost as long as that due to exogenous peptide application (Fig. 2A). Furthermore, the HFS induced depression was also blocked by MC indicating HFS produced a large release of peptide that overwhelmed the peptidases to produce excitatory synaptic depression (Fig. 2C). In addition to the above HFS effect, it was also possible to produce adequate release of peptide from a single MCN, enough to cause synaptic depression. This was achieved by injecting adequate positive current into MCNs to make them fire several action potentials (up to 50 Hz) for a second or more (Fig. 2B). The synaptic depression following this depolarization was also blocked by MC. All these findings indicate that peptides released from the somatodendritic region of the MCN act to decrease excitatory synaptic transmission. The consistent depression of excitatory synaptic transmission observed for OXT and VP contrasts with the effects of these peptides on GABA-mediated inhibitory transmission. While Brussaard et al., 1996

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Fig. 1. (A) Exogenous application of OXT or AVP or endogenous enhancement of neurohypophyseal hormone levels (with appropriate enzymes) leads to a reversible decrease in evoked excitatory synaptic transmission. This effect of peptide can be blocked by Manning compound (MC), a non-selective OXT/AVP receptor antagonist. (B) Histogram showing average effects of peptides and antagonism by MC. * P < 0.05.

reported that OXT depressed the amplitude of both evoked and spontaneous IPSCs recorded in the SON, we (Kombian et al., 2000) did not observe an effect of VP on evoked IPSCs recorded in this nucleus. Given that VP can bind OXT receptors (Mühlethaler and Dreifuss, 1983), it is surprising that we did not observe an effect of VP on evoked IPSCs recorded in the SON. Furthermore, Hermes et al. (2000) reported that VP enhanced spontaneous IPSCs in the PVN indirectly, possibly through the excitation of GABAergic interneurons located in the perinuclear zone of

the PVN. Since Brussaard et al., 1996 showed that the effect of OXT was postsynaptically mediated, and different MCNs may express different peptide receptors (Hurbin et al., 1998), difference in cell sampling in the two studies may be responsible for these discordant observations. Nonetheless, they suggest that spontaneous and evoked IPSCs may selectively respond to only OXT but not VP. Taken together, these indicate that peptide modulation of inhibitory transmission in the SON is complex.

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Fig. 2. Biophysical enhancement of peptide release is accompanied by a decrease in evoked EPSC. (A) High frequency stimulation (HFS) of afferents is accompanied by a decrease in evoked excitatory synaptic transmission. (B) Injection of positive current into a single MCN causes synaptic depression. (Insert is membrane response to current injection in current clamp mode.) (C) The effect of HFS-induced synaptic depression is blocked by MC pretreatment.

Locus of action of OXT/VP in the SON To alter synaptic transmission, neuromodulators can alter either: (1) the responsiveness of the postsynaptic cell to the same amount of transmitter; (2) the background or intrinsic currents in the postsynaptic

Fig. 3. OXT-induced synaptic depression is through a presynaptic action. (A,B) OXT-induced depression is accompanied by an increase in paired-pulse ratio. (C) OXT does not induced a change in the postsynaptic current over a wide range of voltage. * P < 0.05.

cell that will change its response to a transmitter; or (3) the amount of transmitter released from the afferent terminal. The former two are postsynaptic mechanisms while the latter is presynaptic. We used several biophysical tests to determine the locus of action of OXT and VP to decrease EPSC amplitude. Application of two successive synaptic stimuli (separated by 50 ms, Fig. 3A) was most often followed by a much larger EPSC on the second stimulus resulting in a ratio between the second and first response (2/1) of greater than unity (paired-pulse facilitation, PPF).

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This change has been shown to be due, predominantly, to changes in presynaptic terminal calcium dynamics (Zucker, 1989). OXT-induced EPSC reduction was most often followed by a change in paired-pulse ratio (PPR; Fig. 3B) suggesting that it may be acting on presynaptic glutamate terminals to decrease glutamate release. OXT did not appear to affect postsynaptic voltage-activated intrinsic currents as measured by the membrane response to voltage steps and ramps ranging from −120 to −30 mV (Fig. 3C). Furthermore, it did not change AMPAinduced postsynaptic currents in MCNs indicating it does not affect the postsynaptic responsiveness of non-NMDA receptors that mediate evoked EPSCs. By contrast, VP appears to affect evoked EPSCs in the SON by a more complex mechanism that may involve both pre- and post-synaptic mechanisms (unpublished observation). In some cells VP decreased the evoked EPSC and this was via a presynaptic mechanism as the depression was often, but not always, followed by a change in PPR. In other cells, VP increased the evoked EPSC and this was without changing the PPR while enhancing AMPA-induced currents, suggesting a postsynaptic action to enhance evoked EPSCs (unpublished observation). These actions of VP may reflect the presence of different types of MCNs in the SON that may respond differently to VP (Stern and Armstrong, 1995; Leng et al., 1999). These dichotomous actions of VP would enable it to selectively gate certain afferent inputs into the SON by its retrograde action. The currently available evidence is that OXT and/ or VP reduce excitatory synaptic transmission by a presynaptic action where they decrease glutamate release (Kombian et al., 1997, 2000). To determine the mechanism by which OXT/VP may decrease glutamate release, we asked if the afferent terminal calcium, a critical trigger for transmitter release (Sabatini and Regehr, 1997; Wu and Saggau, 1997) was altered by the peptides. First we demonstrated that evoked EPSCs in the SON were calcium-dependent by completely abolishing them with a non-selective calcium channel blocker, Cd2+ (Fig. 4). Next we determined the types of calcium channels that mediate EPSCs in this nucleus by applying relatively selective calcium channel blockers and tested their effects on evoked EPSCs (Hirasawa et al., 2001). We found that saturating concentrations of ω-conotoxin

GVIA, an N-type calcium channel blocker, reduced the evoked EPSC by about 60% while ω-agatoxin TK, a P/Q-type channel blocker decreased it by about 40%. When both these antagonists were present, the evoked EPSC was almost completely abolished (Fig. 4). Other types of calcium channel blockers such as nickel (T- and R-type) and nicardipine (Ltype) were without effect on evoked EPSCs. Since calcium channels are present on both afferent terminals and MCNs (Fisher and Bourque, 1996), it is possible that these channel blockers may produce their effects on the evoked EPSC by altering both the preand post-synaptically located channels. A battery of tests, including PPR, kinetics of evoked currents and responses to bath applied AMPA, all indicated that these substances had very little effect on the soma of MCNs in the voltage range tested. Thus, the results from these tests were all consistent with a presynaptic action of the blockers to decrease evoked EPSC amplitude (Hirasawa et al., 2001). Now if the peptides were interacting with calcium channels, specifically the N- and P/Q-type channels, to decrease glutamate release and hence evoked EPSC amplitude, then the presence of the channel blockers should occlude the actions of OXT/VP. We therefore performed experiments where OXT was applied in the presence of ω-conotoxin or ω-agatoxin (Fig. 5). In the presence of each of these compounds, the synaptic depressant effect of OXT was significantly attenuated (Fig. 5B). This attenuation was much greater in the presence ω-conotoxin than was the case with ωagatoxin suggesting that OXT predominantly affects N-type channels and to a lesser extent P/Q-type channels in this nucleus. These occluding effects of the Nand P/Q-type channel blockers were also observed when the level of endogenous peptide (OXT/VP) was enhanced by the aminopeptidase enzyme inhibitor amastatin (Hirasawa et al., 2001). Thus, the occlusion of OXT effects by certain calcium channel blockers strongly suggest that OXT acts at the same site as the blockers to decrease synaptic transmission. More evidence in support of this fact, though indirect, is that both OXT and these channel blockers, in parallel, also do not affect tetrodotoxin-resistant, extracellular calcium-independent spontaneous EPSCs in this SON (Fig. 6). The mechanism by which these peptides may affect the calcium channels is not known right now.

241

Fig. 4. Excitatory synaptic transmission in the SON is mediated by N- and P/Q-type calcium channels. (A) A time–effect graph of a typical cell showing the effect of two calcium channel blockers on evoked EPSC amplitude. (B) Summary histograms showing the effects of several calcium channel blockers on evoked EPSC. *,** P < 0.05.

However, as most OXT effects have been shown to be G-protein-mediated (Thibonnier et al., 1998) and calcium channels, especially the N-type channels that are affected most by OXT, are known to be modulated by G-proteins (Currie and Fox, 1997), it is plausible that OXT depresses these channels via its G-protein coupling. In addition to the modulation of calcium channels to decrease transmitter release, another possible means by which OXT may affect transmitter release is through processes downstream of calcium entry. These downstream processes may control both evoked and spontaneous EPSCs, the latter in the SON being independent of action potentials and

extracellular calcium (Kabashima et al., 1997; Kombian et al., 2000). The modulation of any processes downstream of calcium entry by OXT is, however, unlikely as OXT does not affect the frequency or amplitude of spontaneous EPSCs (Fig. 6). This lack of effect of OXT on spontaneous EPSC contrasts with its inhibition of evoked EPSCs in the SON and points to the fact that spontaneous and evoked EPSCs may employ different processes to produce transmitter release. The lack of effect of OXT on spontaneous EPSCs frequency and amplitude also contrast with its reported depression of the amplitude of spontaneous IPSCs (Brussaard et al., 1996). These diverse actions of OXT on synaptic responses

242

Fig. 5. ω-CTx and ω-Aga actions occlude OXT effects on excitatory synaptic transmission. (A) Time–effect plot in a typical cell showing the effect of OXT in the presence of calcium channel blockers. (B) Summary histogram showing the effect of OXT in the presence of maximal concentrations of ω-CTx and ω-Aga. * P < 0.05.

suggests that spontaneous EPSCs and IPSCs that are action potential independent (asynchronous) and evoked EPSCs and IPSCs that are produced by the invasion of terminals by action potentials leading to synchronous release of transmitters, may perform different functions in the SON and may be differentially modulated.

Fig. 6. Calcium channel antagonists and OXT have no effect on miniature EPSCs in the SON. (A) Sample traces showing the effect of OXT and ω-CTx on evoked EPSC. (B) In these same cells OXT and ω-CTx have no effect on the amplitude or frequency of miniature EPSCs. (C) Summary histogram showing the lack of effect of OXT or ω-CTx on miniature EPSCs.

Conclusion The dendrites and soma of MCNs of the SON are endowed with peptide containing vesicles and calcium channels (Fisher and Bourque, 1996), two important cellular components that are necessary for the release of peptide into the extracellular space. Release

profiles in the form of membrane plebs have been observed in the somatodendrites of MCNs and peptides have routinely been recovered from SON using microdialysis probes. Since axon collaterals and peptidergic afferents to SON are sparse, the main source of OXT and VP in the SON is thus, through so-

243

Fig. 7. A schematic showing possible mechanisms by which OXT/AVP modulate synaptic transmission in the supraoptic nucleus. Following a burst of APs the MCN releases peptide from the soma/dendrites which cross the synaptic cleft to activate receptors located on excitatory but not inhibitory terminals. This leads to an interaction with N- and P/Q-type calcium channels causing a decrease in evoked, but not spontaneous glutamate release. Dendritically released peptides can also activate autoreceptors located on the MCN leading to a decrease in the response of the MCN to GABA (Brussard et al., 1995).

matodendritic release. Currently available evidence indicate that, at the cellular level, the excitability of MCNs is controlled both by intrinsic conductances as well as afferent excitation/inhibition. During increased peripheral demand for peptide, both afferent excitation and intrinsic conductances may cooperate to cause MCNs to switch their action potential firing pattern from routine baseline patterns and rates to those that enhance peptide release in the periphery. This switch in pattern to optimize peripheral peptide release causes MCNs to fire more action potentials. These potentials that can be initiated in the dendrites (Bains and Ferguson, 1999) or the axon hillock (that can back-propagate to the dendrites, (Stuart et al., 1997)) trigger OXT/VP release from the dendritic stores (Fig. 7). These intra-SON peptides, then act on

receptors located on excitatory, but not inhibitory afferent terminals, to inhibit evoked glutamate release. This is achieved mainly by activation of intracellular processes that affect the function of high voltage activated calcium channels, mainly the N-type and to a lesser extent the P/Q-type. This retrograde excitatory synaptic depressant effect of the peptides will ensure that excessive afferent excitation is curtailed using the MCN dendrite as a sensor. The peptides also act directly on autoreceptors located on the MCNs to increase intracellular calcium mobilization (Lambert et al., 1994; Dayanithi et al., 1997) as well as to modulate the GABAA receptor–chloride channel complex leading to a decrease in the amplitude of both evoked and spontaneous IPSCs. Both the latter two postsynaptic effects of the peptides are func-

244

tionally excitatory and will provide a counterbalance allowing the dendrite to fine tune excitation and inhibition to arrive at an appropriate level and pattern of activity. Thus, the dendrite of the MCN of the SON, in addition to its routine, classic function of reception, integration and relay of afferent information, may also play a novel and unique function of sensing excitatory and inhibitory inputs unto themselves as well as producing retrograde/auto-transmitters that help shape these inputs and hence determine their output of peptide in the periphery. This evolving concept of bidirectional communication between neurons is not unique to the SON as numerous brain regions and synapses in the CNS are now known to engage in this type of communication (Cheramy et al., 1981; Glitsch et al., 1996; Zilberter et al., 1999; Kreitzer and Regehr, 2001). However, the MCNs of the SON have the unique cytoarchitecture that would enable thorough examination of the functional roles of retrograde neurotransmission in the neurohypophysis. Numerous aspects of the processes leading up to dendritic release and the role of these peptides in modulating afferent inputs are still unknown. First of all, we know very little about how dendritic release is organized and controlled. For example it is known that OXT/VP are synthesized as precursor molecules which then undergo a maturational process en route to the posterior pituitary (Dreifuss, 1975; Brownstein et al., 1980; Robinson et al., 1989; Roberts et al., 1991). Is the same process functional in the dendrites? Are the carrier neurophysin molecules released on a one-to-one basis with the neuropeptides as is seen at the axon terminals (Brownstein et al., 1980). We know now that the distribution of calcium channel subtypes on the axon terminals and the soma are different (Fisher and Bourque, 1996). It would be interesting to know if the roles of the various subtypes in dendritic release are similar to those that have been elegantly worked out at the axon terminal (Lemos et al., 1994; Wang et al., 1997, 1999). There is evidence that the release from these two sites can be differentially controlled, (Neumann et al., 1993, 1996), suggesting that there are unique modulatory sites at the dendritic release site. Indeed it would be interesting to know if the synaptic release proteins participating in peptide release at the dendritic and axonal endings are similar. With the availability of

specific blocking molecules, genetically modified animals and perhaps more in depth anatomical studies, these questions can be answered. Abbreviations AMPA EPSC GABA HFS IPSC MC MCN NMDA OXT PPF PPR PVN SON VP

α-Amino-3-hydroxy-5-methyl isoxazolepropionic acid excitatory postsynaptic current γ-aminobutyric acid high-frequency stimulation inhibitory postsynaptic current Manning compound magnocellular nucleus N-methyl-D-aspartate oxytocin paired-pulse facilitation paired-pulse ratio paraventricular nucleus supraoptic nucleus vasopressin

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 19

The active role of dendrites in the regulation of magnocellular neurosecretory cell behavior Mike Ludwig 1,∗ , Nancy Sabatier 1 , Govindan Dayanithi 2, John A. Russell 1 and Gareth Leng 1 1

Department of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, UK 2 Department of Neurobiology, U432, INSERM, University of Montpellier II, Place Eugene Bataillon, F-34094 Montpellier Cedex 5, France

Abstract: The interactions of the dendritically released neuropeptides vasopressin and oxytocin with co-released neuroactive substances such as opioids and nitric oxide are reviewed. Endogenous opioids regulate magnocellular neurons at the level of the supraoptic nucleus and the relationship of dendritically released peptides and co-released opioids seems to be dependent on the stimulus given and the physiological state of the animal. Nitric oxide has a prominent inhibitory action on supraoptic neurons and these actions are predominantly mediated indirectly by GABA inputs. The role of these co-released neuroactive substances in differentially regulated release of neuropeptides from dendrites versus distant axon terminals has to be determined in more detail. A picture emerges in which release of vasopressin and oxytocin from different anatomical compartments of a single neuron may arise from different intracellular secretory pools and their preparation before release. Keywords: Supraoptic nucleus; Hypothalamus; Vasopressin; Oxytocin; Nitric oxide; Opioid; Dendritic release

Introduction Classically, neurotransmission is achieved by release of transmitters from axon terminals to activate postsynaptic receptors. However, it has recently become clear that neurotransmitters are also released from dendrites to produce a local modulation of synaptic transmission. Dendritic release of neurotransmitters, such as glutamate (Cox et al., 1998), GABA (Zilberter et al., 1999), dopamine (Geffen et al., 1976), serotonin (Hery and Ternaux, 1981) and dynorphin (Simmons et al., 1995), has been shown for several neuronal populations. ∗ Correspondence

to: M. Ludwig, Department of Biomedical Sciences, University of Edinburgh Medical School, George Square, Edinburgh EH8 9XD, UK. Tel.: +44131-650-3275; Fax: +44-131-650-3711; E-mail: mike. [email protected]

The cell bodies and dendrites of the magnocellular vasopressin and oxytocin neurons are located within the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN). Dendritic release of their neuropeptides has been demonstrated in vitro and in vivo (for review see Landgraf, 1995; Ludwig, 1998; Morris et al., 2000), resulting in pre- and postsynaptic regulation of the electrical activity (Brussaard et al., 1996; Kombian et al., 1997; Hermes et al., 2000), a cell-type specific receptor-mediated rise in intracellular Ca2+ (Dayanithi et al., 2000) and reorganization of the ultrastructure of the nuclei (Theodosis et al., 1986a). Several recent studies indicate striking spatial and temporal differences between the release profiles from axon terminals and dendrites (Moos et al., 1989; Wotjak et al., 1998; Engelmann et al., 1999). However, the mechanisms of this differentially regulated release from dendrites are not well understood. This dendritic release seems

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to be of great importance in the regulation of the milk ejection reflex, since it has been shown that oxytocin release from dendrites occurs before the onset of milk ejection bursts (Moos et al., 1989). The rate of neurohypophysial peptide secretion is not only regulated by locally released vasopressin and oxytocin but also by co-localized and co-released neuroactive substances such as opioids and nitric oxide (NO). Regulation of activity by dendritic release of co-peptides has been observed for several classes of neuron, such as serotonin neurons within the raphe nuclei (Hery et al., 1982) and dopamine neurons of the substantia nigra (Cheramy et al., 1981). In the latter case, modulation of the activity of these neurons seems to depend on co-released factors since acetylcholinesterase, which is also secreted by nigrostriatal cells (Llinas and Greenfield, 1987), hyperpolarizes these cells by a mechanism independent of acetylcholine hydrolysis (Greenfield et al., 1988). Role of co-released opioids Oxytocin cells synthesize enkephalins and both vasopressin and oxytocin neurons synthesize the κ-opioid agonist, dynorphin. (Watson et al., 1982). Dynorphin is found co-packaged with vasopressin in neurosecretory vesicles in the neural lobe (Whitnall et al., 1983). κ-Receptors are expressed in both oxytocin and vasopressin cell bodies. The receptor at which dynorphin is active has been visualized in the membrane of vasopressinergic neurosecretory vesicles in the cell bodies and axon terminals (Shuster et al., 1999, 2000). Thus it seems probable that dynorphin is co-released upon dendritic exocytosis of vasopressin into the SON. We used direct osmotic stimulation via microdialysis to elicit intranuclear and systemic oxytocin release in the presence and absence of the μ-agonist morphine, or the general opioid antagonist, naloxone (Munro et al., 1994). We showed that dendritic oxytocin release in response to direct osmotic stimulation is unaffected by intravenous morphine, a μ-opioid agonist, or the opioid receptor antagonist naloxone, while oxytocin release into the systemic circulation is inhibited by the former and potentiated by the latter. The failure of the opioid antagonist to modify the intranuclear release of oxytocin in re-

sponse to direct osmotic stimulation indicates that dendritic oxytocin release is not markedly restrained by any co-released endogenous opioid peptide. However, the presence of an endogenous opioid inhibition of dendritic release has been shown in isolated SON and PVN in vitro (Ingram et al., 1996). Furthermore, local application of naloxone has been shown to increase oxytocin release measured in microdialysis samples from the SON of late pregnant rats (Douglas et al., 1995). Thus, the relationship between dendritically released peptides and co-released opioids seems to be dependent on the stimulus given and the physiological state of the animal. Oxytocin release from dendrites within the SON appears to interact with at least some of the effects of opioids on oxytocin neurons. Upon chronic administration of morphine, oxytocin neurons become both tolerant to, and dependent upon, this opiate alkaloid (Russell et al., 1995). Tolerance is seen as a reduction in the effectiveness of morphine inhibition over time, while dependence is manifested as increased activity following withdrawal of morphine. Intranuclear oxytocin release is increased following morphine withdrawal induced by intravenous administration of naloxone to morphine-dependent rats (Russell et al., 1992) and i.c.v. administration of an oxytocin antagonist reduces the magnitude of this excitation (Brown et al., 1997), indicating that dendritic release of oxytocin may contribute to the magnitude of the withdrawal excitation of oxytocin neurons. To investigate the potential importance of this intranuclear release of oxytocin in the generation of the withdrawal excitation of oxytocin neurons, we studied whether the mechanisms which generate morphine dependence reside within the oxytocin neurons themselves or whether these neurons simply follow excitation of their afferent inputs upon morphine withdrawal. We have shown that withdrawal excitation of oxytocin cells can be evoked by administration of naloxone directly into the SON of morphine-dependent rats (Ludwig et al., 1997). Thus, at least some of the mechanisms which generate morphine withdrawal excitation are localized to the SON. However, these effects may be postsynaptic upon the oxytocin neurons themselves, or pre-synaptic upon the axon terminals of their afferent inputs. Oxytocin neurons receive a prominent noradrenergic projection from the A2 cell group in

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the nucleus of the solitary tract (NTS) (Raby and Renaud, 1989) as well as other non-noradrenergic inputs from forebrain regions (Hatton, 1990; Renaud and Bourque, 1991). However, very few of the cells that project to the SON from forebrain regions are activated by morphine withdrawal, while approximately 10% of NTS neurons are activated at this time (Murphy et al., 1997). We have shown that acute pharmacological blockade of noradrenergic transmission can reduce the magnitude of withdrawal excitation in oxytocin neurons (Brown et al., 1998a). Superficially, this provides powerful evidence that a noradrenergic drive on oxytocin neurons is essential to evoke morphine withdrawal excitation in these neurons. However, neurotoxic destruction of noradrenergic systems does not alter the magnitude of the withdrawal excitation in oxytocin neurons (Brown et al., 1998a). The most probable explanation of these apparently inconsistent results is that oxytocin neurons develop morphine dependence separately from their afferent inputs but that full expression of morphine withdrawal excitation requires excitatory synaptic inputs to maintain the underlying excitability of the oxytocin neurons. Thus, it appears that the mechanisms which generate morphine dependence in oxytocin neurons reside in the SON and that dendritic release of oxytocin may contribute to the full expression of morphine withdrawal excitation by oxytocin neurons. After demonstrating that i.v. injection of the μagonist morphine and the κ-agonist U50488 reduced the firing rate of identified oxytocin neurons and that this inhibition was completely reversed by retrodialysis of the opioid antagonist naloxone, indicating that local opioids could inhibit within the SON (Ludwig et al., 1997; Fig. 1), we focused our research on opioid effects on vasopressin cells. The dendritic neurosecretory vesicles of vasopressin neurons contain the same opioid peptide, dynorphin, as found in their neurohypophysial vesicles. Dendritically released vasopressin is predominantly inhibitory to vasopressin neurons (Ludwig and Leng, 1997; Pittman et al., 2000). We studied the contribution which the co-localized dynorphin may make to the autoregulation of vasopressin neuronal activity by dendritic release of vasopressin (Fig. 1). Retrodialysis of U50488 inhibits vasopressin neurons and retrodialysis of a κ-receptor antagonist into the SON

increases the activity of phasic vasopressin neurons by increasing the activity quotient and the intra-burst firing rate (Brown et al., 1998b). This, coupled with the recent observation that κ-receptors are localized to the membranes of vasopressin-containing vesicles within the SON (Shuster et al., 1999, 2000), indicates that dendritically released dynorphin may be a potent regulator of activity in vasopressin neurons. However, the pre- and post-synaptic actions of vasopressin seem to be independent of co-released endogenous opioid peptides, since vasopressin-induced inhibition of vasopressin neurons is not reversed by the opioid antagonist, naloxone, even at high concentrations (Ludwig and Leng, 1998). Thus, it is probable that dendritically released vasopressin and dynorphin act in parallel to curtail the activity of vasopressin neurons. κ-Opioid activation reduces the amplitude of the depolarizing after-potential in SON neurons and this may underlie the reduction in burst duration of vasopressin cells by dendritic release of an endogenous κ-agonist (Brown et al., 1999). In conclusion, it appears that the actions of dendritically released vasopressin do not rely upon co-released endogenous opioid peptides. The role of nitric oxide There is growing evidence that nitric oxide (NO) also functions as a local modulator of magnocellular neuronal activity. Neuronal NO synthase (NOS) is expressed densely in the SON and PVN (Arevalo et al., 1992), where it is colocalized with oxytocin- and vasopressin-synthesizing neurons, and its expression is functionally regulated (Sagar and Ferriero, 1987; Bredt et al., 1990; Pasqualotto et al., 1991; Vincent and Kimura, 1992). In the SON and PVN, the expression of neuronal NOS mRNA is increased in response to osmotic stimuli (Kadowaki et al., 1994; Villar et al., 1994; Ueta et al., 1995) and hypovolemia (Ueta et al., 1998), and staining for NADPH-diaphorase changes during late pregnancy and parturition (Okere and Higuchi, 1996). In rats and in humans, NO inhibits oxytocin secretion from the posterior pituitary (Chiodera et al., 1994), however direct effects of NO on dendritic release have not been studied yet. We have shown that systemic administration of NOS inhibitors led to a facilitation of oxytocin release evoked by hyperosmotic stimu-

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Fig. 1. Local opioid inhibition of SON neurons. (A) Intravenous (i.v.) injection of morphine (MOR, 1 mg/kg) markedly reduced the spontaneous activity of an identified (excited by CCK, 20 μg/kg, i.v.) oxytocin neuron. Retrodialysis of naloxone (NLX, 2 mM) onto the supraoptic nucleus restored the spontaneous activity of this neuron and reduced the inhibitory effect of subsequent morphine injection. (B) Microdialysis administration (retrodialysis) of the κ-opioid agonist U50488 (1 mM) completely inhibits the activity of the recorded vasopressin neuron. Retrodialysis of naloxone (2 mM) onto the SON restores the spontaneous activity, indicating that the opioid inhibition occurs within the SON. (C) Retrodialysis of the κ-antagonist nor-binaltorphimine (BNI, 200 μg/ml) onto the SON increases the burst duration indicating tonic effects on vasopressin cells by endogenous κ-opioids. Adapted from Ludwig et al., 1997; Brown et al., 1998b.

lation. Direct application of the NO donor sodium nitroprusside to the supraoptic nucleus by in vivo microdialysis inhibited the electrical activity of both oxytocin neurons and vasopressin neurons, whereas direct application of an NOS inhibitor increased electrical activity, indicating that endogenous NO acts within the supraoptic nucleus to inhibit neuronal activity (Fig. 2). However, during late pregnancy, the influence of endogenous NO is dramatically downregulated, reflected by a reduced expression of nNOS mRNA in these neurons and a loss of efficacy of NOS inhibitors on stimulus-evoked oxytocin release. This down-regulation may cause the oxytocin system to become more excitable at term, resulting in

the capacity for greater release of oxytocin during parturition (Srisawat et al., 2000). Electrophysiological studies in vitro (Liu et al., 1997; Ozaki et al., 2000) and as mentioned above in vivo (Srisawat et al., 2000) have shown that the NO donor sodium nitroprusside (SNP) and the NO precursor L-arginine inhibit SON neurons, whereas the NOS inhibitor L-NAME and the NO scavenger hemoglobin excite them. One mechanism by which NO influences signaling in the central nervous system is by modulating neurotransmitter release, including, in particular, the inhibitory neurotransmitter — GABA (Segovia et al., 1994; Ohkuma et al., 1996). GABAergic synapses comprise about 40% of

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Fig. 2. In vivo and in vitro recordings indicating inhibitory action of nitric oxide (NO) on vasopressin and oxytocin neurons via their GABAergic inputs. (A) Inhibition of a continuously firing putative vasopressin neuron during microdialysis administration (retrodialysis) of the NO precursor L-arginine (100 mM); (B) Retrodialysis of the NO synthase inhibitor, L-NNA (10 mM) induced an increase in the firing rate of this vasopressin neuron (inhibited by CCK) in vivo; (C) the NO donor SNP (50 mM) retrodialysed onto the SON also inhibits the activity of the identified (CCK, 20 μg/kg, i.v.) oxytocin neuron and (D) pre-treatment by retrodialysis of the GABAA antagonist bicuculline (2 mM) results in a reduced inhibitory effect of SNP. GABAA miniature inhibitory postsynaptic currents (mIPSCs) in a vasopressin cell in vitro before (E) and during (F) bath application of SNP (100 μM). Data modified from Srisawat et al., 2000; Stern and Ludwig, 2001.

all synaptic contacts in the SON (Theodosis et al., 1986b) and GABA plays a key role in controlling the firing activity of both oxytocin and vasopressin neurons (Wuarin and Dudek, 1993; Moos, 1995). The NO and GABA systems seem to interact strongly in the PVN (Krukoff, 1999). For instance, perfusion of the PVN with NO-containing medium by microdialysis or microinjection of SNP increases local GABA release (Horn et al., 1994). NO has also been shown to inhibit renal sympathetic outflow by modulating local GABA activity within the PVN (Zhang and Patel, 1998). Similarly, NMDA receptor activation in the PVN increased GABAergic activity on magnocellular neuroendocrine neurons, an effect mediated by local NO production (Bains and Ferguson, 1997). To study further NO–GABA interactions, and their

physiological relevance in modulating the activity of SON magnocellular neurons, we combined in vitro and in vivo electrophysiological studies on identified magnocellular neurons. The results suggest that nitric oxide inhibition of neuronal excitability in oxytocin and vasopressin neurons involves preand post-synaptic potentiation of GABAergic synaptic activity in the SON (Stern and Ludwig, 2001; Fig. 2). The mechanisms of opioid dependence and withdrawal excitation in magnocellular neurons are not well understood. One possible adaptation involves attenuation of inhibitory mechanisms: NO generation is a candidate. To investigate the involvement of endogenous NO in morphine dependence of SON oxytocin neurons we completed a series of blood

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sampling and electrophysiology experiments. The data indicate that NO mechanisms restraining oxytocin neurons are unchanged in morphine dependence. During withdrawal excitation, endogenous NO restrains oxytocin neurons; however, the restraint may be predominantly post-synaptic rather than on GABA terminals, as in naïve rats. Although there is a GABA inhibition of oxytocin neurons during withdrawal excitation, this loss of NO action on GABA terminals may contribute to excitation (Ludwig et al., 2001). Vasopressin and oxytocin It is now well accepted that both peptides are released from the dendrites of the magnocellular neurons. However, depending upon the stimulus given, neurons within the magnocellular nuclei and other brain areas are apparently capable of regulating their local dendritic and distant axonal terminal release of

neuroactive substances in either a co-ordinated or independent manner. Differential release of neurotransmitters from different compartments of a single neuron requires subtle regulatory mechanisms and the contributions of nitric oxide and endogenous opioids to the regulation of these mechanisms is currently not well understood. Part of these differences may be explained by local feedback mechanisms induced by the peptides themselves. Autoregulation of magnocellular neurons and a number of post- and, recently, pre-synaptic effects have been reported (Fig. 3). For dendritically released oxytocin modulation of glutamatergic inputs seems to be pre-synaptic, acting to suppress glutamate-mediated EPSPs on oxytocin neurons. Interestingly, vasopressin acts mainly post-synaptically, enhancing EPSPs in oxytocin neurons and depressing them in vasopressin cells (Kombian et al., 1997; Pittman et al., 2000). The in vitro effect of vasopressin is consistent with recent in vivo reports showing

Fig. 3. Dendritic release of neuroactive substances; oxytocin, opioids and nitric oxide. (1) Dendritic oxytocin release results, at least in part, from a receptor-mediated (OTR), G protein-coupled positive autoregulatory action of oxytocin. (2) Dendritic exocytosis of oxytocin contributes to the coordination of the electrical activity within the four nuclei during birth and lactation. (3) Oxytocin also modulates pre- and post-synaptically afferent GABAergic and glutamatergic inputs. (4) Furthermore, oxytocin induces changes in the intracellular calcium concentration resulting in vesicle priming. (5) In combination with other factors, such as steroids, oxytocin induces morphological changes (e.g. retraction of glial cells between the neurons, increases dendrodendritic interactions) within the nuclei which favors synchronization of oxytocin neurons during reproduction. Pre- and post-synaptic regulation of the electrical activity of the magnocellular neurons also occurs through dendritically released endogenous opioids (enkephalin, dynorphin) co-packaged with oxytocin, and which bind to specific μ- and κ-receptors (OR), respectively. Finally, nitric oxide released from dendrites/cell bodies acts as a local autoregulatory signal.

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that vasopressin predominantly suppresses the firing rate of vasopressin neurons (Ludwig and Leng, 1997). Interestingly, Gouzenes et al. have shown that vasopressin inhibits or excites vasopressin neurons depending on their ongoing electrical activity, so that fast-firing neurons are slowed, and slow-firing neurons are excited (Gouzenes et al., 1998). This may be due to activation of different intracellular secondmessenger pathways, since intracellular mechanisms activated by binding of vasopressin to its receptors increases intracellular Ca2+ concentrations via both adenylate cyclase- and phospholipase C-coupled intracellular transduction pathways (Sabatier et al., 1998). An alternative explanation for the excitatory effects of vasopressin could be in the modulation of different synaptic inputs. We have recently shown that dendritically released vasopressin facilitates noradrenaline release from terminals in the supraoptic nucleus (Ludwig et al., 2000). Alternatively, differences in peptide release from dendrites versus axon terminals may be due to: (1) independent peptide biosynthesis and storage of neurosecretory granules in dendrites; (2) site-specific synaptic contacts on dendrites or soma; (3) compartmentalization of receptor populations and/or second-messenger systems between dendrites and soma; (4) mediation of changes in intracellular Ca2+ via second-messenger systems in dendrites and by voltage-dependent channels in the soma; and (5) dissociation of electrical activity in axons and dendrites. We have focused our research on the possibility that dendritic release is dissociated from the electrical activity of magnocellular neurons and mediated by large changes in intracellular Ca2+ . Antidromic activation of magnocellular SON neurons has shown that even intense spike activity does not lead to vasopressin and oxytocin release from dendrites. Therefore we are currently focusing on the effects of intracellular calcium changes on dendritic peptide release. Researchers in Montpellier have shown that application of oxytocin or vasopressin to isolated magnocellular neurons produces a cell-type specific rise in [Ca2+ ]i . Oxytocin mobilizes Ca2+ mainly from thapsigargin-sensitive intracellular Ca2+ stores, whereas the response induced by vasopressin requires an influx of external Ca2+ as well as mobilization of thapsigargin-sensitive intracellular Ca2+ stores (for review see Dayanithi et al., 2000). The

consequences of peptide-induced changes in [Ca2+ ]i in magnocellular neurons are not known, but may have rapid effects on both systemic peptide secretion (by altering electrical activity) and dendritic peptide secretion (Moos et al., 1984; Wotjak et al., 1994). A rise in [Ca2+ ]i may also have sustained effects, e.g. inducing the accumulation of peptidecontaining vesicles beneath the plasma membrane and subsequently priming release (Ashery et al., 2000). Priming is an essential and rate-limiting step in secretion from neurons and neuroendocrine cells (Sudhof, 1995; Brose et al., 2000) and may be required before action potentials that propagate back into the dendrites can trigger substantial peptide release. Ongoing experiments are investigating this hypothesis. Abbreviations GABA NO NOS NTS PVN SON

γ-aminobutyric acid nitric oxide nitric oxide synthase nucleus of the solitary tract paraventricular nucleus supraoptic nucleus

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vasopressin-sensitive neurones of the rat supraoptic nucleus. J. Physiol., 513: 699–710. Sagar, S.M. and Ferriero, D.M. (1987) NADPH diaphorase activity in the posterior pituitary: relation to neuronal function. Brain Res., 400: 348–352. Segovia, G., Porras, A. and Mora, F. (1994) Effects of a nitric oxide donor on glutamate and GABA release in striatum and hippocampus of the conscious rat. NeuroReport, 5: 1937– 1940. Shuster, S.J., Riedl, M., Li, X., Vulchanova, L. and Elde, R. (1999) Stimulus-dependent translocation of k opioid receptors to the plasma membrane. J. Neurosci., 19: 2658–2664. Shuster, S.J., Riedl, M., Li, X., Vulchanova, L. and Elde, R. (2000) The kappa opioid receptor and dynorphin co-localize in vasopressin magnocellular neurosecretory neurons in guineapig hypothalamus. Neuroscience, 96: 373–383. Simmons, M.L., Terman, G.W., Gibbs, S.M. and Chavkin, C. (1995) L-type calcium channels mediate dynorphin neuropeptide release from dendrites but not axons of hippocampal granule cells. Neuron, 14: 1265–1272. Srisawat, R., Ludwig, M., Bull, P.M., Douglas, A.J., Russell, J.A. and Leng, G. (2000) Nitric oxide and the oxytocin system in pregnancy. J. Neurosci., 20: 6721–6727. Stern, J.E. and Ludwig, M. (2001) NO inhibits supraoptic oxytocin and vasopressin neurons via activation of GABAergic synaptic inputs. Am. J. Physiol., 280: R1815–R1822. Sudhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature, 375: 645–653. Theodosis, D.T., Montagnese, C., Rodriguez, F., Vincent, J.D. and Poulain, D.A. (1986a) Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system. Nature, 322: 738–740. Theodosis, D.T., Paut, L. and Tappaz, M.L. (1986b) Immunocytochemical analysis of the GABAergic innervation of oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus. Neuroscience, 19: 207–222. Ueta, Y., Levy, A., Chowdrey, H.S. and Lightman, S.L. (1995) Water deprivation in the rat induces nitric oxide synthase (NOS) gene expression in the hypothalamic paraventricular and supraoptic nuclei. Neurosci. Res., 23: 317–319. Ueta, Y., Levy, A., Lightman, S.L., Hara, Y., Serino, R., Nomura, M., Shibuya, I., Hattori, Y. and Yamashita, H. (1998) Hypovolemia upregulates the expression of neuronal nitric oxide synthase gene in the paraventricular and supraoptic nuclei of rats. Brain Res., 790: 25–32. Villar, M.J., Ceccatelli, S., Ronnqvist, M. and Hokfelt, T. (1994) Nitric oxide synthase increases in hypothalamic magnocellular neurons after salt loading in the rat. An immunohistochemical and in situ hybridization study. Brain Res., 644: 273–281. Vincent, S.R. and Kimura, H. (1992) Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience, 46: 755– 784. Watson, S.J., Akil, H., Fischli, W., Goldstein, A., Zimmerman, E., Nilaver, G. and van Wimersma Greidanus, T.B. (1982) Dynorphin and vasopressin: common localization in magnocellular neurons. Science, 216: 85–87. Whitnall, M.H., Gainer, H., Cox, B.M. and Molineaux, C.J.

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 20

Cardiovascular regulation of supraoptic vasopressin neurons J. Thomas Cunningham ∗ , Stacy B. Bruno, Regina R. Grindstaff, Ryan J. Grindstaff, Karen H.R. Higgs, Danilo Mazzella and Margaret J. Sullivan Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri – Columbia, Research Park, Columbia, MO 65211, USA

Abstract: A number of laboratories have identified several key areas in the central nervous system that relay information from arterial baroreceptors to the supraoptic nucleus. Two of these regions are the diagonal band of Broca and the perinuclear zone of the supraoptic nucleus. Recent findings suggest that the inhibition of vasopressin neurons in the SON by caval–atrial stretch may also involve the perinuclear zone. Using Fos immunocytochemistry in combination with volume expansion in unanesthetized rats, we observed that volume expansion activates a number of regions in the CNS including the area postrema, the nucleus of the solitary tract, the caudal ventrolateral medulla, the paraventricular nucleus, the perinuclear zone and oxytocin neurons in the supraoptic nucleus. Further experiments using pericardial catheters demonstrate that the activation of the nucleus of the solitary tract, the ventrolateral medulla, the paraventricular nucleus and the perinuclear zone by volume expansion is dependent on cardiac afferents. However, the Fos in the area postrema and oxytocin neurons of the supraoptic nucleus is not affected by removal of cardiac afferents. Similarly, electrophysiological experiments show that stimulation of cardiac receptors in the caval–atrial junction inhibits supraoptic vasopressin neurons but does not significantly affect the activity of supraoptic oxytocin neurons. These experiments suggest that while the inhibition of supraoptic vasopressin neurons during volume expansion is mediated by cardiac afferents, the activation of supraoptic oxytocin is independent of cardiac afferents and may be mediated by other visceral afferents or humoral factors. Additional electrophysiological experiments examined the importance of the perinuclear zone in cardiopulmonary regulation of vasopressin. Excitotoxin lesions of the perinuclear zone region block the inhibitory effects of caval–atrial stretch on supraoptic vasopressin neurons. This lesion has previously been shown to block the inhibitory effects of arterial baroreceptor stimulation on supraoptic vasopressin neurons. Thus, the neural pathways that inhibit vasopressin release in response to an increase in blood pressure and an increase in blood volume may overlap at the perinuclear zone of the supraoptic nucleus. Also while the inhibition of supraoptic vasopressin neurons during volume expansion is mediated by cardiac afferents, the activation of supraoptic oxytocin neurons is independent of cardiac afferents and may be mediated by other visceral afferents or hormonal factors. Keywords: Baroreceptors; Cardiopulmonary receptors; Diagonal band of Broca; Oxytocin; Volume expansion; Congestive heart failure

∗ Correspondence to: J.T. Cunningham, Dalton Cardiovascular Research Center, Research Park, Columbia, MO 65211, USA. Tel.: +1-573-884-7229; Fax: +1-573-884-4232; E-mail: [email protected]

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Introduction The supraoptic nuclei (SON) in the rat are located on the ventral surface of the hypothalamus lateral and dorsal to the optic chiasm and optic tracts. The magnocellular neurons located in the SON project to the posterior pituitary along with magnocellular neurons from the paraventricular (PVN) nuclei of the hypothalamus and accessory regions in the hypothalamus (Armstrong, 1985). These neurons release peptide hormones into the systemic circulation. The two primary secretory products of these cells are vasopressin and oxytocin. Vasopressin, one of the major hormonal factors involved in body fluid homeostasis, is a potent vasoconstrictor, increases water reabsorption by the collecting duct of the kidneys and alters arterial baroreceptor and cardiopulmonary reflex control of the sympathetic nervous system (Cunningham and Sawchenko, 1991). Oxytocin stimulates lactation and parturition (Cunningham and Sawchenko, 1991) and has been suggested to have natriuretic effects. Previous studies have demonstrated that vasopressin and oxytocin are synthesized (Xi et al., 1999) and released by separate populations of neurosecretory cells (Cunningham and Sawchenko, 1991; Renaud and Bourque, 1991). Vasopressin neurons can be distinguished from oxytocin neurosecretory neurons based on their spontaneous activity and their responsiveness to various peripheral stimuli (Bourque and Renaud, 1990; Renaud and Bourque, 1991). For example, vasopressin neurons in the SON have either phasic or continuous patterns of spontaneous activity (Bourque and Renaud, 1990; Renaud and Bourque, 1991) and selectively inhibited by baroreceptor afferent stimulation, and are activated by chemoreceptor afferent input (Harris, 1979). Oxytocin neurons, on the other hand, are selectively activated by gastric distension and peripherally injected CCK (Renaud et al., 1987; Renaud and Bourque, 1991). Neuroanatomical experiments have detailed the afferent input to the SON and the magnocellular PVN. Regions projecting to the hypothalamic magnocellular neurosecretory cells include the caudal ventrolateral medulla (CVL), the nucleus of the solitary tract (NTS), the raphe nuclei, the A14 and A15 dopamine cell groups, the tuberomammillary nuclei, the arcuate nucleus, the subfornical organ, the median preoptic nucleus, the organum vascu-

losum of the lamina terminalis, and the olfactory bulb (Miselis, 1981; Silverman et al., 1981; Tribollet and Dreifuss, 1981; Sawchenko and Swanson, 1982; Tribollet et al., 1985; Wiess et al., 1989; Wilkin et al., 1989). The supraoptic nucleus also receives direct retinal hypothalamic projections (Levine et al., 1994). In addition, the SON is also innervated by the perinuclear zone (PNZ) of the SON, which lies in the lateral hypothalamus dorsal to the SON (Tribollet et al., 1985; Jhamandas et al., 1989a,b). The putative neurotransmitters used by many of these neuronal projections have been described. For example, the hypothalamic neurosecretory neurons receive prominent noradrenergic (Sawchenko and Swanson, 1982), dopaminergic (Van Vulpen et al., 1999), serotinergic (Sawchenko et al., 1983), histaminergic (Panula et al., 1984), peptidergic (Jhamandas et al., 1989a,b; Sawchenko et al., 1990), and GABAergic projections (Tappaz et al., 1983; Van Den Pol, 1988; Theodosis et al., 1986). Classic studies done in the 1970s demonstrated that the electrical activity of magnocellular neurons in the SON and the PVN is strongly related to the plasma levels of vasopressin and oxytocin (Dyball, 1971; Dyball and Dyer, 1971; Dutton and Dyball, 1979; Poulain and Wakerly, 1982; Renaud and Bourque, 1991; Armstrong, 1995; Leng et al., 1999). Both the frequency of activity and the patterning of action potentials have been related to hormone release (Poulain and Theodosis, 1988). The patterning of actions potentials in magnocellular neurons is determined by the currents expressed by the cells and these properties can be influenced by the physiological state of the animal (Armstrong and Stern, 1998). Thus, the synaptic input to SON neurons interacts with the membrane properties of the cells to determine the circulation levels of vasopressin and oxytocin. There is also evidence that many of these pathways may interact with each other in the SON through presynaptic mechanisms and the dendritic release of vasopressin and oxytocin (Ludwig, 1998; Pittman et al., 1998, 2000). Traditionally, vasopressin release has been described as being controlled by osmotic and nonosmotic signals. Studies on the osmotic regulation of vasopressin release have focused on the intrinsic osmosensitivity of magnocellular neurons themselves (Bourque and Oliet, 1997; Bourque, 1998) and osmotically sensitive circumventricular organs in the

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forebrain (Bourque, 1998) and the hindbrain (Curtis et al., 1999). Peripheral sodium receptors may also contribute to the osmotic regulation of vasopressin secretion (Huang et al., 2000a,b). Non-osmotic signals that regulate vasopressin release include all other factors that alter vasopressin release such as changes in blood pressure and blood volume and stress (Cunningham and Sawchenko, 1991; Share, 1996; Leng et al., 1999). The neural projections to the magnocellular neurons in the hypothalamus provide the framework for these different signals to reach the neurosecretory cells, influence their activity and consequently alter circulating levels of vasopressin and oxytocin. While earlier reviews have examined general aspects of the neurophysiology and pharmacology of the magnocellular system (Armstrong, 1995; Hatton and Li, 1998; Leng et al., 1999), this review will focus on arterial baroreceptor and cardiac receptor pathways that inhibit vasopressin SON neurons. Arterial baroreceptor pathways to vasopressin neurons in the SON Arterial baroreceptor neurons are primary sensory cells that convert the distention of major arteries into electrochemical signals that are relayed to the central nervous system. These cells have their sensory nerve endings embedded in the adventitia of either the aortic arch or the carotid sinuses while their primary central target is the nucleus of the solitary tract (NTS). Baroreceptor neurons, their role in reflex control of the autonomic nervous system and their interaction with NTS neurons have been the topic of many recent review articles (Andresen and Kunze, 1994; Andresen and Mendelowitz, 1996; Drummond et al., 2001). Another function of arterial baroreceptors is their role in regulating vasopressin release and the baroreceptor-mediated inhibition of SON neurons (Share, 1988, 1996; Kumada et al., 1990; Cowely, 1992; Dampney, 1994). The influence of peripheral baroreceptors on vasopressin release has been described in whole animal experiments that examined the effects of peripheral nerve section on circulating vasopressin levels. Share and Levy (1962) demonstrated that circulating levels of vasopressin in the dog are elevated by vagotomy and carotid occlusion. Similar experiments also in

the dog have demonstrated that plasma vasopressin can be elevated by the removal of carotid, aortic, and cardiopulmonary baroreceptors (Thames and Schmid, 1979; Share, 1988). In the rat, baroreceptor denervation produces an acute elevation in circulating levels of vasopressin (Alexander and Morris, 1986; Morris and Alexander, 1989) and oxytocin (Morris and Alexander, 1989). The results of these studies indicate that baroreceptors tonically inhibit vasopressin release (Share, 1988). Electrophysiological studies also have demonstrated that changes in blood pressure, atrial pressure and the stimulation of peripheral baroreceptor afferents directly influence the spontaneous activity of magnocellular neurons. Stimulation of the carotid sinus nerve in the cat produces excitatory and inhibitory effects on supraoptic neurons that are intensity-dependent (Yamashita, 1977). Yamashita suggested that the excitatory effects of carotid sinus nerve stimulation were due to chemoreceptor stimulation while the inhibitory effects were produced by the activation of baroreceptor nerves. Unfortunately, this study could not determine if the effect of carotid nerve stimulation was selective for vasopressin neurons or common to both types of magnocellular neurons. In the rat, Harris (1979) observed that an increase in arterial pressure or stretch of the isolated carotid sinus interrupts the spontaneous activity of phasic SON neurons. He also showed that chemoreceptor stimulation with CO2 -saturated saline increases the activity of phasic neurons. Based on these results, Harris suggested that the activation of arterial baroreceptors inhibits the activity of vasopressin neurons in the SON. Since the publication of Yamashita and Harris’ work it has been established that phasic activity is a property of vasopressin neurons in the rat (Cobbett et al., 1986; Yamashita et al., 1983; Renaud and Bourque, 1991). Subsequently, Day and Sibbald (1993a) have shown that aortic depressor nerve stimulation inhibits the activity of vasopressin neurons in the rat. These data suggest that baroreceptor activation inhibits the activity of vasopressin neurons in the supraoptic nucleus. An early study by McAllen and Harris (1988) indicated that this pathway is probably polysynaptic. Subsequent studies by Renaud and colleagues (Renaud et al., 1993; Renaud, 1996), Day and Sibbald (1990, 1993a,b) and now in this laboratory by

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Grindstaff et al. (2000a,b), have concentrated on the central pathways that bring arterial baroreceptor information to the SON. The results of these studies have described a complex polysynaptic pathway as predicted by McAllen and Harris (1988). A number of studies have provided evidence that the neurotransmitter GABA mediates the baroreceptor inhibition of SON neurons. Pretreating either the SON (Jhamandas and Renaud, 1986a, 1987) or the PVN (Kasai et al., 1987) with the GABA-A receptor antagonist bicuculline prevents the inhibition of vasopressin neuron associated with the stimulation of peripheral baroreceptors. Increased blood pressure produced by phenylephrine increases extracellular GABA in the SON region (Voisin et al., 1994). Results from in vitro studies demonstrate that magnocellular neurons are postsynaptically inhibited by GABA acting via GABA-A receptors (Renaud and Bourque, 1991). The SON receives a dense GABA input (Tappaz et al., 1983; Theodosis et al., 1986) and much of this appears to be of local origin (Meyer et al., 1980). The diagonal band of Broca (DBB), which is located in the forebrain anterior to the lamina terminalis region, has been identified as a critical component of the pathway that brings baroreceptor information to the SON (Jhamandas and Renaud, 1986a,b). The DBB contains a sizable population of GABAergic neurons and electrical stimulation of the DBB selectively inhibits the vasopressin neurons of the SON (Jhamandas and Renaud, 1986b). Electrical stimulation of the DBB also increases the extracellular concentrations of GABA in the SON (Voisin et al., 1994). Ibotenic acid lesions of the DBB prevent the baroreceptor-mediated inhibition of SON vasopressin neurons (Cunningham et al., 1992a). However, anatomical studies indicate that the DBB does not project directly to the SON but rather to the PNZ (Tribollet et al., 1985; Jhamandas et al., 1989a,b). Therefore, it was suggested that the DBB exerted its inhibitory influence on the SON not directly but through these local GABAergic interneurons (Jhamandas et al., 1989a,b). The PNZ region has previously been shown to contain GABA neurons (Tappaz et al., 1983; Theodosis et al., 1986) although this finding is not universally supported (Roland and Sawchenko, 1993). Functional evidence for the existence of the PNZ

interneurons has been provided by a series of experiments that have taken advantage of the resistance of SON neurons to excitotoxins (Hastings et al., 1985; Herman and Wiegand, 1986). In vitro studies have demonstrated that the electrophysiological properties of SON neurons are not affected by ibotenic acid injections into the SON-PNZ region that destroy the surrounding neuropile including the GABAergic neurons in the PNZ (Hu et al., 1992). Lateral hypothalamic lesions involving the DBB terminal field in the PNZ significantly decreased the number of vasopressin SON neurons that are inhibited by increases in arterial pressure (Nissen et al., 1993). Moreover, the DBB-evoked inhibition of the same vasopressin SON neurons was also abolished in rats with PNZ lesions (Nissen et al., 1993). However, these lesions did not block the effects of either electrical stimulation of the median preoptic nucleus or peripherally injected angiotensin II on vasopressin SON neurons indicating that fibers of passage remained intact. These results demonstrate that both the baroreceptor-mediated inhibition and the DBBevoked inhibition of vasopressin SON neurons are dependent on the integrity of PNZ neurons located in the lateral hypothalamus. This supports the hypothesis that PNZ neurons are a necessary component of the neural system mediating the baroreceptorinduced inhibition of vasopressin SON neurons. The source of the baroreceptor information to the DBB has also been the focus of recent investigation. Several studies suggest that the noradrenergic innervation of the DBB is involved in the baroreceptormediated inhibition of vasopressin releasing neurons in the SON. First, catecholamine depletion of the DBB blocks the effects of baroreceptor stimulation on vasopressin neurons in the SON (Cunningham et al., 1992b). Also, norepinephrine injected into the DBB inhibits the activity of vasopressin neurons in the SON without significantly influencing the activity of oxytocin neurons (Cunningham et al., 1993). Finally, Bealer (1997) has shown that increases in blood pressure produced with a peripherally acting vasoconstrictor are associated with an increase in the norepinephrine content of the DBB. Neuroanatomical studies of the DBB suggest that it receives afferents from the locus coeruleus (LC), the parabrachial nucleus and the A1 region of the ventrolateral medulla (Jones and Moore, 1977; Lind-

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vall and Stenevi, 1978; Saper and Loewy, 1980; Vertes, 1988; Zaborszky and Cullinan, 1996; Senatorov and Renaud, 1999). The existence of a direct pathway from the NTS to the DBB is less established (Ricardo and Koh, 1978; Vertes, 1988; Senatorov and Renaud, 1999). Several studies suggest that the parabrachial nucleus (Ohman et al., 1990; Jhamandas et al., 1991a,b) and the LC region (Kannan et al., 1981; Sved, 1986) are involved in regulating the activity of SON neurons. The A1 region of the ventrolateral medulla has been shown to mediate vasopressin release produced by hemorrhage (Blessing and Willoughby, 1985; Head et al., 1989). Electrophysiological studies of LC neurons suggest that a majority of its cells are activated by decreases in blood pressure and inhibited by increases in blood pressure (Elam et al., 1985; Olpe et al., 1985), although one study did report that a subpopulation of LC neurons was activated by increased blood pressure (Olpe et al., 1985). However, a recent study showed that electrical stimulation of the aortic depressor nerve produces excitatory and inhibitory effects on the LC neurons (Murase et al., 1994), and another study demonstrated Fos activation in the LC after aortic depressor nerve stimulation (McKitrick et al., 1992). Banks and Harris (1984) showed that lesions of the LC would disrupt the baroreceptormediated inhibition of SON vasopressin neurons. Yet, studies by Day suggest that the A1 region of the ventrolateral medulla (Day and Sibbald, 1990), the parabrachial nucleus (Day and Sibbald, 1993a), the LC (Day and Sibbald, 1993b) are not involved in transmitting baroreceptor information to the SON. Recently, we have addressed this issue by combining retrograde tract tracing with Fos immunocytochemistry (Grindstaff et al., 2000b). Fos is the protein product of the early active gene c-fos and its expression has been widely used as an indicator of neuronal activation (Morgan and Curran, 1991; Curran and Morgan, 1995). This approach has several limitations. First, it has been shown that not all cells in the CNS express Fos after they are stimulated (Dampney et al., 1995). Second, Fos will only be expressed in neurons that are activated and not by cells that are inhibited (Dampney et al., 1995). Finally, immunocytochemical detection of Fos requires a one to two hour time period for expression of the Fos protein to reach detectable levels (Morgan and Curran,

1991; Dampney et al., 1995). On the positive side, the Fos technique allows us to perform experiments on unanesthetized rats free from the confounding effects of anesthetics. It can be combined with retrograde tract tracing and immunocytochemical staining for other proteins that are expressed in the CNS. Previous studies have successfully used Fos immunocytochemistry to determine which regions of the CNS are activated by baroreceptor stimulation produced by infusing peripheral vasoconstrictors (Badoer et al., 1994; Li and Dampney, 1994; Graham et al., 1995; Potts et al., 1997). Initially, we looked at Fos staining in the major noradrenergic cell groups that are reported to project to the DBB after baroreceptor stimulation produced by phenylephrine infusions (Fig. 1; Grindstaff et al., 2000b). Our results indicated that of these regions only the LC contained a significant population of the DBH-positive cells that stained for Fos after phenylephrine infusion. Sino-aortic denervation blocked this increase in Fos demonstrating that it was mediated by arterial baroreceptors (Grindstaff et al., 2000b). In addition, we injected retrograde tract tracers into the DBB and found that LC neurons that were activated by baroreceptor stimulation did project to the DBB (Grindstaff et al., 2000b). Finally, ibotenic acid lesions of the LC blocked the baroreceptor-mediated inhibition in a significant number of phasic vasopressin neurons in the SON (Grindstaff et al., 2000). These data indicate that the LC may provide the DBB with baroreceptor information. How baroreceptor reaches the LC remains controversial. Although there is evidence that the LC receives projections from the NTS (Cedarbaum and Aghajanian, 1978; Ter Horst et al., 1989), another study found no find evidence for such a projection (Aston- Jones et al., 1986). It is possible that information from peripheral baroreceptors may reach the LC via alternative projections from the ventrolateral medulla (Aston-Jones et al., 1986; Pieribone and Aston-Jones, 1991; Valentino et al., 1992). We have also conducted experiments to further characterize the role of the DBB in the regulation of vasopressin release. Electrophysiological experiments have described several different cell types in DBB (Griffith and Matthews, 1986; Griffith, 1988; Markram and Segal, 1990; Matthews and Lee, 1991). One cell type that has broad action potentials and

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Fig. 1. Changes in the mean number of Fos and Fos + DBH cells in sham SAD rats (top) and SAD rats (bottom) after either saline or phenylephrine infusions which significantly increased blood pressure. In sham SAD rats, phenylephrine significantly increases Fos above control levels in each region presented (* P < 0.05 from saline control). Only in the locus coeruleus did the baroreceptor stimulation produced by phenylephrine increase the number of Fos + DBH neurons. In SAD rats the phenylephrine infusions did not significantly increase Fos staining in any of the regions examined. This suggests that activation of arterial baroreceptors activated noradrenergic neurons in the locus coeruleus. (From Grindstaff et al., 2000a.)

large after-hyperpolarizations has been characterized as cholinergic (Griffith and Matthews, 1986; Griffith, 1988; Markram and Segal, 1990; Matthews and Lee, 1991). We have used retrograde tract tracing from the PNZ and immunocytochemistry for choline acetyltransferase (ChAT) and acetylcholine-specific immunotoxin lesions to evaluate the contribution of cholinergic DBB neurons to the baroreceptormediated inhibition of vasopressin SON neurons (Grindstaff et al., 2000a). The results of this study

demonstrated that cholinergic neurons do not contribute to this system. DBB neurons that were retrogradely labeled from the PNZ were not positive for ChAT immunofluorescence. Similarly, injections of a cholinergic-specific immunotoxin into the DBB, which significantly depleted the cholinergic neurons of the DBB did not significantly affect the inhibition of vasopressin SON neurons by baroreceptor stimulation. This indicates that a different population of DBB neurons project to the PNZ. The DBB

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contains a number of other cell types including neurons that produce luteinizing hormone releasing hormone, gonadotropin-releasing hormone, calretinin, vasopressin, substance P, neurotensin, and other neuropeptides (Caffe et al., 1989; Kiss et al., 1997; Ebling et al., 1998; Gonzalo-Ruiz et al., 1999). In addition, the DBB contains glutamatergic neurons (Gonzalo-Ruiz et al., 1999). These cells may be the most likely source of DBB innervation to the PNZ. The DBB has also been studied to determine its role in autonomic regulation. Kirouac and Ciriello (1997) report that glutamate injections into the horizontal limb of the DBB of anesthetized rats produces sympatho-inhibition. Abdelmalek et al. (1999) report that injections of beta-adrenergic agonists into the DBB in conscious rats produced a depressor response. In this study the authors also observed a pressor response to alpha2-adrenoceptor stimulation of the DBB and the magnitude of the pressor response was affected by anesthesia. Injections of colchicines into the DBB also influence blood pressure (Abdelmalek et al., 1994). The colchicine lesions of the DBB result in hypotension without affecting plasma vasopressin or the vasopressin response to hemorrhage. The authors suggest that the lesion of the DBB produces a sympatho-inhibition that is not compensated for by vasopressin release. Additional experiments have used ibotenic acid lesions of the DBB to examine drinking behavior (Sullivan et al., 1991), cardiovascular parameters, and vasopressin release (Mazzella et al., 2000). Rats with ibotenate lesions of the DBB drank significantly more water following angiotensin II or polyethylene glycol injections than sham-lesioned rats (Sullivan et al., 1991). The lesions do not significantly affect their drinking responses to hypertonic saline and water deprivation, indicating that the DBB may be selectively involved in drinking behavior related to extracellular dehydration. The resting blood pressure of DBB lesioned rats is not significantly different from rats with control injections (Mazzella et al., 2000). The rats with DBB lesions did have resting heart rates that were significantly lower compared to controls and basal plasma vasopressin levels were significantly higher than controls (Fig. 2; Mazzella et al., 2000). Our preliminary data suggest that at the time point when we measured the plasma vasopressin the DBB-lesioned rats have a normal fluid

intake, food intake and water balance (Fig. 2). Exogenously administered vasopressin has been shown to act at the area postrema to suppress baroreflex function (Hasser et al., 1997). However, this effect has not been consistently observed in the rat. The decrease in resting heart rate that was observed in the DBB-lesioned rats could be due to an inhibition of the sympathetic nervous system produced by the increase in circulating vasopressin. The magnitude of the change in circulating vasopressin may not be sufficient to produce the change in heart rate that we observed in the DBB-lesioned animals. Nevertheless, this hypothesis could be easily tested by infusing DBB-lesioned rats with a vasopressin antagonist. These data and the results from other laboratories suggest that the DBB is involved in more than just vasopressin release and its role in body fluid balance and autonomic regulations should be explored in greater detail. Cardiopulmonary baroreceptor pathways to vasopressin neurons in the SON Fewer studies have addressed the role of cardiac receptors or atrial receptors in the control of neurons in the SON (Schmid et al., 1984). Activation of cardiac receptors by atrial distension inhibits the activity of magnocellular neurons in the SON of the dog (Koizumi and Yamashita, 1978) and the cat (Koizumi and Yamashita, 1978; Menninger, 1979). In the rat, stimulation of stretch receptors in the right atria inhibits drinking behavior (Kaufman, 1984), vasopressin release (Bennett et al., 1983; Kaufman, 1987) and renal sympathetic nerve activity (Hines et al., 1994; Hines and Mifflin, 1995). Similarly, isotonic increases in plasma volume, which could also stimulate cardiopulmonary baroreceptors, inhibit the activity of vasopressin neurons while oxytocin neurons show an initial excitation followed by inhibition (Pendlebury et al., 1992). These data suggest that increased plasma volume inhibits the release of both vasopressin and oxytocin. Other data suggest that volume expansion may increase vasopressin and oxytocin release. Using Fos immunocytochemistry in urethane-anesthetized rats, Naraveaz et al. (1993) suggested that volume expansion activates vasopressin neurons in the SON. In this study the anesthetized rats were injected with either

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Fig. 2. Basal plasma AVP levels are significantly increased in rats with ibotenic acid lesions of the DBB (DBX, n = 6) compared to rats injected with vehicle (sham, n = 6). Basal heart rate is significantly decreased in rats with DBB lesions (DBX, n = 14) as compared to control (n = 14). These observations were made 5–8 days following lesion. In a separate group of rats, metabolism cage studies were used to examine the water balance of DBB-lesioned rats (DBX, n = 11) and control (CON, n = 9). Immediately after surgery (S) both groups showed a significant change from baseline that has recovered to control levels by day 10.

0.5 or 5 ml of saline i.v. Both protocols increased Fos in vasopressin neurons in the SON by approximately 25%, which they attribute to the activation of atrial receptors. These results appear to contradict the earlier studies cited above which indicate that volume expansion and the stimulation of atrial receptors inhibits vasopressin release. A series of studies by Antunes-Rodrigues et al. (Antunes-Rodrigues et al., 1993; Reis et al., 1994; Haanwinckel et al., 1995)

indicate that volume expansion is associated with a significant increase in circulating oxytocin. They suggest that volume expansion activates baroreceptors and renal afferents to stimulate oxytocin release from the hypothalamus via the LC and by cholinergic and ANP neurons in the lamina terminalis region (Haanwinckel et al., 1995). Oxytocin release stimulated by volume expansion is hypothesized to increase sodium excretion by acting directly on the

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kidney and by facilitating ANP release from the atria. Thus, it appears that the role of atrial receptors in the regulation of vasopressin and oxytocin is perhaps not as clear as earlier work in this field suggested. In earlier electrophysiological studies that examined the influence of cardiac receptors on the activity of SON neurons, the authors did not determine whether the effects that they observed were selective to vasopressin or oxytocin neurons (Koizumi and Yamashita, 1978; Menninger, 1979). In part this was due to the fact that these studies were completed before differences in firing pattern and differential responses to physiological stimuli among vasopressin and oxytocin neurons had been characterized. Another factor is the species used. Vasopressin magnocellular neurons in the cat do not have a regenerative component of their whole-cell calcium current that is necessary for phasic activity (Fagan and Andrew, 1991). Therefore it would not have been possible for Koizumi and Yamashita (1978) and Menninger (1979) to discriminate vasopressin neurons from oxytocin neurons based on their patterns of activity. Although Pendlebury et al. (1992) did characterize their cells as vasopressin and oxytocin, the nature of the stimulus that they used and its time course were more consistent with determining the effects of humoral mechanisms on the activity of SON neurons. In order to better characterize the specific effects of cardiac receptor stimulation on magnocellular neurons in the SON, we used a technique that was first used to study drinking behavior. As initially described by Kaufman (1984), a latex balloon catheter was placed at the junction of the right atrium and the vena cava of anesthetized rats (Grindstaff et al., 2000). This catheter was used to stimulate cardiac receptors while extracellular recordings were obtained from characterized SON neurons. Short periods of stimulation were used to avoid any confounding effects of hormone release (Garcia et al., 1987). In these experiments, 63/84 vasopressin neurons were inhibited by stimulation of the caval–atrial junction and this inhibitory effect was blocked by bilateral vagotomy (Fig. 3). Oxytocin neurons, on the other hand, were not significantly affected by caval–atrial stretch. Stimulation of cardiopulmonary baroreceptors by atrial distention influences the activity of vasopressin

releasing neurons in the SON. However, there is little information to describe how cardiopulmonary baroreceptor information reaches the SON. Electrophysiological studies indicate that neurons in the NTS (Hines et al., 1994; Hines and Mifflin, 1995) and the LC (Jhamandas et al., 1988) are activated by atrial distention but it has not been demonstrated that these neurons project to the hypothalamus. In Fos experiments with isotonic volume expansion in unanesthetized rats, we have observed significant Fos activation in the PNZ, the CVL, the area postrema and the NTS (Randolph et al., 1998). In this experiment, we also observed by isotonic volume expansion selectively activated oxytocin neurons in the SON. In order to determine whether these responses were dependent on cardiac receptors, rats were instrumented with intrapericardial catheters (Cunningham et al., 2000). This technique permits substances to be injected into the pericardial space of unanesthetized animals (Bell et al., 1993). We used it to inject procaine into the pericardial space to block cardiac afferents prior to isotonic volume expansion. Pericardial pretreatment with procaine blocked the increase in Fos staining seen in the PNZ, CVL and NTS following volume expansion. A significant increase in Fos staining was observed in the area postrema and the oxytocin neurons of the SON in the volume-expanded group that received pericardial procaine. Thus, the activation of neurons in the PNZ, CVL and NTS following volume expansion depends on the integrity of cardiac afferents while the increased Fos staining in the area postrema and the SON oxytocin neurons do not require cardiac afferent input. This suggests that a humoral factor (Garcia et al., 1987; Chenault et al., 1992) or a non-cardiac afferent is primarily responsible for activating the area postrema and the SON oxytocin neurons during volume expansion. Functionally, the PNZ plays a major role in the arterial baroreceptor-mediated inhibition of vasopressin neurons in the SON. Our Fos data suggest that the PNZ could play a similar role in the inhibitory effects of atrial distention on vasopressin releasing neurons in the SON. Previously, excitotoxic lesions of the PNZ were used to test its involvement in the baroreceptor-mediated inhibition of vasopressin releasing neurons in the SON (Nissen et al., 1993). This technique has also been employed

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Fig. 3. Continuous rate meter recording from a phasic vasopressin neuron in the SON of a normal rat instrumented with a caval–atrial balloon catheter showing the effects of bilateral cervical vagotomy of baroreceptor and cardiac receptor inhibition. Inflation of the balloon (Balloon) and i.v. injection of phenylephrine (Phe) both inhibit the cell prior to vagotomy. After sectioning of the left (L) and right (R) vagi the inhibitory effects of the balloon are blocked.

to evaluate the contribution of the PNZ to the inhibition of vasopressin neurons by caval–atrial stretch (Grindstaff and Cunningham, 2001). The results of this study showed that PNZ lesions that blocked the baroreceptor-mediated inhibition of vasopressin SON neurons also blocked the inhibitory effects of caval–atrial stretch (Fig. 4). This indicates that these two pathways overlap in the PNZ, but there is insufficient information to speculate whether or not the same population of PNZ neurons mediates baroreceptor and cardiac receptor inhibition of vasopressin neurons. In the Fos experiments with volume expansion, we did not observe an increase in Fos in the DBB. This finding suggests that while the PNZ is activated by volume expansion the DBB is not. Although the failure to observe Fos expression in a region does not

necessarily mean that the cells were not activated, we hypothesize that the differences in Fos expression observed in the DBB and PNZ do represent differences in neural activation associated with volume expansion. Thus, the pathways for baroreceptor-mediated inhibition of vasopressin neurons may overlap only at the level of the PNZ in the forebrain. This topic will be the focus of future investigations. Summary The maintenance of body fluid and electrolyte balance requires the coordination of physiological, behavioral and hormonal systems, which include vasopressin and oxytocin release. This organization necessarily involves a complex neural network to sense changes in hydromineral balance and initiate the ap-

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Fig. 4. (A) Rate meter recordings of phasic vasopressin neurons from rats injected into the PNZ with either vehicle (Con) or ibotenic acid (PNZX). Note that in the rate meter record from the control animal acute increases in blood pressure produced by i.v. phenylephrine (filled boxes) and caval–atrial stretch (open bars) inhibit the activity of the cell. In a rat with a lesion of the PNZ neither stimulus interrupts the activity of the cell. (B) An example of a PNZ lesion (gray shaded area) that blocked both arterial baroreceptor and cardiac receptors inhibition of phasically active vasopressin neurons.

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propriate short-term and long-term adjustments to maintain body fluid homeostasis. While this area has been the subject of considerable study (Abboud and Thames, 1982; Share, 1988; Kumada et al., 1990; Cowely, 1992; Dampney, 1994), much of the central network involved in the maintenance of body fluid balance remains to be defined. Recent studies by Schrier indicate that vasopressin may play a significant role in congestive heart failure (Schrier et al., 1998). Specifically, non-osmotic regulation of vasopressin secretion may contribute to high circulating levels of vasopressin in end-stage heart failure (Martin and Schrier, 1997; Schrier et al., 1998). Norsk (1996) has proposed that changes in arterial baroreceptor and atrial receptor control of vasopressin release may contribute to changes in extracellular fluid volume observed during space flight and prolonged bed rest. Alterations in control of vasopressin release have also been observed during pregnancy (Lindheimer et al., 1995). Understanding the neural pathways involved in the cardiovascular regulation of vasopressin release is an important first step that is needed to address the role of vasopressin in each of these altered physiological states. Research in the neurohypophysial system is unique in that it provides the opportunity to study fundamental neuroscience within an important integrative physiological context. Abbreviations ChAT CO2 CNS CVL DBB GABA LC NTS PNZ PVN SON

choline acetyl transferase carbon dioxide central nervous system caudal ventrolateral medulla diagonal band of Broca gamma-amino butyric acid locus coeruleus nucleus of the solitary tract perinuclear zone of the supraoptic nucleus paraventricular nucleus of the hypothalamus supraoptic nucleus

Acknowledgements This work was supported by National Heart, Lung and Blood Institute Grants R29-HL55692 (JTC),

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 21

The central vasopressinergic system in experimental left ventricular hypertrophy and dysfunction Frank Muders 1,∗ , Günter A.J. Riegger 1 , Udo Bahner 2 and Miklos Palkovits 3 1

Klinik und Poliklinik für Innere Medizin II, University of Regensburg, Regensburg, Germany 2 Medizinische Universitätklinik, Würzburg, Germany 3 Laboratory of Neuromorphology, Semmelweis University, Budapest, Hungary

Introduction While the pathophysiological role of compensatory neurohumoral mechanisms in heart failure has been widely investigated, the significance of the centrally acting cardiovascular neuropeptide systems remains largely unclear. Measurements of metabolic markers such as hexokinase, cFos and NADPH-diaphorase suggest increased neuron activity in hypothalamic areas of the brain, the locus coeruleus and in other nuclei of the brain involved in central circulatory regulation in rats 6 weeks after myocardial infarction (Patel et al., 1993, 2000; Zhang et al., 1998). There have been only isolated investigations of the functional state of central neuropeptide systems involved in cardiocirculatory regulation in heart failure. With regard to the central renin–angiotensin system, there have been two experimental studies suggesting stimulation. Intracerebroventricular administration of the AT1 receptor antagonist, losartan, in sheep with heart failure due to rapid ventricular pacing led to hemodynamic changes, including a fall in blood pressure, which did not occur in healthy animals (Rademaker

∗ Correspondence

to: F. Muders, Klinik und Poliklinik für Innere Medizin II, Universitätsklinikum, FranzJosef-Strauss-Allee 11, 93053 Regensburg, Germany. Tel.: +49-941-9447211; Fax: +49-941-9447213; E-mail: [email protected]

et al., 1995). In rats with an aortocaval fistula, there was increased AT1 receptor expression in the nucleus paraventricularis, nucleus tractus solitarius and subfornical organ (Yoshimura et al., 2000). Investigations of the central vasopressinergic system in heart failure have not been conducted to date. Our aim, therefore, was to investigate the central vasopressinergic system in a model of myocardial hypertrophy and left ventricular dysfunction (supravalvular aortic stenosis model). Banding of the ascending aorta in 8-weeks-old Wistar rats produces severe ventricular hypertrophy after 12 weeks of pressure overload. At this stage, this model is characterized by a transition of LV hypertrophy into cardiac failure, as suggested by functional and molecular studies, as well as by an increased mortality (Weinberg et al., 1994; Bruckschlegel et al., 1995). Neurohumoral vasoconstrictor systems were stimulated in this model in order to maintain blood pressure in the chronic aortic stenosis with a reduced cardiac index. In comparison to healthy control animals, vasopressin plasma levels and plasma renin activities were significantly increased, while levels of plasma norepinephrine and 24-h urinary excretion were unchanged (Muders et al., 1995), parameters both indicating the activity of the sympathetic nervous system (Goldstein et al., 1983). Increased levels of plasma vasopressin can be attributed to non-osmotic stimuli in this model since plasma osmolality was unchanged in comparison to

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control animals. In an earlier investigation of the same animal model, at a later time (after 15 weeks), enhanced vasopressin plasma levels, not related to plasma osmolality, were found in animals which had developed a marked left ventricular dysfunction (Riegger et al., 1988). Our studies support the hypothesis that non-osmotic stimuli play an important role in the regulation of vasopressin in cardiovascular disorders. In the model of supravalvular aortic stenosis, baroreceptor stimulation and/or angiotensin IImediated stimulation of vasopressin release is possible: the supravalvular position of the silver clip leads to a weakened signal to baroreceptors in the aorta and carotid artery and, consequently, to stimulation of vasopressin secretion. In contrast, cardiac baroreceptors are exposed to increased left ventricular filling pressure and, consequently, counteract increased vasopressin release. However, cardiopulmonary receptors play a subordinate role in the regulation of vasopressin release under physiological conditions in the presence of intact arterial baroreceptors (Chen et al., 1991). In order to test the hypothesis that levels of vasopressin change centrally as well as peripherally, levels of vasopressin were measured in 20 different areas of the brain involved in central cardiocirculatory regulation. The brain areas were obtained by the micropunch technique of Palkovits (Palkovits, 1973). In the hypothalamus, different changes were found in the areas producing vasopressin. While vasopressin content in the paraventricular and suprachiasmatic nuclei was significantly raised in rats with aortic stenosis, no changes were found in the supraoptic nucleus, compared to healthy control animals. This aspect is of particular interest, since the paraventricular and suprachiasmatic nuclei are involved in central cardiocirculatory regulation through their vasopressinergic connections to the brainstem while the supraoptic nucleus does not have these connections. These different changes in vasopressin content in these hypothalamic brain areas indicate an alteration in the central vasopressinergic cardiocirculatory control in animals with supravalvular aortic stenosis. In addition, there was a significantly increased content of vasopressin in the median eminence, an area which is part of the hypothalamo-hypophysial axis. This implies that stimulation of the axis in animals with aortic stenosis consequently raises plasma levels of vasopressin.

The vasopressin content was also significantly increased in four other ‘extrahypothalamic’ brain areas involved in central cardiocirculatory control. Interestingly, vasopressin content in the locus coeruleus was markedly diminished compared to control animals. In association with the unchanged norepinephrine plasma levels and urinary excretion in rats with aortic stenosis, the results suggest an important central circulatory regulating mechanism, in which a reduced vasopressin concentration in the locus coeruleus may counteract activation of the sympathetic nervous system. In summary, our studies of the central vasopressinergic system in experimental left ventricular hypertrophy and dysfunction show that, in addition to stimulation of peripheral neurohumoral factors, a central neuropeptide system is altered which, in rats, is involved with supravalvular aortic stenosis through its cardiovascular regulatory effects (Muders et al., 1995). Modulation of the central vasopressinergic system by blockade of the renin–angiotensin system In the 1980s, an association was found between the renin–angiotensin system and vasopressin in patients with heart failure. In these patients, who had plasma levels that were increased inappropriately relative to the plasma osmolality, chronic treatment with the ACE inhibitor, Captopril, led to a significant reduction in the vasopressin plasma levels and to a restoration of the relationship between plasma osmolality and vasopressin (Lee and Packer, 1986; Riegger and Kochsiek, 1986). Nowadays, blockade of the renin– angiotensin system by ACE inhibitors and AT1 receptor antagonists is established in the treatment of heart failure, left ventricular hypertrophy and hypertension. The therapeutic efficacy of these medications, including a reduction in the mortality of these cardiovascular disorders, has been confirmed in animal models as well as in patients (The SOLVD Investigators, 1992; The Acute Infarction Ramipril Efficacy Study Investigators, 1993; Remme, 1995). This also includes investigations that were performed using the model of supravalvular aortic stenosis in the rat. Despite persisting strain on the left ventricle, treatment with an ACE inhibitor and AT1 receptor antagonist, carried out from the 6th postoperative week, led to a signif-

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icant regression of left ventricular hypertrophy and to a reduction in mortality, in both treatment groups (Bruckschlegel et al., 1995). It is not known whether chronic treatment with an ACE inhibitor and AT1 receptor antagonist, respectively, also produces modulation of central neuropeptide systems involved in cardiocirculatory regulation, like the vasopressinergic system. In order to pursue this question, we measured vasopressin in plasma and in individual brain areas in rats with supravalvular aortic stenosis, which were treated chronically either with an ACE inhibitor or with an AT1 receptor antagonist. Twelve weeks after placement of the aortic clip, plasma vasopressin levels were reduced in both treatment groups compared to untreated aortic stenosis animals and showed no significant differences with healthy animals. Treatment with the ACE inhibitor and AT1 receptor antagonist also led to a reduction in vasopressin concentrations in specific areas of the hypothalamus and brainstem. These effects were different in some brain areas, which can be attributed to a different distribution of ACE and angiotensin II receptors in these areas (Muders et al., 1999). The fact that treatment of rats with supravalvular aortic stenosis with an ACE inhibitor and AT1 receptor antagonist, respectively, led not to activation but to suppression of the vasopressinergic system, despite a further fall in blood pressure, is of particular interest pathophysiologically and can be explained by (1) modulation of the baroreceptor reflex, and (2) direct peripheral or central inhibition of vasopressin synthesis and release. As explained above, the dysregulation of baroreceptors with consequent restriction of sympathoadrenal and humoral inhibition is a pathophysiological characteristic of chronic heart failure, which includes increased vasopressin synthesis and release (Zucker and Gilmore, 1985; Hirsch et al., 1987). The ability of the baroreceptors to shift their baseline in the direction of the surrounding pressure (reflex resetting) is absent and the sensitivity of the baroreceptors is reduced, as we also found in the model of supravalvular aortic stenosis (unpublished data). As has already been shown in rabbits with experimental heart failure and in patients, treatment with an ACE inhibitor or AT1 receptor antagonist leads to an improvement in the sensitivity of the arterial baroreceptors and restores the ability of the pressure

discharge curve to be readjusted (Murakami et al., 1996; Grassi et al., 1997). Whether chronic blockade of the renin–angiotensin system in the model of supravalvular aortic stenosis leads to an improvement in the baroreceptor reflex is unknown and should be investigated further. In addition to these effects mediated by the baroreceptor reflex, a ‘direct’ effect of the ACE inhibitor and AT1 receptor antagonist, respectively, on the vasopressinergic system is possible, in which vasopressin synthesis and release are suppressed either through angiotensin II receptors of the circumventricular organs or through direct inhibition of central ACE or angiotensin II receptors in specific areas of the brain. Central action of ACE inhibitors and AT1 receptor antagonists: in vitro autoradiography of the brain The existence of a central renin–angiotensin system and the significance of central angiotensin II in cardiocirculatory regulation are recognized today. In addition to synaptic inhibition of the baroreceptor reflex, central angiotensin II causes stimulation of sympathetic nervous activity and vasopressin synthesis and release, which can be suppressed by prior administration of an AT1 receptor antagonist (Unger et al., 1988; Wright and Harding, 1995). On the other hand, the central efficacy of systemically administered ACE inhibitors and AT1 receptor antagonists is controversial. Since chronic oral treatment with an ACE inhibitor and AT1 receptor antagonist in rats with supravalvular aortic stenosis produces suppression of central vasopressin and may be attributed to a central effect of these drugs, we studied the central efficacy of an ACE inhibitor and AT1 receptor antagonist, using in vitro autoradiography after systemic administration for 2 and 4 weeks, respectively (Muders et al., 1997, 2001). We were able to show that both medications caused significant ACE inhibition and angiotensin II receptor blockade in specific brain areas. Moreover, we recorded diminished ACE activities and angiotensin II receptor densities in brain areas that have no blood–brain barrier (and were thus accessible to the drug circulating in the blood) as well as in areas situated inside the brain.

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A precondition for the central efficacy of an orally administered medication in specific brain areas is penetration of the blood–brain barrier. Studies of the distribution of orally administered radioactively marked drugs often show an absence of accumulation of the ACE inhibitor and AT1 receptor antagonists after a single dose. However, autoradiographic investigations that employed chronic administration as in this investigation, revealed a central blockade of the renin–angiotensin system. Chronic therapy with an ACE inhibitor or AT1 receptor antagonist (as is usual in the treatment of cardiovascular diseases) therefore includes central effects (Gohlke et al., 1989; Song et al., 1991; Polidori et al., 1996). There has been evidence of a central action, even for treatment with Captopril, the most hydrophilic drug of all the ACE inhibitors. Neurophysiological investigations showed for Captopril a marked improvement in cognitive abilities in comparison with placebo-treated patients (Zubenko and Nixon, 1984). In summary, mechanisms of the blood–brain barrier are complex and the penetration of a drug is dependent on many factors, including the duration of use, lipophilia of individual drugs and dosage. The central efficacy of ACE inhibitors and AT1 receptor antagonists is of interest insofar as the modulation of central cardiocirculatory mechanisms contributes to the therapeutic effects of these medications in the treatment of cardiovascular diseases.

centrations in the locus coeruleus, an important regulatory area of sympathetic nervous activity, suggest a central regulatory mechanism through which stimulation of the sympathetic nervous activity can be prevented. Our investigations showed that non-osmotic factors like the baroreceptor reflex and angiotensin II, are important stimuli of the vasopressinergic system. We were also able to show that the central vasopressinergic system in rats with experimental heart failure and myocardial hypertrophy is inhibited by treatment with an ACE inhibitor and AT1 receptor antagonist. As seen with autoradiography, this effect is mediated by a central effect of the drugs. Research into central regulatory mechanisms in cardiovascular diseases is, on the one hand, of crucial importance to our understanding of complex pathophysiological processes, and on the other hand, it serves the development of new therapeutic approaches with the goal of influencing these mechanisms directly pharmacologically and for the elucidation of central, currently unknown effects of cardiovascular drugs. Abbreviations ACE AT LV NADPH

angiotensin converting enzyme angiotensin left ventricular nicotinamide adenine dinucleotide phosphate

Summary References In the course of cardiac diseases, various neurohormonal systems in the plasma are activated. So far there have been only isolated results of investigations about the functional state of central neuropeptide systems in cardiac diseases and, in particular, in heart failure. We investigated, therefore, the central vasopressinergic system, an important neuropeptide system in cardiocirculatory regulation in a model of myocardial hypertrophy and left ventricular dysfunction, a model of supravalvular aortic stenosis. In addition to increased vasopressin concentrations in plasma, central vasopressin is also altered in this model. A differential stimulation of vasopressin in the hypothalamic areas and in the areas of the brain stem that are involved in central cardiocirculatory regulation was detected. Reduced vasopressin con-

Bruckschlegel, G., Holmer, S.R., Jandeleit, K., Grimm, D., Muders, F., Kromer, E.P., Riegger, G.A. and Schunkert, H. (1995) Blockade of the renin–angiotensin system in cardiac pressureoverload hypertrophy in rats. Hypertension, 25: 250–259. Chen, J.S., Wang, W., Bartholet, T. and Zucker, I.H. (1991) Analysis of baroreflex control of heart rate in conscious dogs with pacing-induced heart failure. Circulation, 83: 260–267. Gohlke, P., Scholkens, B., Henning, R., Urbach, H. and Unger, T. (1989) Inhibition of converting enzyme in brain tissue and cerebrospinal fluid of rats following chronic oral treatment with the converting enzyme inhibitors ramipril and Hoe 288. J. Cardiovasc. Pharmacol., 14(Suppl. 4): S32–36. Goldstein, D.S., Mc Carty, R., Polinsky, R.J. and Kopin, I. (1983) Relationship between plasma norepinephrine and sympathetic neuronal activity. Hypertension, 5: 552–559. Grassi, G., Cattaneo, B.M., Seravalle, G., Lanfranchi, A., Pozzi, M., Morganti, A., Carugo, S. and Mancia, G. (1997) Effects of chronic ACE inhibition on sympathetic nerve traffic and

279 baroreflex control of circulation in heart failure. Circulation, 96: 1173–1179. Hirsch, A.T., Dzau, V.J. and Creager, M.A. (1987) Baroreceptor function in congestive heart failure: effect on neurohumoral activation and regional vascular resistance. Circulation, 75: 1136–1148. Lee, W.H. and Packer, M. (1986) Prognostic importance of serum sodium concentration and its modification by convertingenzyme inhibition in patients with severe chronic heart failure. Circulation, 73: 257–267. Muders, F., Kromer, E.P., Bahner, U., Elsner, D., Ackermann, B., Schunkert, H., Palkovits, M. and Riegger, G.A. (1995) Central vasopressin in experimental aortic stenosis in the rat. Cardiovasc. Res., 29: 416–421. Muders, F., Elsner, D., Jandeleit, K., Bahner, U., Kromer, E.P., Kirst, I., Riegger, G.A. and Palkovits, M. (1997) Chronic ACE inhibition by quinapril modulates central vasopressinergic system. Cardiovasc. Res., 34: 575–581. Muders, F., Elsner, D., Schunkert, H., Riegger, G.A. and Palkovits, M. (1999) Central vasopressin is modulated by chronic blockade of the renin–angiotensin system in experimental left ventricular hypertrophy. Am. J. Hypertens., 12: 311–314. Muders, F., Palkovits, M., Bahner, U., Kirst, I., Elsner, D. and Jandeleit-Dahm, K. (2001) Central inhibition of AT1 receptors by eprosartan — in vitro autoradiography in the brain. Pharmacol. Res., 43: 251–255. Murakami, H., Liu, J.L. and Zucker, I.H. (1996) Blockade of AT1 receptors enhances baroreflex control of heart rate in conscious rabbits with heart failure. Am. J. Physiol., 271: R303–R309. Palkovits, M. (1973) Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res., 59: 449–450. Patel, K.P., Zhang, P.L. and Krukoff, T.L. (1993) Alterations in brain hexokinase activity associated with heart failure in rats. Am. J. Physiol., 265: R923–R928. Patel, K.P., Zhang, K., Kenney, M.J., Weiss, M. and Mayhan, W.G. (2000) Neuronal expression of Fos protein in the hypothalamus of rats with heart failure. Brain Res., 865: 27– 34. Polidori, C., Ciccocioppo, R., Pompei, P., Cirillo, R. and Massi, M. (1996) Functional evidence for the ability of angiotensin AT1 receptor antagonists to cross the blood–brain barrier in rats. Eur. J. Pharmacol., 307: 259–267. Rademaker, M.T., Fitzpatrick, M.A., Charles, C.J., Frampton, C.M., Richards, A.M., Nicholls, M.G. and Espiner, E.A. (1995) Central angiotensin II AT1-receptor antagonism in normal and heart-failed sheep. Am. J. Physiol., 269: H425–H432.

Remme, W.J. (1995) Neurohormonal modulation in heart failure: ACE inhibition and beyond. Eur. Heart J., 16(Suppl. N): 73– 78. Riegger, G.A. and Kochsiek, K. (1986) Vasopressin, renin and norepinephrine levels before and after captopril administration in patients with congestive heart failure due to idiopathic dilated cardiomyopathy. Am. J. Cardiol., 58: 300–303. Riegger, G.A., Wolf, P. and Kochsiek, K. (1988) Vasoconstrictor role of vasopressin and angiotensin in experimental aortic stenosis in the rat. J. Cardiovasc. Pharmacol., 11: 538–542. Song, K.F., Zhuo, J.L. and Mendelsohn, F.A. (1991) Access of peripherally administered DuP 753 to rat brain angiotensin II receptors. Br. J. Pharmacol., 104: 771–772. The Acute Infarction Ramipril Efficacy (AIRE) Study Investigators (1993) Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure. Lancet, 342: 821–828. The SOLVD Investigators (1992) Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fraction. N. Engl. J. Med., 327: 685–691. Unger, T., Badoer, E., Ganten, D., Lang, R.E. and Rettig, R. (1988) Brain angiotensin: pathways and pharmacology. Circulation, 77: 140–154. Weinberg, E.O., Schoen, F.J., George, D., Kagaya, Y., Douglas, P.S., Litwin, S.E., Schunkert, H., Benedict, C.R. and Lorell, B.H. (1994) Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis. Circulation, 90: 1410–1422. Wright, J.W. and Harding, J.W. (1995) Brain angiotensin receptor subtypes AT1, AT2, and AT4 and their functions. Regul. Pept., 59: 269–295. Yoshimura, R., Sato, T., Kawada, T., Shishido, T., Inagaki, M., Miyano, H., Nakahara, T., Miyashita, H., Takaki, H., Tatewaki, T., Yanagiya, Y., Sugimachi, M. and Sunagawa, K. (2000) Increased brain angiotensin receptor in rats with chronic highoutput heart failure. J. Card. Fail., 6: 66–72. Zhang, K., Zucker, I.H. and Patel, K.P. (1998) Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure. Brain Res., 786: 219–225. Zubenko, G.S. and Nixon, R.A. (1984) Mood-elevating effect of captopril in depressed patients. Am. J. Psychiatry, 141: 110– 111. Zucker, I.H. and Gilmore, J.P. (1985) Aspects of cardiovascular reflexes in pathologic states. Fed. Proc., 44: 2400–2407.

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Elsevier Science B.V. All rights reserved

CHAPTER 22

Cardiovascular effects of oxytocin Maria Petersson * Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Institutet, S-171 77 Stockholm, Sweden

Abstract: The well known effects of oxytocin on uterine contraction and milk ejection were found as early as the beginning of the 20th century. Since then many other effects of oxytocin have been found and among them a great number of effects on the cardiovascular system. Oxytocin is released from the neurohypophysis into the circulation and from parvocellular neurons within the paraventricular nucleus (PVN) to many areas within the central nervous system (CNS). Indeed, oxytocin may modify blood pressure as well as heart rate both through effects within the CNS and through effects in other organs, such as the heart, blood vessels and kidney. Oxytocin may also cause cardiovascular effects by affecting other mediators, such as atrial natriuretic peptide (ANP), nitric oxide (NO) and alpha 2-adrenoreceptors. Keywords: Blood pressure; Heart rate; Kidney; alpha 2-adrenoreceptor

Introduction The nonapeptide oxytocin is produced mainly in the paraventricular (PVN) and supraoptical nuclei (SON) in the hypothalamus. Oxytocinergic magnocellular neurons in the PVN and the SON project to the neurohypophysis whence oxytocin is released into the circulation whereas oxytocinergic parvocellular neurons in the PVN project to several areas within the brain. For example the amygdala, hippocampus, olfactory bulb, bed nucleus of the stria terminalis, substantia nigra, raphe nuclei, locus coeruleus (LC), vagal nuclei, the median eminence and spinal cord are all reached by oxytocinergic neurons (Buijs, 1983; Sofroniew, 1983). Oxytocin synthesis has also been demonstrated outside the brain, for example, in the uterus, ovaries, testis, thymus, adrenal gland, pancreas and, interestingly, in both

∗ Correspondence

to: M. Petersson, Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Institutet, S-171 77 Stockholm, Sweden. Tel.: +46-851770326; Fax: +46-8-51773096; E-mail: [email protected]

the heart and several of the large vessels (Wathes and Swann, 1982; Ang and Jenkins, 1984; Nicholson et al., 1984; Geenen et al., 1986; Amico et al., 1988; Lefebvre et al., 1992; Jankowski et al., 1998; Gutkowska et al., 2000). In 1906, Dale found that an extract from the neurohypophysis could induce uterine contraction in cats (Dale, 1906) and four years later Ott and Scott showed that this extract also could induce milk ejection (Ott and Scott, 1910). The oxytocinergic system is equally developed in males and females, although it is under strong influence of female steroid hormones. Increased osmotic pressure, hemorrhage, feeding, gastric distension and mating stimulate the release of oxytocin in both males and females (Fox and Knaggs, 1969; McNeilly and Ducker, 1972; Tindal, 1974; Weitzman et al., 1978; Uvnäs-Moberg et al., 1985; Hughes et al., 1987; Renaud et al., 1987). Oxytocin has many other effects besides uterine contraction and milk ejection. Many of them are exerted within the CNS. Oxytocin influences a variety of behaviors such as maternal, social, sexual and feeding behavior (Carter, 1998). For example, it participates in osmoregulation, memory and learning, tolerance and dependence, antinociception, ther-

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moregulation, immunological processes and in the modification of the release of several other hormones (see for example reviews by: Argiolas and Gessa, 1991; Richard et al., 1991). In addition, oxytocin can influence cardiovascular regulation in several ways. Below is an overview of the different reported effects of oxytocin on the cardiovascular system, in the periphery and within the CNS. Oxytocin and the vasculature Oxytocin can induce both vasodilatation and vasoconstriction, depending on the type of vasculature and the degree of tone in the vasculature (Altura and Altura, 1984). In the placental–umbilical vasculature, oxytocin is a more potent vasoconstrictor than vasopressin (Altura and Altura, 1984). In female rats, oxytocin induces vasoconstriction during estrus, whereas during diestrus it mediates vasodilatation (Lloyd and Pickford, 1961). In dogs, oxytocin causes a decrease in blood pressure accompanied by an increase in heart rate and the effect has been suggested to be mediated by the vasodilator action of oxytocin (Nakano and Fisher, 1963). In humans, the most frequently reported effect of oxytocin is a slight and short decrease (5–10 min) in blood pressure. Interestingly, this decrease has been reported to be more pronounced in non-pregnant women, compared to pregnant women (Brotanek and Kazda, 1965). Oxytocin administered peripherally to rats causes a short-lasting increase in blood pressure which, in females, is most pronounced during proestrus and estrus, when progesterone and estrogen levels are highest (Petersson et al., 1999d). In ovariectomized (OVX) rats, estrogen and progesterone treatment potentiates the increase in blood pressure induced by oxytocin (Lloyd, 1959). The increase in blood pressure is accompanied by a decrease in heart rate and the effects are partially antagonized by an oxytocin antagonist. This suggests that the effects are induced through oxytocin receptors in the vasculature and, because the counteraction by the oxytocin antagonist is not total, perhaps also through binding to vasopressin receptors of the V1a-type. When oxytocin is given in large amounts, it can bind to vasopressin receptors (Jard et al., 1987; Barberis and Tribollet, 1996; Petersson et al., 1999d). In some studies, the oxytocin-induced rise in blood pressure in rats is fol-

lowed by a decrease in blood pressure, together with an increase in cardiac output, which reaches its nadir at about 30 min after the injection. In other studies, a decrease in blood pressure was seen to occur several hours after the injection (Petty et al., 1985; Petersson et al., 1999c). One explanation for the different and opposite reported effects of oxytocin may be that oxytocin receptors are located both in endothelial and vascular smooth muscle cells where they mediate opposite effects. Oxytocin receptors in endothelial cells have been found to stimulate NO and, thereby, induce vasodilatation, whereas oxytocin receptors in smooth muscle cells are thought to mediate vasoconstriction (Yazawa et al., 1996; Thibonnier et al., 1999). In addition, as mentioned above, the binding of oxytocin to vasopressin receptors may also contribute to differences in effects. Recently, oxytocin synthesis was found in several large vessels, including the aorta and vena cava (Gutkowska et al., 2000). Thus, oxytocin may act as an autocrine/paracrine hormone in the vasculature. Oxytocin and the heart An increase in cardiac output together with a decrease in blood pressure in humans was demonstrated as early as 1959 (Kitchin et al., 1959). In dogs, intravenous (i.v.) oxytocin causes an increase in heart rate, cardiac output and myocardial contractile force together with a decrease in blood pressure. These changes have been thought to be secondary to the vasodilatation and decrease in blood pressure induced by oxytocin and thereby a decreased return of venous blood (Nakano and Fisher, 1963). However, oxytocin synthesis as well as oxytocin receptors are present in the heart, and oxytocin has also been suggested to decrease heart rate and the force of contraction through these receptors (Jankowski et al., 1998; Gutkowska et al., 2000). Oxytocin may also stimulate the release of ANP through the oxytocin receptors within the heart and via ANP-induced diuresis, natriuresis and vasodilatation. Oxytocin injected systemically stimulates ANP release from the isolated perfused rat heart, an effect which can be blocked by an oxytocin antagonist (Gutkowska et al., 2000).

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Oxytocin and the kidney Oxytocin receptors in the kidney are located in the collecting ducts and in the macula densa (Stoeckel and Freund-Mercier, 1990; Inoue et al., 1993) and both antidiuretic and diuretic effects of oxytocin have been reported. Oxytocin causes an increase in glomerular filtration and natriuresis. Hypophysectomized rats retain sodium, an effect that can be counteracted by oxytocin (Forsling and Brimble, 1985). In rats, oxytocin was found to produce natriuresis even at physiological concentrations and the effects were antagonized by an oxytocin antagonist. In addition, postnephrectomy natriuresis in rats was attenuated in response to infusion of an oxytocin antagonist (Huang et al., 1994, 1995). Infusion of oxytocin, in amounts within the physiological range, induced natriuresis in humans as well (KostoglouAthanassiou et al., 1994). Moreover, oxytocin, as mentioned above, stimulates the release of ANP which also mediates natriuretic effects (Gutkowska et al., 2000). Oxytocin and cardiovascular effects within the CNS Oxytocin fibers project from the PVN to a number of regions in the CNS which are of importance for cardiovascular regulation. For example, the locus coeruleus (LC), nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus nerve (DMX), rostral ventrolateral medulla (RVLM), dorsal raphe nucleus and intermediolateral cell column of the spinal cord are all reached by oxytocinergic neurons (Buijs, 1983; Sofroniew, 1983). Stimulation of the PVN with L-glutamate or weak current decreases blood pressure in rats and it has been suggested that the effect is caused by inhibition of spinal sympathetic preganglionic neurons (Yamashita et al., 1987; Kannan et al., 1988). Indeed, oxytocin is thought to decrease blood pressure through inhibition of these neurons as well as through depression of neurons in the caudal medulla (Morris et al., 1980; Gilbey et al., 1982). Furthermore, oxytocin administered into the DMX causes bradycardia which can be blocked by an oxytocin antagonist and by atropine (Rogers and Hermann, 1985). In contrast, oxytocin injected into the NTS of rats anesthetized with urethane increases

both blood pressure and heart rate (Matsuguchi et al., 1982), whereas in another study, with lower doses of oxytocin injected into the NTS, no effect on blood pressure or heart rate was seen (Vallejo et al., 1984). An involvement of oxytocin neurons in the PVN in the increase in blood pressure and heart rate in response to substance P has also been suggested, since central pretreatment with an oxytocin antisense oligodeoxynucleotide attenuates the effect of substance P on blood pressure and heart rate in rats (Maier et al., 1998). Moreover an interaction between oxytocin and vasopressin in central cardiovascular regulation has been proposed since the increase in blood pressure and heart rate in response to vasopressin given intracerebroventricularly (i.c.v.) is larger if rats are preinjected with oxytocin (Poulin et al., 1994). In contrast, i.c.v. oxytocin has been reported to attenuate the increase in blood pressure induced by electrical stimulation of the mesencephalic reticular formation in anaesthetized male rats (Versteeg et al., 1982). Oxytocin administered intracisternally (i.c.) to anaesthetized dogs increases blood pressure without a change in heart rate (Tran et al., 1982). When it is administered i.c. to rats, no effects on blood pressure or heart rate are induced, but there is a decrease in the baroreceptor reflex sensitivity in response to changes in blood pressure (Petty et al., 1985). Oxytocin administered intrathecally (i.t.) has also been found to attenuate the exercise pressor reflex (Stebbins and Ortiz-Acevedo, 1994). In cats, oxytocin administered i.c.v. may decrease or increase blood pressure depending on the dose given, with lower doses inducing a reduction in blood pressure and higher doses inducing an increase in blood pressure (Nashold et al., 1962). In rats, no acute change in blood pressure or heart rate is seen in response to oxytocin administered i.c.v. (Feuerstein et al., 1984; Petersson et al., 1996b). However, 6–10 h after the injection blood pressure is decreased and is still significantly lower 24 h after the injection (Petersson et al., 1996b, 1999c). I.t. administration of oxytocin in cats preferentially increases heart rate and not blood pressure (Yashpal et al., 1987). In rats, oxytocin i.t. (although in a lower dose than the one tested in the cat study) influenced neither blood pressure nor heart rate in one study (Porter and Brody, 1986), whereas in an-

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other, an increase in blood pressure, which in some rats was preceded by a short decrease in blood pressure, was reported (Tan and Tsou, 1985). Long-term effects of oxytocin When oxytocin (0.3 μg) is administered i.c.v. to rats, no immediate effects on blood pressure or heart rate are detected. However, several hours after the injection, blood pressure decreases and is still significantly lower 24 h later. When oxytocin was given repeatedly once a day during 5 days to male rats blood pressure gradually decreased (105 ± 4.6 com-

Fig. 1. Systolic blood pressure and heart rate in male rats treated with oxytocin 1 μg/kg i.c.v. for 5 days compared to controls given saline for 5 days. Each point shows the mean ± SD. Circle, oxytocin (n = 5); square, NaCl (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 oxytocin compared to controls. These data are printed with the kind permission of Elsevier Science B.V. where they were first published (Petersson et al., 1996b).

pared to 122 ± 2.6 mmHg (systolic) in saline-treated controls). The heart rate was unchanged. A significant difference between oxytocin-treated rats and controls persisted for 8 days after the end of the oxytocin treatment period but disappeared at 10 days (Fig. 1). An even more long-lasting effect was found in female rats where blood pressure stayed low for 3 weeks after a 5-day treatment with oxytocin. The effect of oxytocin on blood pressure seems to be potentiated by the female steroid hormones since in OVX rats the effect on blood pressure was similar to

Fig. 2. Systolic blood pressure and heart rate in spontaneously hypertensive male rats treated with oxytocin 1 mg/kg s.c. for 5 days compared to controls given saline for 5 days. Each point shows the mean ± SD. Circle, oxytocin (n = 5); square, NaCl (n = 5). ** p < 0.01, *** p < 0.001 oxytocin compared to controls. These data are printed with the kind permission of Elsevier Science B.V. where they were first published (Petersson et al., 1997).

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that found in male rats. When the duration of oxytocin treatment was doubled, the reduction in blood pressure persisted for 3 weeks in the OVX rats. When oxytocin was injected in high doses s.c. (1 mg/kg) for 5 days, an effect similar to that after i.c.v. treatment was found, that is, a decrease in blood pressure without a change in heart rate in both male and female rats (Petersson et al., 1996b, 1999d). Since oxytocin in high amounts may pass the blood– brain barrier (Jones and Robinson, 1982; Ermisch et al., 1985) and since we also found increased concentrations of oxytocin in the cerebrospinal fluid from rats injected with oxytocin (1 mg/kg s.c.), we have suggested that this long-lasting decrease in blood pressure is mediated within the CNS. Oxytocin

also decreases blood pressure in male, but surprisingly, not in female spontaneously hypertensive rats (SHR), though the effect is not as long-lasting as in the normotensive rats (Fig. 2). Interestingly, brain oxytocin concentrations are decreased in the SHR (Möhring et al., 1983). There were additional effects in rats treated for 5 days with oxytocin (1 μg/kg i.c.v. or 1 mg/kg s.c.). They had significantly decreased corticosterone levels, increased nociceptive thresholds, changed spontaneous motor activity, decreased plasma levels of gastrin, insulin and cholecystokinin and increased plasma levels of insulin-like growth factors and increased activity in the alpha 2-adrenoreceptors in the CNS (Petersson et al., 1996a, 1998a, 1999a,b, 1999c).

Fig. 3. Representative recording illustrating clonidine-induced inhibition of a single noradrenergic neuron in the locus coeruleus for rats treated with oxytocin (1 mg/kg s.c.) for 5 days (top) and for controls injected with saline s.c. (bottom). Each vertical arrow indicates 0.5 μg/kg of clonidine given intravenously. These data are printed with the kind permission of Elsevier Science B.V. where they were first published (Petersson et al., 1998b).

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Oxytocin and alpha 2-adrenoreceptors The long-lasting decrease in blood pressure induced by oxytocin treatment is not reversed by an oxytocin antagonist. However, idazoxan i.c.v., an alpha 2-adrenoreceptor antagonist which increases blood pressure, does not induce this effect in rats treated with oxytocin for five days. In addition, the effects of clonidine, an alpha 2-adrenoreceptor agonist, on both blood pressure and spontaneous motor activity are enhanced in oxytocin-treated rats (Petersson et al., 1999c). Indeed, oxytocin-treated rats have increased alpha 2-adrenoreceptor activity in several brain areas. In the hypothalamus, amygdala, NTS and the thalamic paraventricular nucleus, the number of alpha 2-adrenoreceptors, as measured by autoradiography, is increased in response to oxytocin (Diaz-Cabiale et al., 2000b). In the LC, the responsiveness of the alpha 2-adrenoreceptors, as measured by electrophysiological single cell recording, is significantly enhanced (Fig. 3) (Petersson et al., 1998b). In contrast to the long-lasting decrease in blood pressure in response to oxytocin and the enhanced effect of clonidine administered to oxytocin-pretreated rats, oxytocin injected into the NTS counteracts the vasodepressor and bradycardic effects of clonidine, and in vitro oxytocin decreases the affinity while increasing the number of alpha 2-adrenoreceptors within the NTS (Diaz-Cabiale et al., 2000a). Thus the immediate effect of oxytocin may be to antagonize alpha 2-adrenoreceptor function, whereas in a more longterm perspective oxytocin seems to enhance alpha 2-adrenoreceptor activity. Summary In this review, we have tried to summarize the most important findings concerning oxytocin and cardiovascular regulation. Obviously, oxytocin can affect this system in several ways. In the periphery, for example, by effects on the kidney, heart and blood vessels where oxytocin can interact with NO and ANP. In the CNS, where oxytocinergic fibers reach important regions for cardiovascular regulation, such as the LC and the vagal nuclei, interactions with alpha 2-adrenoreceptors might possibly be of importance for the effects of oxytocin on blood pressure in rats. Several questions concerning the cardiovas-

cular effects of oxytocin remain to be answered. For example, we still do not know if oxytocin is of any importance for cardiovascular regulation in humans. Hopefully, we will obtain the answers in the future and with them, probably many new interesting questions. Abbreviations ANP CNS DMX i.c. i.c.v. i.t. i.v. LC NO NTS OVX PVN RVLM s.c. SHR SON

atrial natriuretic peptide central nervous system dorsal motor nucleus of the vagus nerve intracisternally intraventricularly intrathecally intravenous locus coerulus nitric oxide nucleus of the solitary tract ovariectomized paraventricular nucleus rostral ventrolateral medulla subcutaneous spontaneously hypertensive rats supraoptic nucleus

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D. Poulain, S. Oliet and D. Theodosis (Eds.) Progress in Brain Research, Vol. 139 © 2002 Published by Elsevier Science B.V.

CHAPTER 23

Treatment of the diabetic patient: focus on cardiovascular and renal risk reduction Kevin C. Abbott 1 and George L. Bakris 2,* 2

1 Nephrology Service, Walter Reed Army Medical Center, Washington, DC, USA Rush University Hypertension Center, Department of Preventive Medicine, Rush Presbyterian–St. Luke’s Medical Center, Chicago, IL 60612, USA

Abstract: Diabetes mellitus increases the risk for hypertension and associated cardiovascular diseases, including coronary, cerebrovascular, renal and peripheral vascular disease. The risk for developing cardiovascular disease is increased when both diabetes and hypertension co-exist; in fact, over 11 million Americans have both diabetes and hypertension. These numbers will continue to climb, internationally, since the leading associated risk for diabetes development, obesity, has reached epidemic proportions, globally. Moreover, the frequent association of diabetes with dyslipidemia, as well as coagulation, endothelial, and metabolic abnormalities also aggravates the underlying vascular disease process in patients who possess these comorbid conditions. The renin–angiotensin–aldosterone system (RAS) and arginine vasopressin (AVP) are overactivated in both hypertension and diabetes. Drugs that inhibit this system, such as ACE inhibitors and more recently angiotensin receptor antagonists (ARBs), have proven beneficial effects on the micro- and macrovascular complications of diabetes, especially the kidney. The BRILLIANT study showed that lisinopril reduces microalbuminuria better than CCB therapy. Numerous other long-term studies confirm this association with ACE inhibitors including the HOPE trial. Furthermore, the European Controlled trial of Lisinopril in Insulin-dependent Diabetes (EUCLID) study, showed that lisinopril slowed the progression of renal disease, even in individuals with mild albuminuria. In fact, there are now five appropriately powered randomized placebo-controlled trials to show that both ACE inhibitors and ARBs slow progression of diabetic nephropathy in people with type 2 diabetes. These effects were shown to be better than conventional blood pressure lowering therapy, including dihydropyridine CCBs. In patients with microalbuminuria, ACE inhibitors and ARBs reduce the progression of microalbuminuria to proteinuria and provide a risk reduction of between 38 and 60% for progression to proteinuria. This is important since microalbuminuria is known to be associated with increased vascular permeability and decreased responsiveness to vasodilatory stimuli. Recently, increased AVP levels have been lined to microalbuminuria and hyperfiltration in diabetes. The microvascular and macrovascular benefits of ACE inhibition, ARBs and possible role of AVP antagonists in diabetic patients will be discussed, as will be recommendations for its clinical use.

Introduction Diabetes is one of the strongest risk factors for cardiovascular disease and renal disease. Diabetes is the ∗ Correspondence

to: G.L. Bakris, Rush University Hypertension Center, Department of Preventive Medicine, Rush Presbyterian – St. Luke’s Medical Center, 1700 W. Van Buren, Suite 470, Chicago, IL 60612, USA. Tel.: +1-312563-2195; Fax: +1-312-942-8119; E-mail: [email protected]

single most common cause of end-stage renal disease (ESRD) in the United States, and at its current rate of increase will account for a majority of all incident cases of ESRD in the near future (http://www.usrds.org). Chronic renal disease and cardiac disease track together. Hence, one cannot develop chronic renal disease without the presence of either clinically apparent or occult cardiac disease. Over half of all deaths observed in people with ESRD requiring dialysis are attributable to cardiovascular causes (Vincent and Kimura, 1992; El-Reshaid et al.,

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1994; Foley et al., 1994). Chronic renal insufficiency is traditionally defined as reduction in glomerular filtration rate to levels of ≤70 ml/min in persons aged ≤70 years. Clinically, this is manifest as an elevation in serum creatinine concentration above the normal rage (≥1.2 mg/dl in women or ≥1.4 mg/dl in men). This decrease in renal function along with either a decrease in total plasma protein or albumin or an increase in daily microalbuminuria (>30 mg/dl < 300 mg/dl) or albuminuria (>300 mg/dl) clearly indicates the presence of renal dysfunction. Left ventricular hypertrophy and evidence of endothelial damage, including microalbuminuria, can manifest even in normotensive diabetics (Hirayama et al., 2000) and are nearly universal when diabetic patients develop hypertension (Okin et al., 2000). The numbers of patients with diabetes who develop cardiovascular and renal disease will doubtless continue to climb since the leading cause of type II diabetes, obesity, is increasing at epidemic proportions (Randomized Aldactone Evaluation Study, 1996). This chapter will focus on the key hormone systems that increase cardiovascular and renal risk in diabetic patients, and targeted pharmacotherapy to reduce these risks. Since the role of hormones has been established most clearly in arterial hypertension or the development of microalbuminuria, this chapter will primarily discuss their role in these settings. Autonomic neural signals A progressive fall in cardiac output activates the sympathetic neural system. The sympathetic nerves impinge upon the efferent arteriole of the glomerulus, and increased sympathetic activity increases efferent arteriolar vasoconstriction (mediated through alpha-receptors) (DiBona, 2000). This increased efferent arteriolar vasoconstriction causes a greater fraction of plasma to percolate through the glomerulus and be filtered. This relative increase in filtration of plasma leaves a greater concentration of proteins present when plasma finishes its course through the glomerulus and enters the network of capillaries surrounding the proximal tubule (Schmieder et al., 1997). This plasma has a greater oncotic pressure (because of the protein enrichment) which renders the plasma coursing through the peritubular capillaries more avid for recovering sodium filtered at

the glomerulus and now passing through the tubules, resulting in greater sodium retention as more sodium is now recovered from filtrate. The sympathetic nerves also stimulate renin release (through beta-receptors). Release of renin initiates the well-known cascade of the renin angiotensin system, and results in an increase in angiotensin-II. Angiotensin-II also contributes to increased efferent arteriolar vascular tone and, increases the filtration fraction, thereby rendering plasma more enriched with protein and therefore, more avid in sodium recovery. Plasma norepinephrine levels are frequently increased in patients with end-stage renal disease and chronic renal failure (Campese et al., 1981; Cuche et al., 1986; Campese and Kogosov, 1995; Rohmeiss et al., 1999; Grekas et al., 2001). In animal models of experimentally induced chronic renal failure, central nervous system sympathetic activity is increased, associated with increased blood pressure (Ye et al., 1997; Campese, 2000). Local administration of selective neurotoxins reduced blood pressure in such models (Bigazzi et al., 1994). The importance of renal afferent impulses in the generation and maintenance of blood pressure differs according to the hypertensive model. For instance, renal afferent impulses are significant in models of renovascular hypertension, but not in DOCA salt hypertension (Jing-Yun et al., 1985; Katholi et al., 1980, 1981). The decreased blood pressure associated with bilateral nephrectomy has been associated with lower sympathetic nerve firing and regional vascular resistance (Converse et al., 1992). Nitric oxide synthase (NOS) may be a critical mediator of neurogenic control of blood pressure (Bredt et al., 1990; Vincent and Kimura, 1992). Campese and colleagues have shown that expression of neuronal NOS is increased in the brains of rats with chronic renal failure and may attenuate the rise in sympathetic nerve firing (Campese et al., 1981). The precise molecular pathways mediating this pathway are still under active investigation. Angiotensin-II Several processes other than direct sympathetic beta1-receptor stimulation enhance renin release, and thereby, angiotensin-II production. As sodium absorp-

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tion in the more proximal renal tubule increases, the amount of sodium present in the distal parts of the nephron diminishes. This fall in distal nephron sodium concentration is an additional stimulus of renin release. Afferent arteriole stretch also falls as renal perfusion diminishes in the face of a falling cardiac output and this fall in afferent arteriolar tone represents another renin-release signal (Ichikawa et al., 1990). In addition to effects on efferent arteriolar tone, angiotensin-II also stimulates proximal tubule cells directly (Ito and Abe, 1996) to recover filtered sodium through enhancement of activity in the Na/H antiporter on the luminal side of the epithelia. Angiotensin-II is a potent stimulus to aldosterone production and release, and angiotensin-II, indirectly, stimulates distal tubule sodium recovery by stimulating aldosterone whose primary sodium reabsorption action is at these distal sites. The increased activity of the renin–angiotensin–aldosterone system (RAS) has been long noted in chronic renal failure (Rosenberg et al., 1994). The utility of angiotensin converting enzyme inhibitors (ACE) and angiotensin receptor blockers (ARB) has been voluminously described (Ichikawa et al., 1990). Aldosterone Aldosterone is produced and released under several regulatory circumstances. ACTH from the pituitary gland is a major regulator of aldosterone production. Angiotensin-II is probably more potent in its effects on aldosterone production and release, and appears to be more important then ACTH in aldosterone control in congestive heart failure. Increases in potassium intake and falls in sodium levels are additional factors, which increase aldosterone production and release. Aldosterone stimulates the activity of the sodium– potassium ATPase enzyme on the basolateral side of epithelium, and thereby prompts transporting epithelial cells, like those in the distal nephron and the cortical collecting duct of the kidney, to increase sodium reabsorption. As aldosterone increases sodium uptake into cells, potassium or hydrogen ions are extruded into the urinary lumen to replace the recovered sodium and balance the residual negative charges, which leads to hypokalemia and alkalosis (Gomez-Sanchez et al., 2001).

Vasopressin Arginine vasopressin (AVP) is produced in the posterior pituitary gland, and principally functions to control the osmolality of plasma and thirst. AVP release is increased in heart failure through non-osmolality related mechanisms. AVP works in the collecting ducts of the kidney through the V2-receptor to generate maximal urinary concentration by promoting water reabsorption. In congestive heart failure, the non-osmotic release of AVP causes the excretion of inappropriately concentrated urine, with the result that ingested water is retained generating hyponatremia because the retained water dilutes total body sodium (Schrier et al., 2001). Sodium is also retained, out of proportion to body needs and results in edema of the lungs and dependent regions. When congestive heart failure progresses to the level of stimulating the non-osmotic release of AVP, the relative water retention exceeds the relative sodium retention and hyponatremia develops. Levels of AVP are increased in chronic renal failure (Nonoguchi et al., 1996) and resistance to AVP has also been reported in CRF (Teitelbaum and McGuinness, 1995). These may account for the defective hydroosmotic response in chronic renal failure (Osorio and Teitelbaum, 1997). These developments have been associated with the loss of nocturnal drop in blood pressure (‘dipping’), which may precede the development of clinically evident hypertension (Jensen and Pedersen, 1997). An underlying mechanism for both the increase in AVP levels and AVP resistance may be decreased aquaporin levels, which has been noted in animal models (Kwon et al., 1998). Because the kidney may not respond adequately to AVP levels, levels are increased, with toxicity perhaps similar to other hormonal models (Bardoux et al., 1999; Bouby et al., 1999; Kwon et al., 2001). Aquaporins At least seven types of aquaporins are expressed in the kidney at distinct sites (Schrier et al., 1998a). In conditions of water retention including heart failure, both aquaporin-2 expression and apical membrane targeting is increased (Martin, 1998; Schrier and Martin, 1998; Schrier et al., 1998b; Martin et al.,

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Left Ventricular Hypertrophy Damaged Microvasculature Reduced capillary density (early finding)

Diabetes Smoking Anemia (Hct