Primer on the Autonomic Nervous System (Third Edition)

PRIMER ON THE AUTONOMIC NERVOUS SYSTEM THIRD EDITION PRIMER ON THE AUTONOMIC NERVOUS SYSTEM THIRD EDITION Editor In C...

Author: Italo Biaggioni | David W. Robertson | Geoffrey Burnstock | Phillip A. Low | Julian F.R. Paton

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PRIMER ON THE AUTONOMIC NERVOUS SYSTEM THIRD EDITION

PRIMER ON THE AUTONOMIC NERVOUS SYSTEM THIRD EDITION Editor In Chief

David Robertson Vanderbilt University

Editors

Italo Biaggioni Vanderbilt University

Geoffrey Burnstock

University College Medical School

Phillip A. Low

Mayo College of Medicine

Julian F.R. Paton University of Bristol

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 225 Wyman Street, Waltham, MA 02451, USA 525 B Street, Suite 1800, San Diego, CA 92101-4495, USA First edition 1996 Second edition 2004 Third edition 2012 Copyright © 2012 Elsevier Inc. All rights reserved Except the figures of chapter 76 for which the author retains copyright Cover Image: The human autonomic nervous system, which in large part lies deep within the body, is readily accessible through biopsies of the skin. In the cover image, using the cholinergic marker, vasoactive intestinal peptide (shown in red), there is clear visualization of the sympathetic cholinergic innervation running between the endothelial and basal lamina layers of cutaneous blood vessels in a skin biopsy of the distal thigh. The blood vessels are visualized with the endothelial vascular marker CD31 (an endothelial vascular marker that highlights blood vessels – shown in green) and the vascular basal lamina with collagen type IV (a marker that highlights the basal lamina layer of the extracellular matrix – shown in blue). Image courtesy of Ningshan Wang, Christopher Gibbons and Roy Freeman. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (44) (0) 1865 843830; fax (44) (0) 1865 853333; email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information 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 drug dosages should be made British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN : 978-0-12-386525-0 For information on all Academic Press publications visit our website at elsevierdirect.com Typeset by MPS Limited, a Macmillan Company, Chennai, India www.macmillansolutions.com Printed and bound in United States of America 12

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Primer on the Autonomic Nervous System (Third Edition) Copyright © 2012 Elsevier Inc. All rights reserved ISBN: 978-0-12-386525-0 About this Book | Shortcut link to this Title Add to Favorites

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Table of Contents Front-matter, Pages i,iii Show preview |

PDF (187 K) | Related articles | Related reference work articles

Copyright, Page iv Show preview |


Preface, Page xix, David Robertson, Italo Biaggioni, Geoffrey Burnstock, Phillip A. Low, Julian F.R. Paton Show preview |


List of Contributors, Pages xxi-xxv, Ana P.L. Abdala, David H. Adams, Marlies Alvarenga, Amy C. Arnold, Felicia B. Axelrod, Franca Barbic, Peter J. Barnes, Deborah Bauer, Christopher Bell, Eduardo E. Benarroch, Elizabeth M. Berry-Kravis, Luciano Bernardi, Italo Biaggioni, Lori Birder, Virginia L. Brooks, Joan Heller Brown, Geoffrey Burnstock, Michael Camilleri, J.Preston Campbell, Robert M. Carey, et al. Show preview |


Part I: Introduction Part II: Biochemical and Pharmacological Mechanisms Part III: Autonomic Physiology Part IV: Stress Part V: Pathophysiological Mechanisms Part VI: Cardiovascular Disorders Part VII: Catecholamine Disorders Part VIII: Autonomic Synucleinopathies Part IX: Peripheral Autonomic Disorders Part X: Autonomic Disorders Part XI: Orthostatic Intolerance Part XII: Special Clinical Conditions Part XIII: Management of Autonomic Disorders Part XIV: Experimental Autonomic Neuroscience Index, Pages 693-703 Show preview |


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Primer on the Autonomic Nervous System (Third Edition) Copyright © 2012 Elsevier Inc. All rights reserved ISBN: 978-0-12-386525-0 About this Book | Shortcut link to this Title Add to Favorites

Font Size: = Full-text available

= Abstract only

Table of Contents Front-matter, Pages i,iii Show preview |


Copyright, Page iv Show preview |


Preface, Page xix, David Robertson, Italo Biaggioni, Geoffrey Burnstock, Phillip A. Low, Julian F.R. Paton Show preview |


List of Contributors, Pages xxi-xxv, Ana P.L. Abdala, David H. Adams, Marlies Alvarenga, Amy C. Arnold, Felicia B. Axelrod, Franca Barbic, Peter J. Barnes, Deborah Bauer, Christopher Bell, Eduardo E. Benarroch, Elizabeth M. Berry-Kravis, Luciano Bernardi, Italo Biaggioni, Lori Birder, Virginia L. Brooks, Joan Heller Brown, Geoffrey Burnstock, Michael Camilleri, J.Preston Campbell, Robert M. Carey, et al. Show preview |


Part I: Introduction Chapter 1 - Development and Differentiation of Autonomic Neurons, Pages 3-8, Chun-Hyung Kim, Kwang-Soo Kim Show preview |


Chapter 2 - Central Autonomic Control, Pages 9-12, Eduardo E. Benarroch Show preview |


Chapter 3 - Imaging of Brainstem Sites Involved in Cardiovascular Control, Pages 13-16, Vaughan G. Macefield, Luke A. Henderson Show preview |


Chapter 4 - Peripheral Autonomic Nervous System, Pages 17-26, Robert W. Hamill, Robert E. Shapiro, Margaret A. Vizzard Show preview |


Chapter 5 - Cotransmission, Pages 27-33, Geoffrey Burnstock Show preview |


Part II: Biochemical and Pharmacological Mechanisms Chapter 6 - Noradrenergic Neurotransmission, Pages 37-43, David S. Goldstein Show preview |


Chapter 7 - Tyrosine Hydroxylase, Pages 45-47, Kazuto Kobayashi, Toshiharu Nagatsu Show preview |


Chapter 8 - Antidepressant-Sensitive Norepinephrine Transporters: Structure and Regulation, Pages 49-50, Maureen K. Hahn Show preview |


Chapter 9 - α -Adrenergic Receptors, Pages 51-54, Marion C. Mohl, Robert M. Graham 1 Show preview |


Chapter 10 - α -Adrenergic Receptors, Pages 55-58, Qin Wang 2 Show preview |


Chapter 11 - β-Adrenergic Receptors, Pages 59-61, C.Michael Stein Show preview |


Chapter 12 - Dopaminergic Neurotransmission, Pages 63-65, Graeme Eisenhofer, Heinz Reichmann Show preview |


Chapter 13 - Dopamine Receptors, Pages 67-70, Sean M. Peterson, Nikhil Urs, Marc G. Caron Show preview |


Chapter 14 - Cholinergic Neurotransmission, Pages 71-74, Brett A. English, Carrie K. Jones Show preview |


Chapter 15 - Acetylcholine and Muscarinic Receptors, Pages 75-78, Joan Heller Brown, Nora Laiken Show preview |


Chapter 16 - Nicotinic Receptors, Pages 79-82, Palmer Taylor Show preview |


Chapter 17 - Serotonin Receptors and Neurotransmission, Pages 83-86, Elaine Sanders-Bush, Charles D. Nichols Show preview |


Chapter 18 - Purinergic Neurotransmission and Nucleotide Receptors, Pages 87-93, Geoffrey Burnstock Show preview |


Chapter 19 - Adenosine Receptors and Autonomic Regulation, Pages 95-97, Italo Biaggioni Show preview |


Chapter 20 - Nitric Oxide and Autonomic Regulation, Pages 99-101, Alfredo Gamboa Show preview |


Chapter 21 - Glutamatergic Neurotransmission, Pages 103-107, Deborah Bauer, Michael Robinson Show preview |


Chapter 22 - GABAergic Neurotransmission, Pages 109-111, Andre H. Lagrange, Mark D. Grier Show preview |


Chapter 23 - Renin-Angiotensin, Pages 113-116, Amy C. Arnold, Debra I. Diz Show preview |


Chapter 24 - Aldosterone and the Mineralocorticoid Receptor, Pages 117-119, J. Howard Pratt Show preview |


Chapter 25 - Vasopressin and Disorders of Water Homeostasis, Pages 121-125, Joseph G. Verbalis Show preview |


Chapter 26 - Calcitonin Gene-Related Peptide and Adrenomedullin, Pages 127-129, Donald J. DiPette, Scott C. Supowit Show preview |


Chapter 27 - Leptin Signaling and Energy Homeostasis, Pages 131-134, Kamal Rahmouni Show preview |


Chapter 28 - The Endothelin System, Pages 135-139, Ernesto L. Schiffrin Show preview |


Chapter 29 - Pharmacology of the Nucleous Tractus Solitarii, Pages 141-144, Ching-Jiunn Tseng, Pei-Wen Cheng, Che-Se Tung Show preview |


Part III: Autonomic Physiology Chapter 30 - Entrainment of Sympathetic Rhythms, Pages 147-149, Michael P. Gilbey Show preview |


Chapter 31 - Cross-talk Between Body Systems: Respiratory-Cardiovascular Coupling in Health and Disease, Pages 151-155, Julian F.R. Paton, Anthony E. Pickering Show preview |


Chapter 32 - Circadian Rhythms and Autonomic Function, Pages 157-159, Diego A. Golombek Show preview |


Chapter 33 - Baroreceptor Reflexes, Pages 161-165, Mark W. Chapleau Show preview |


Chapter 34 - Genetic Determinants of Baroreflex Function, Pages 167-169, Italo Biaggioni Show preview |


Chapter 35 - Cardiac and Other Visceral Afferents, Pages 171-176, John C. Longhurst, Liang-Wu Fu Show preview |


Chapter 36 - Autonomic Control of the Heart, Pages 177-180, Rachel C. Drew, Lawrence I. Sinoway Show preview |


Chapter 37 - Cardiac Vagal Ganglia, Pages 181-185, Javier G. Castillo, David H. Adams Show preview |


Chapter 38 - Neural Control of Blood Vessels, Pages 187-191, Julian H. Lombard, Allen W. Cowley Jr. Show preview |


Chapter 39 - Physiology of Upright Posture, Pages 193-195, Wouter Wieling, Jan T. Groothuis Show preview |


Chapter 40 - Cerebral Circulation, Pages 197-200, Ronald Schondorf Show preview |


Chapter 41 - Autonomic Control of the Lower Airways, Pages 201-204, Peter J. Barnes Show preview |


Chapter 42 - Gastrointestinal Function, Pages 205-209, Michael Camilleri Show preview |


Chapter 43 - The Splanchnic Circulation, Pages 211-213, Gregory D. Fink, John W. Osborn Show preview |


Chapter 44 - Autonomic Control of the Kidney, Pages 215-220, Edwin K. Jackson Show preview |


Chapter 45 - Dopamine Mechanisms in the Kidney, Pages 221-223, Robert M. Carey Show preview |


Chapter 46 - Autonomic Control of the Lower Urinary Tract, Pages 225-228, Lori Birder, William C. de Groat Show preview |


Chapter 47 - Bladder Function in Health and Disease, Pages 229-233, Marcus J. Drake, Brian A. Parsons Show preview |


Chapter 48 - Physiology and Pathophysiology of Female Sexual Function, Pages 235-238, Max J. Hilz Show preview |


Chapter 49 - Control of the Pupil, Pages 239-242, Karen M. Joos, Mark R. Melson Show preview |


Chapter 50 - Central Thermoregulation, Pages 243-247, Shaun F. Morrison Show preview |


Chapter 51 - Sweating, Pages 249-251, Phillip A. Low Show preview |


Chapter 52 - Regulation of Metabolism, Pages 253-255, Christopher Bell Show preview |


Chapter 53 - Autonomic Innervation of the Skeleton, Pages 257-259, Florent Elefteriou, J.Preston Campbell Show preview |


Chapter 54 - Sex Differences in Autonomic Function, Pages 261-264, Emma C. Hart, Nisha Charkoudian, Michael J. Joyner Show preview |


Chapter 55 - Autonomic Control During Pregnancy, Pages 265-268, Virginia L. Brooks, Belinda H. McCully, Priscila A. Cassaglia Show preview |


Part IV: Stress Chapter 56 - Aging and the Autonomic Nervous System, Pages 271-273, Lewis A. Lipsitz, Vera Novak Show preview |


Chapter 57 - Exercise, Pages 275-279, Qi Fu, Benjamin D. Levine Show preview |


Chapter 58 - Effects of High Altitude, Pages 281-282, Luciano Bernardi Show preview |


Chapter 59 - Space Physiology, Pages 283-285, Gilles Clément, Scott Wood Show preview |


Chapter 60 - Hypothermia and Hyperthermia, Pages 287-289, Nisha Charkoudian Show preview |


Chapter 61 - Psychological Stress and the Autonomic Nervous System, Pages 291-293, Michael G. Ziegler Show preview |


Chapter 62 - Mind–Body Interactions, Pages 295-299, Calvin Carter, Daniel Tranel Show preview |


Part V: Pathophysiological Mechanisms Chapter 63 - Alpha-Synuclein and Neurodegeneration, Pages 303-305, Kiren Ubhi, Leslie Crews, Eliezer Masliah Show preview |


Chapter 64 - Insulin Resistance and the Autonomic Nervous System, Pages 307-312, Megan S. Johnson, Vincent G. DeMarco, Adam WhaleyConnell, James R. Sowers Show preview |


Chapter 65 - Salt Sensitivity of Blood Pressure, Pages 313-318, Cheryl L. Laffer, Fernando Elijovich Show preview |


Chapter 66 - Endothelial Dysfunction, Pages 319-324, Julian P.J. Halcox Show preview |


Chapter 67 - Inflammation, Immunity and the Autonomic Nervous System, Pages 325-329, Paul J. Marvar, David G. Harrison Show preview |


Chapter 68 - Oxygen Sensing, Pages 331-333, Nanduri R. Prabhakar Show preview |


Chapter 69 - Reactive Oxygen Species and Oxidative Stress, Pages 335-338, Rhian M. Touyz Show preview |


Part VI: Cardiovascular Disorders Chapter 70 - Neurally Mediated Syncope, Pages 341-343, Satish R. Raj Show preview |


Chapter 71 - Sympatho-Vagal Imbalance in Hypertension, Pages 345-348, Guido Grassi, Gino Seravalle Show preview |


Chapter 72 - Baroreflex Failure, Pages 349-353, Jens Jordan Show preview |


Chapter 73 - Blood Pressure Variability, Pages 355-357, Stanley Fernandez, Sirisha Srikakarlapudi, Joseph L. Izzo Show preview |


Chapter 74 - Obesity-Associated Hypertension, Pages 359-361, Cyndya Shibao Show preview |


Chapter 75 - Orthostatic Hypertension, Pages 363-365, David Robertson Show preview |


Chapter 76 - Heart Failure, Pages 367-370, John S. Floras Show preview |


Chapter 77 - Stress Cardiomyopathy and Takotsubo Syndrome, Pages 371-375, David Robertson, Kyoko Sato Show preview |


Chapter 78 - Clinical Evaluation of Autonomic Disorders, Pages 377-382, Paola Sandroni Show preview |


Chapter 79 - Tilt Table Studies, Pages 383-387, Satish R. Raj Show preview |


Chapter 80 - Sympathetic Microneurography, Pages 389-392, B. Gunnar Wallin Show preview |


Chapter 81 - Clinical Applications of Microneurography, Pages 393-397, Tadaaki Mano Show preview |


Chapter 82 - Clinical Sympathetic Imaging, Pages 399-403, David S. Goldstein Show preview |


Chapter 83 - Assessment of the Autonomic Control of the Cardiovascular System by a Frequency Domain Approach, Pages 405-408, Raffaello Furlan, Franca Barbic Show preview |


Chapter 84 - Assessment of Sudomotor Function, Pages 409-411, Ronald Schondorf Show preview |


Chapter 85 - Cutaneous Autonomic Innervation: Assessment by Skin Biopsy, Pages 413-417, Christopher H. Gibbons, Roy Freeman Show preview |


Part VII: Catecholamine Disorders Chapter 86 - Pheochromocytoma, Pages 421-425, Graeme Eisenhofer, Jacques W.M. Lenders, William M. Manger Show preview |


Chapter 87 - Deficiencies of Tetrahydrobiopterin, Tyrosine Hydroxylase and Aromatic L-Amino Acid Decarboxylase, Pages 427-430, Keith Hyland, Lauren A. Hyland Show preview |


Chapter 88 - Dopamine β-Hydroxylase Deficiency, Pages 431-434, Emily M. Garland Show preview |


Chapter 89 - Menkes Disease and Other ATP7A-Related Phenotypes, Pages 435-437, Stephen G. Kaler Show preview |


Chapter 90 - Norepinephrine Transporter Deficiency, Pages 439-442, Maureen K. Hahn Show preview |


Chapter 91 - Monoamine Oxidase Deficiency, Pages 443-444, Jacques W.M. Lenders, Graeme Eisenhofer Show preview |


Chapter 92 - Congenital Central Hypoventilation Syndrome (CCHS) and PHOX2B Mutations, Pages 445-449, Debra E. Weese-Mayer, Pallavi P. Patwari, Casey M. Rand, André Diedrich, Nancy L. Kuntz, Elizabeth M. Berry-Kravis Show preview |


Part VIII: Autonomic Synucleinopathies Chapter 93 - Multiple System Atrophy, Pages 453-457, David Robertson, Sid Gilman Show preview |


Chapter 94 - Parkinson’s Disease, Pages 459-461, John Y. Fang, Thomas L. Davis Show preview |


Chapter 95 - Dementia with Lewy Bodies, Pages 463-466, Sylvia Stemberger, Michaela Stampfer, Gregor K. Wenning Show preview |


Chapter 96 - Pure Autonomic Failure, Pages 467-469, Horacio Kaufmann, Irwin J. Schatz Show preview |


Part IX: Peripheral Autonomic Disorders Chapter 97 - Diagnostic Workup of Peripheral Neuropathies with Dysautonomia, Pages 473-475, Amanda C. Peltier Show preview |


Chapter 98 - Diabetic Autonomic Dysfunction, Pages 477-481, Amanda C. Peltier, Stephen N. Davis Show preview |


Chapter 99 - Amyloidotic Autonomic Failure, Pages 483-487, Yadollah Harati, Cecile L. Phan Show preview |


Chapter 100 - Autoimmune Autonomic Ganglionopathy, Pages 489-492, Steven Vernino, Phillip A. Low Show preview |


Chapter 101 - Guillain–Barré Syndrome, Pages 493-494, Phillip A. Low, James G. McLeod Show preview |


Chapter 102 - Hereditary Autonomic Neuropathies, Pages 495-498, Yadollah Harati, Shahram Izadyar Show preview |


Chapter 103 - Familial Dysautonomia (Riley–Day Syndrome), Pages 499-502, Horacio Kaufmann, Lucy Norcliffe-Kaufmann, Felicia B. Axelrod Show preview |


Part X: Autonomic Disorders Chapter 104 - Autonomic Disturbances in Spinal Cord Injuries, Pages 505-509, Christopher J. Mathias, David A. Low Show preview |


Chapter 105 - Drug-Induced Autonomic Dysfunction, Pages 511-514, James M. Luther Show preview |


Part XI: Orthostatic Intolerance Chapter 106 - Postural Tachycardia Syndrome (POTS), Pages 517-519, Phillip A. Low, Paola Sandroni Show preview |


Chapter 107 - Mechanisms of Postural Tachycardia Syndrome, Pages 521-523, Satish R. Raj Show preview |


Chapter 108 - Symptoms and Signs of Postural Tachycardia Syndrome (POTS), Pages 525-528, Julian Stewart Show preview |


Chapter 109 - Delayed Orthostatic Hypotension, Pages 529-530, Christopher H. Gibbons, Roy Freeman Show preview |


Chapter 110 - Chronic Fatigue Syndrome and the Autonomic Nervous System, Pages 531-534, Luis E. Okamoto, Satish R. Raj, Italo Biaggioni Show preview |


Chapter 111 - Joint Hypermobility Syndrome and Dysautonomia, Pages 535-537, Giris Jacob, Blair P. Grubb Show preview |


Part XII: Special Clinical Conditions Chapter 112 - Neuroleptic Malignant Syndrome, Pages 541-544, Fenna T. Phibbs, P. David Charles Show preview |


Chapter 113 - Migraine and the Autonomic Nervous System, Pages 545-547, Pietro Cortelli Show preview |


Chapter 114 - Epilepsy and Autonomic Regulation, Pages 549-552, Matthias Dütsch, Max J. Hilz Show preview |


Chapter 115 - Disorders of Sweating, Pages 553-559, Robert D. Fealey Show preview |


Chapter 116 - Male Erectile Dysfunction, Pages 561-564, Douglas F. Milam Show preview |


Chapter 117 - Sleep Apnea, Pages 565-569, Tomas Konecny, Virend K. Somers Show preview |


Chapter 118 - Altered Adrenal Function and the Autonomic Nervous System, Pages 571-574, Subbulaxmi Trikudanathan, Gordon H. Williams Show preview |


Chapter 119 - Mastocytosis, Pages 575-576, L. Jackson Roberts II Show preview |


Chapter 120 - Cocaine Overdose, Pages 577-581, Andrew Kontak, Wanpen Vongpatanasin, Ronald G. Victor Show preview |


Chapter 121 - Complex Regional Pain Syndrome, Pages 583-587, Wilfrid Jänig Show preview |


Chapter 122 - Carcinoid Tumors, Pages 589-591, Kenneth R. Hande Show preview |


Chapter 123 - Paraneoplastic Autonomic Dysfunction, Pages 593-596, Ramesh K. Khurana Show preview |


Chapter 124 - Abdominal Pain and Cyclic Vomiting, Pages 597-600, Gisela Chelimsky, Thomas Chelimsky Show preview |


Chapter 125 - Fecal Incontinence, Pages 601-602, James F.X. Jones Show preview |


Part XIII: Management of Autonomic Disorders Chapter 126 - Panic Disorder, Pages 605-608, Murray Esler, Marlies Alvarenga, David Kaye, Gavin Lambert, Jane Thompson, Jacqui Hastings, Rosemary Schwarz, Margaret Morris, Jeff Richards Show preview |


Chapter 127 - Physical Measures, Pages 609-612, Wouter Wieling, Roland D. Thijs Show preview |


Chapter 128 - Water and the Osmopressor Response, Pages 613-615, Jens Jordan Show preview |


Chapter 129 - Droxidopa (L-DOPS), Pages 617-620, Horacio Kaufmann Show preview |


Chapter 130 - Midodrine, Adrenergic Agonists and Antagonists, Pages 621-625, Janice L. Gilden Show preview |


Chapter 131 - Agents Potentiating Sympathetic Tone, Pages 627-630, Cyndya Shibao, Luis Okamoto Show preview |


Chapter 132 - Acetylcholinesterase and its Inhibitors, Pages 631-633, Brett A. English, Andrew A. Webster Show preview |


Chapter 133 - Fludrocortisone, Pages 635-637, David Robertson, Rose Marie Robertson Show preview |


Chapter 134 - Acarbose, Pages 639-641, Cyndya Shibao Show preview |


Chapter 135 - Erythropoietin in Autonomic Failure, Pages 643-644, Italo Biaggioni Show preview |


Chapter 136 - Somatostatin Agonists, Pages 645-648, Robert Hoeldtke Show preview |


Chapter 137 - Harnessing the Autonomic Nervous System for Therapeutic Intervention, Pages 649-652, Murray Esler Show preview |


Chapter 138 - Acupuncture Regulation of Cardiovascular Function, Pages 653-657, John C. Longhurst Show preview |


Chapter 139 - Bionic Baroreflex, Pages 659-662, Takayuki Sato, André Diedrich, Kenji Sunagawa Show preview |


Part XIV: Experimental Autonomic Neuroscience Chapter 140 - Anesthetic Management in Autonomic Disorders, Pages 665-668, Chih Cherng Lu, Shung Tai Ho, Che Se Tung Show preview |


Chapter 141 - Evolution of the Cardiovascular Autonomic Nervous System in Vertebrates, Pages 669-673, Tobias Wang Show preview |


Chapter 142 - Human Physiome, Pages 675-680, Peter Hunter Show preview |


Chapter 143 - Modeling the Autonomic Nervous System, Pages 681-687, Ilya A. Rybak, Yaroslav I. Molkov, Julian F.R. Paton, Ana P.L. Abdala, Daniel B. Zoccal Show preview |


Chapter 144 - Optogenetics, Pages 689-691, Sergey Kasparov Show preview |


Index, Pages 693-703 Show preview |

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Preface

The Primer on the Autonomic Nervous System aims to provide a concise and accessible overview of autonomic neuroscience for students, scientists, and clinicians. In spite of its compact size, its 144 chapters draw on the expertise of more than 250 scientists and clinicians. We thank the American Autonomic Society for its continued interest and moral support of this project. We especially express appreciation to our contributors, who, along with the editors, prepared their chapters without compensation in order to keep the cost of the Primer within the reach of students and trainees. We are delighted with the enthusiastic reception of the first and second English editions, and the Japanese edition of the Primer, which has sold more copies than any previous text on autonomic neuroscience. With this edition we welcome Julian F.R. Paton as a new editor. The third edition of the Primer would not have been possible without Mrs. Sonja Campbell, whose efficiency

and wisdom, combined with her mastery of lucid English prose, facilitated the preparation of this substantially enlarged edition. We also thank Mica Haley and Melissa Turner at Academic Press, who kept all of us on track and on schedule. In earlier editions, readers were encouraged to email their criticisms and advice for improving the text. We thank the many of you who took time to do just that. Several new sections and a number of clarifications based on these suggestions have been implemented. If you have comments or advice for improving future editions please send them to [email protected].

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David Robertson Italo Biaggioni Geoffrey Burnstock Phillip A. Low Julian F.R. Paton

List of Contributors

Ana P.L. Abdala School of Physiology and Pharmacology, Bristol Heart Institute, University of Bristol, Bristol, UK David H. Adams Guggenheim Pavillion, New York, NY, USA Marlies Alvarenga Baker IDI Heart and Diabetes Institute, Melbourne, Australia Amy C. Arnold Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA Felicia B. Axelrod Professor, Pediatrics and Neurology, New York University School of Medicine, New York, NY 10016, USA Franca Barbic Neuroscience Research Association, Bolognini Hospital, Seriate (Bg), Italy Peter J. Barnes Department of Thoracic Medicine, National Heart and Lung Institute, London, UK Deborah Bauer Departments of Pediatrics and Pharmacology, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA Christopher Bell Department of Health and Exercise Science, Colorado State University, Fort Collins, CO, USA Eduardo E. Benarroch Department of Neurology, Mayo Clinic, Rochester, MN, USA Elizabeth M. Berry-Kravis Professor of Pediatrics, Neurology, and Biochemistry at Rush University Medical Center, Chicago, IL 60612, USA Luciano Bernardi Clinica Medica 2 – Dipartimento Medicina Interna, IRCCS S. Matteo, Universita’ di Pavia, 27100 Pavia, Italy Italo Biaggioni Professor of Medicine and Pharmacology, Vanderbilt University, Nashville, TN 37212, USA Lori Birder University of Pittsburgh School of Medicine, Departments of Medicine and Pharmacology, Pittsburgh PA 15261, USA Virginia L. Brooks Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA Joan Heller Brown Department of Pharmacology, University of California, San Diego School of Medicine, La Jolla, CA, USA Geoffrey Burnstock Autonomic Neuroscience Centre, University College Medical School, London NW3 2PF, UK Michael Camilleri Mayo Clinic, Rochester, MN, USA J. Preston Campbell Vanderbilt University Medical Center, Nashville, TN 37232, USA Robert M. Carey Division of Endocrinology, University of Virginia Health Systems, Charlottesville, VA, USA Marc G. Caron Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA Calvin Carter Department of Neurology, University of Iowa, College of Medicine, Iowa City, IA, USA Priscila A. Cassaglia Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA Javier G. Castillo Resident Physician, Department of Cardiothoracic Surgery, The Mount Sinai School of Medicine, New York, NY, USA

Primer on the Autonomic Nervous System. DOI: 10.1016/B978-0-12-386525-0.00149-9

Mark W. Chapleau Departments of Internal Medicine, and Molecular Physiology and Biophysics, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA, USA Nisha Charkoudian Department of Anesthesiology and Department of Physiology, and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA P. David Charles Movement Disorders Clinic, Medical Center South, Vanderbilt University, Nashville, TN, USA Gisela Chelimsky Department of Pediatrics, Rainbow Babies and Children’s Hospital, and University Hospitals Case Medical Center, Cleveland, OH 44106, USA Thomas Chelimsky Department of Neurology, University Hospitals Case Medical Center, Cleveland, OH 44106, USA Pei-Wen Cheng Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC Gilles Clément International Space University, Strasbourg, France Pietro Cortelli Department of Neurological Sciences, Alma Mater Studiorum, University of Bologna, 40123 Bologna, Italy Allen W. Cowley Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA Leslie Crews Department of Pathology, University of California, San Diego/La Jolla, CA, USA Stephen N. Davis Chair, Internal Medicine, University of Maryland, Baltimore, MD, USA Thomas L. Davis Clinical Research Center, Vanderbilt University, Nashville, TN, USA William C. de Groat University of Pittsburgh School of Medicine, Departments of Medicine and Pharmacology, Pittsburgh, PA 15261, USA Vincent G. DeMarco University of Missouri, Diabetes and Cardiovascular Center, and the Harry S. Truman VA Medical Center, Columbia, MO, USA André Diedrich Autonomic Dysfunction Center, Department of Medicine and Department of Biomedical Engineering, Vanderbilt University School of Medicine, Nashville, TN, USA Donald J. DiPette Departments of Medicine (DJD), and Cell Biology and Anatomy (SCS), University of South Carolina School of Medicine, Columbia, SC 29208, USA Debra I. Diz Professor and Director, Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1032, USA Marcus J. Drake FRCS(Urol)Bristol Urological Institute, Bristol, UK Rachel C. Drew Heart and Vascular Institute, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, PA, USA Matthias Dütsch Department of Neurology, University of Erlangen-Nuremberg, D-91054 Erlangen, Germany, and Department of Neurology, Rummelsberg Hospital, D-90592 Schwarzenbruck, Germany Graeme Eisenhofer Department of Medicine, and Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Dresden, Dresden, Germany

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© 2012 Elsevier Inc. All rights reserved.

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LIst of ContrIbutors

Florent Elefteriou Assistant Professor and Director Elect, Vanderbilt Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA Fernando Elijovich Professor of Medicine, Texas A&M HSC, College of Medicine and Center for Neuroscience, USA Brett A. English Division of Allergy, Pulmonary and Critical Care Medicine, Drug Discovery, Vanderbilt University Medical Center, Nashville, TN, USA Murray Esler Baker IDI Heart and Diabetes Institute, Melbourne, Australia John Y. Fang Assistant Professor, Department of Neurology, Vanderbilt University, Nashville, TN, USA and Staff Physician, Neurology Service, Tennessee Valley Healthcare System, Nashville, TN, USA Robert D. Fealey Department of Neurology, Mayo Clinic, Rochester, MN, USA Stanley Fernandez Department of Medicine, State University of New York at Buffalo, Buffalo, NY 14215, USA Gregory D. Fink Michigan State University, Department of Pharmacology and Toxicology, East Lansing, MI 48840, USA John S. Floras University Health Network and Mount Sinai Hospital Department of Medicine, University of Toronto, Toronto, Ont., Canada Roy Freeman Center for Autonomic and Peripheral Nerve Disorders, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA Qi Fu Institute for Exercise and Environmental Medicine, Texas Health Presbyterian Hospital Dallas, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Liang-Wu Fu Department of Medicine, School of Medicine, University of California, Irvine, Irvine, CA 92697-4075, USA Raffaello Furlan Internal Medicine, Bolognini Hospital, Seriate (Bg), University of Milan, Milan, Italy Alfredo Gamboa Autonomic Dysfunction Center, Vanderbilt University, Nashville, TN, USA Emily M. Garland Division of Clinical Pharmacology, Medical Center North, Vanderbilt University, Nashville, TN, USA Christopher H. Gibbons Center for Autonomic and Peripheral Nerve Disorders, Beth Israel Deaconess Medical Center, Boston MA 02215, USA Michael P. Gilbey Department of Physiology, University College London, London, UK Janice L. Gilden Professor of Medicine, Rosalind Franklin University of Medicine and Science, James A. Lovell Federal Health Care Center, North Chicago, and Saints Mary and Elizabeth Medical Center, Chicago, IL, USA Sid Gilman Department of Neurology, 300 N. Ingalls St. 3D15, Ann Arbor, MI, USA David S. Goldstein National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA Diego A. Golombek Universidad Nacional de Quilmes/ CONICET, Buenos Aires, Argentina Robert M. Graham Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia Guido Grassi Università Milano-Bicocca, Ospedale San Gerardo, Monza (Milan), Milan, Italy, andIstituto Auxologico Italiano, Milan, Italy

Mark D. Grier Department of Pharmacology, Vanderbilt University, Nashville, TN, USA Jan T. Groothuis Department of Physiology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands, and, Department of Rehabilitation, St Maartenskliniek, 6500 GM Nijmegen, The Netherlands Blair P. Grubb Recanati Autonomic Dysfunction Center, Tel Aviv University, Faculty of Medicine, Tel-Aviv 64239, Israel Maureen K. Hahn Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Julian P.J. Halcox Professor of Cardiology, Cardiff University School of Medicine, Wales Heart Research Institute, Cardiff, CF14 4XN, UK Robert W. Hamill Department of Neurology, University of Vermont, College of Medicine, Burlington, VT, USA Kenneth R. Hande Division of Medical Oncology, Preston Research Building, Nashville, TN, USA Yadollah Harati Department of Neurology, Baylor College of Medicine, Houston, TX, USA David G. Harrison Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, TN, USA Emma C. Hart Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA Jacqui Hastings Baker IDI Heart and Diabetes Institute, Melbourne, Australia Luke A. Henderson Department of Anatomy and Histology, University of Sydney, Sydney, Australia Max J. Hilz Department of Neurology, University of ErlangenNuremberg, D-91054 Erlangen, Germany, and Departments of Neurology, Medicine, Psychiatry, New York University School of Medicine, New York, NY 10016, USA Robert Hoeldtke Division of Endocrinology, West Virginia University, Morgantown, WV, USA Shung Tai Ho Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, ROC Peter Hunter Auckland Bioengineering Institute, University of Auckland, New Zealand Keith Hyland Department of Neurochemistry, Medical Neurogenetics, Atlanta, GA, USA Lauren Hyland Department of Neurochemistry, Medical Neurogenetics, Atlanta, GA, USA Shahram Izadyar Department of Neurology, Baylor College of Medicine, Houston, TX, USA Joseph L. Izzo Department of Medicine, State University of New York at Buffalo, Buffalo, NY 14215, USA Edwin K. Jackson University of Pittsburgh School of Medicine, Department of Pharmacology and Chemical Biology, Pittsburgh, PA 15219, USA Giris Jacob Head of Medicine F, Recanati Autonomic Dysfunction Center, Tel Aviv (Sourasky) Medical Center, Tel Aviv University, Faculty of Medicine, Tel-Aviv 64239, Israel Wilfrid Jänig Physiologisches Institut, Christian-AlbrechtsUniversität zu Kiel, Kiel, Germany Megan S. Johnson University of Missouri, Diabetes and Cardiovascular Center, Columbia, MO, USA Carrie K. Jones Department of Pharmacology, and Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN, USA James F.X. Jones School of Medicine and Medical Sciences, University College Dublin, Ireland

I. INTRODUCTION


Karen M. Joos Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN, USA Jens Jordan Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany Jens Jordan Institute of Clinical Pharmacology, Hannover Medical School, 30625 Hannover, Germany Michael J. Joyner Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA Stephen G. Kaler Program in Molecular Medicine, NICHD, Bethesda, Maryland 20892-1853, USA Sergey Kasparov Professor of Molecular Physiology, University of Bristol, Bristol, BS8 1TD, UK Horacio Kaufmann Professor, Neurology, Pediatrics and Medicine, New York University School of Medicine, New York, NY 10016, USA Horacio Kaufmann New York University School of Medicine, New York, NY 10016, USA David Kaye Baker IDI Heart and Diabetes Institute, Melbourne, Australia Ramesh K. Khurana Division of Neurology, Union Memorial Hospital, Baltimore, MD, USA Chun-Hyung Kim Department of Psychiatry, Harvard Medical School, Boston, MA, USA Kwang-Soo Kim Department of Psychiatry, Harvard Medical School, Boston, MA, USA Kazuto Kobayashi Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan Nancy L. Kuntz Associate Professor of Pediatrics, Northwestern University Feinberg School of Medicine, Center for Autonomic Medicine in Pediatrics at CMH, Chicago, IL 60614, USA Tomas Konecny Assistant Professor of Medicine, Department of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN, USA, and ICRC – Department of Cardiovascular Diseases, St Anne’s University Hospital Brno, Brno, Czech Republic Andrew Kontak Division of Cardiology/Hypertension Section, University of Texas Southwestern Medical Center, Dallas, TX, USA Cheryl L. Laffer Associate Professor of Medicine, Texas A&M HSC College of Medicine, USA Andre H. Lagrange Assistant Professor of Neurology, Epilepsy Division, Vanderbilt University, Nashville, TN, USA Nora Laiken Department of Pharmacology, University of California, San Diego School of Medicine, La Jolla, CA, USA Gavin Lambert Baker IDI Heart and Diabetes Institute, Melbourne, Australia Jacques W.M. Lenders Department of Internal Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, and Department of Medicine, University Hospital Carl Gustav Carus Dresden, Dresden, Germany Benjamin D. Levine Institute for Exercise and Environmental Medicine, Texas Health Presbyterian Hospital Dallas, The University of Texas, Southwestern Medical Center at Dallas, Dallas, TX, USA Lewis A. Lipsitz Institute for Aging Research, Hebrew Senior Life; Division of Gerontology, Beth Israel Deaconess Medical Center; Harvard Medical School, Boston, MA, USA Julian H. Lombard Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA

xxiii

John C. Longhurst Departments of Medicine, Physiology and Biophysics, Pharmacology and Biomedical Engineering, University of California, Irvine, Irvine, CA 92697-4075, USA David A. Low Autonomic and Neurovascular Medicine Unit, Faculty of Medicine, Imperial College London at St Mary’s Hospital London, WZ1NY, UK Phillip A. Low Department of Neurology, Mayo Foundation, Rochester, MN 55905, USA Chih Cherng Lu, MD,MS Department of Anaesthesiology, Departments of Tri-Service General Hospital/National Defense Medical Center, Taipei, Taiwan, ROC James M. Luther Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN, USA Vaughan G. Macefield Professor of Integrative Physiology, School of Medicine, University of Western Sydney, NSW 1797, Australia Belinda H. McCully Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA James G. McLeod Department of Medicine, University of Sydney, Sydney, Australia William M. Manger New York University Medical Center, and National Hypertension Association, New York, NY, USA Tadaaki Mano Tokai Central Hospital, Kakamigahara, Gifu, Japan Paul J. Marvar Department of Psychiatry, and Center of Behavioral Sciences, Vanderbilt University School of Medicine, Nashville, TN, USA Eliezer Masliah University of California-San Diego, La Jolla, CA 92093-0624, USA Christopher J. Mathias Autonomic and Neurovascular Medicine Unit, Faculty of Medicine, Imperial College London at St Mary’s Hospital London, WZ1NY, UK and Autonomic Unit, National Hospital for Neurology and Neurosurgery, Queen Square/Institute of Neurology, University College London, London, UK Mark R. Melson Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN, USA Douglas F. Milam Department of Urologic Surgery, Vanderbilt University, Nashville, TN, USA Marion C. Mohl Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW, 2010, Australia Yaroslav I. Molkov Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA Margaret Morris Baker IDI Heart and Diabetes Institute, Melbourne, Australia Shaun F. Morrison Department of Neurological Surgery, Oregon Health and Science University, Portland, OR 97239, USA Toshiharu Nagatsu Department of Pharmacology, School of Medicine, Fujita Health University, Toyoake 470-1192, Japan Charles D. Nichols Department of Pharmacology, LSU Health Sciences Center, New Orleans, LA, USA Lucy Norcliffe-Kaufmann Instructor, Physiology and Neuroscience, New York University School of Medicine, New York, NY 10016, USA Vera Novak Division of Gerontology, Beth Israel Deaconess Medical Center;, Harvard Medical School, Boston, MA, USA Luis E. Okamoto Department of Medicine, Division of Clinical Pharmacology, and the Autonomic Dysfunction Center, Vanderbilt University School of Medicine, Nashville, TN, USA

I. INTRODUCTION

xxiv


John W. Osborn University of Minnesota, Department of Integrative Biology and Physiology, Minneapolis, MN 55455, USA Brian A. Parsons Bristol Urological Institute, Southmead Hospital, Bristol, UK Julian F.R. Paton School of Physiology and Pharmacology, Bristol Heart Institute, University of Bristol, Bristol, BS8 1TD, UK Pallavi P. Patwari Assistant Professor of Pediatrics, Northwestern University Feinberg School of Medicine, and Assistant Director, Center for Autonomic Medicine in Pediatrics at CMH, Chicago, IL 60614, USA Cecile L. Phan Department of Neurology, Baylor College of Medicine, Houston, TX, USA Fenna T. Phibbs Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA Nanduri R. Prabhakar Institute for Integrative Physiology, and Center for Systems Biology of O2 Sensing, Biological Sciences Division, University of Chicago, IL, USA Amanda C. Peltier Department of Neurology, Division of Neuromuscular, Vanderbilt University, Nashville, TN, USA Sean M. Peterson Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA Anthony E Pickering School of Physiology and Pharmacology, Bristol Heart Institute, University of Bristol, Bristol, BS8 1TD, UK J. Howard Pratt Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA Kamal Rahmouni University of Iowa, Cardiovascular Center, Iowa City, IA, USA Satish R. Raj Autonomic Dysfunction Unit, Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN, USA Casey M. Rand Center for Autonomic Medicine in Pediatrics at CMH, Chicago, IL 60614, USA Heinz Reichmann Department of Neurology, University Hospital Carl Gustav Carus Dresden, Dresden, Germany Jeff Richards Baker IDI Heart and Diabetes Institute, Melbourne, Australia L.Jackson Roberts Division of Clinical Pharmacology, Robinson Research Building, Vanderbilt University, Nashville, TN, USA David W. Robertson Vanderbilt University, Nashville, TN, USA Rose Marie Robertson Vanderbilt University, Nashville, TN, USA Michael Robinson Departments of Pediatrics and Pharmacology, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA Ilya A. Rybak Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA Elaine Sanders-Bush Department of Pharmacology, Vanderbilt University, Nashville, TN, USA Paola Sandroni Deptartment of Neurology, Mayo Clinic, Rochester, MN, USA Kyoko Sato Department of Cardiovascular Control, Kochi Medical School, Nankoku, Japan Takayuki Sato Department of Cardiovascular Control, Kochi Medical School, Japan Irwin J. Schatz John A. Burns School of Medicine, University of Hawaii at Manoa, Department of Medicine, Honolulu, HI, USA

Ernesto L. Schiffrin Department of Medicine, Sir Mortimer B. Davis-Jewish General Hospital, and Lady Davis Institute for Medical Research, McGill University, Montreal, Que., Canada. Ronald Schondorf Department of Neurology, Sir Mortimer B. Davis Jewish General Hospital, Montreal, QC, Canada Rosemary Schwarz Baker IDI Heart and Diabetes Institute, Melbourne, Australia Gino Seravalle Istituto Auxologico Italiano, Milan, Italy. Robert E. Shapiro Department of Neurology, University of Vermont College of Medicine, Burlington, VT, USA Cyndya Shibao Department of Medicine, Division of Clinical Pharmacology, and the Autonomic Dysfunction Center,Vanderbilt University School of Medicine, Nashville, TN, USA Virend Somers Professor of Medicine, Department of Cardiovascular Diseases and Internal Medicine, Mayo Clinic, Rochester, MN, USA Michaela Stampfer Movement Disorders Section, Department of Neurology, University Hospital, Innsbruck, Austria C.Michael Stein Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA Sylvia Stemberger Division of Clinical Neurobiology, Innsbruck Medical University, Innsbruck, Austria Julian Stewart New York Medical College, Hawthorne, NY, USA Lawrence I. Sinoway Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey, PA, USA James R. Sowers University of Missouri, Diabetes and Cardiovascular Center, and the Harry S. Truman VA Medical Center, Columbia, MO, USA Sirisha Srikakarlapudi Department of Medicine, State University of New York at Buffalo, Buffalo, NY 14215, USA Kenji Sunagawa Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Japan Scott C. Supowit Departments of Medicine (DJD), and Cell Biology and Anatomy (SCS), University of South Carolina School of Medicine, Columbia, SC 29208, USA Palmer Taylor Department of Pharmacology, University of California, La Jolla, CA, USA Jane Thompson Baker IDI Heart and Diabetes Institute, Melbourne, Australia Roland D. Thijs Department of Neurology and Clinical Neurophysiology, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands and Department of Neurology, Dutch Epilepsy Clinics Foundation, 2300 RC Hoofddorp, the Netherlands Rhian M Touyz Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ont., Canada Daniel Tranel Department of Neurology, University of Iowa, College of Medicine, Iowa City, IA, USA Subbulaxmi Trikudanathan Endocrinology, Diabetes and Hypertension Division, Brigham and Women’s Hospital, and, Harvard Medical School, Boston, MA, USA Ching-Jiunn Tseng Department of Medical Education and Research, Kaohsiung Veterans’ General Hospital, Kaohsiung, Taiwan, ROC Che-Se Tung Department of Physiology, National Defense Medical Center, Taipei, Taiwan, ROC Kiren Ubhi Department of Neurosciences, University of California, San Diego/La Jolla, CA, USA

I. INTRODUCTION


Nikhil Urs Department of Cell Biology, Duke University, Durham, NC 27710, USA Joseph G. Verbalis Georgetown University, Washington, DC 20007, USA Steven Vernino Department of Neurology, UT Southwestern Medical Center, Dallas, TX 75390-9036, USA Ronald G. Victor Hypertension Center, The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Margaret A. Vizzard University of Vermont College of Medicine, Burlington, VT, USA Wanpen Vongpatanasin Division of Cardiology/Hypertension Section, University of Texas Southwestern Medical Center, Dallas, TX, USA B. Gunnar Wallin Institute of Neuroscience and Physiology, Sahlgrenska Academy at Göteborg University, S-41345 Göteborg, Sweden Tobias Wang Zoophysiology, Department of Biological Sciences, Aarhus University, Denmark Qin Wang Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA Andrew A. Webster Professor and Chair, Department of Pharmaceutical Sciences, Belmont University School of Pharmacy, Nashville, TN, USA

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Debra E. Weese-Mayer Professor of Pediatrics, Northwestern University Feinberg School of Medicine, and Director, Center for Autonomic Medicine in Pediatrics, at Children’s Memorial Hospital (CMH), Chicago, IL 60614, USA Gregor K. Wenning Movement Disorders Section, Department of Neurology, University Hospital, Innsbruck, Austria Adam Whaley-Connell University of Missouri, Diabetes and Cardiovascular Center, and the Harry S. Truman VA Medical Center, Columbia, MO, USA Wouter Wieling Department of Internal Medicine, Academic Medical Centre, 1105 AZ Amsterdam, The Netherlands Gordon H. Williams Endocrinology, Diabetes and Hypertension Division, Brigham and Women’s Hospital, and, Harvard Medical School, Boston, MA, USA Scott Wood Universities Space Research Association, Houston, TX, USA Michael G. Ziegler UCSD Medical Center, San Diego, CA, USA Daniel B. Zoccal Department of Physiological Sciences, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil

I. INTRODUCTION

PA R T I

INTRODUCTION



C H A P T E R

1 Development and Differentiation of Autonomic Neurons Chun-Hyung Kim, Kwang-Soo Kim increasing the sympathetic outflow to the heart and other viscera, the parasympathetic system is responsible for the basal autonomic functions such as heart rate and respiration under normal conditions. The enteric system regulates peristalsis of the gut wall and modulates the activity of the secretary glands. During the last decade, exciting progress has been made with regard to the molecular mechanisms underlying the development of the ANS. Among many different aspects, this review will focus primarily on the transcriptional regulatory code underlying the development and neurotransmitter identity determination of the ANS.

An intricate network of extracellular signals and nuclear transcription factors orchestrates the specification of numerous neuronal phenotypes during development of the vertebrate nervous system. During the last decade or so, impressive progress has been achieved in identifying the extracellular signaling molecules and key transcription factors that critically govern the development and fate determination of the autonomic nervous system (ANS). In particular, the so-called “transcriptional regulatory code” underlying the development and differentiation of the ANS has been elucidated; several key fatedetermining transcription factors such as Mash1, Phox2, AP2 and GATA3, have been identified to be responsible for development of the autonomic nervous system and the neurotransmitter identity specification. One important emerging feature is that those key transcription factors regulate not only development, but also final properties of differentiated neurons such as neurotransmitter identity. In line with this concept, those factors directly or indirectly regulate the expression of both cell type-specific markers as well as pan-neuronal markers. Second, these transcription factors function in an intricate regulatory cascade, starting from key signaling molecules such as bone morphogenic proteins. Finally, as evidenced by the study of dopamine β-hydroxylase gene regulation, multitudes of cell type-specific factors (e.g., Phox2a and 2b) and general transcription factors (e.g., CREB and Sp1) co-operatively regulate the expression of cell type-specific marker genes. This new molecular information will facilitate our understanding of the function of the autonomic nervous system in the normal, as well as in the diseased brain.

THE ANS IS DERIVED FROM NEURAL CREST CELLS During the early developmental period of vertebrate embryo, the neural tube and notochord are formed from the ectodermal and mesodermal layers, respectively. At this time, neural crest cells originate from the dorsolateral edge of the neural plate, and are generated at the junction of the neural tube and the ectoderm (Fig. 1.1). Interactions between the non-neural ectodermal layer and the neural plate critically influence the formation of neural crest cells at their interface [1]. Upon formation, neural crest cells migrate along specific routes to diverse destinations and differentiate into a variety of cell types that include all neurons and glial cells of the peripheral nervous system and the neurons of the gastric mucosal plexi. In addition, some other cell types such as smooth muscle cells, pigment cells, and chromatophores are known to arise from neural crest cells. Much progress has recently been achieved in the identification of signaling molecules and downstream transcription factors that control lineage determination and differentiation of neural crest cells [2,3].

INTRODUCTION The autonomic nervous system (ANS; also called the autonomic division or the autonomic motor system) is one of two major divisions of the peripheral nervous system. The ANS has three major subdivisions that are spatially segregated: the sympathetic, the parasympathetic, and the enteric nervous systems. While the sympathetic system controls the fight-or-flight reactions during emergencies by


SIGNALING MOLECULES REGULATE THE DEVELOPMENTAL PROCESSES OF THE ANS Recent studies have demonstrated that various signaling molecules, e.g., members of the bone morphogenic

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4

1. DEvElOPmENT AND DIffERENTIATION Of AUTONOmIC NEURONs

development, while its robust activation opposes, even in the presence of BMP-2, SA cell development and the expression of SA lineage-determining genes [9]. Finally, during the last decade, numerous studies demonstrated that different neurotrophic factors such as NGF, GDNF, NT3, and/or their receptor signaling, critically regulate the survival and development of all three divisions of the ANS [10,11].

Neural plate Ectodermis Mesoderm

TRANSCRIPTIONAL REGULATORY CODE UNDERLYING THE DEVELOPMENT AND PHENOTYPIC SPECIFICATION OF THE ANS Notochord

Neural crest

Neural tube

Somite Dorsal aorta

FIGURE 1.1 All cell types of the ANS are derived from neural crest cells. Neural crest cells are formed at the dorsal neural tube and migrate along diverse routes. Depending on their specific routes and interactions with the target tissues, they differentiate into a variety of cell types including pigment cells, different types of neurons and glia of the ANS, and parts of the adrenal gland.

protein (BMP) family, Wnt, sonic hedgehog, and fibroblast growth factor, play critical roles in the early formation of neural crest cells as well as for final determination of neuronal identity [1–5]. These signals are often provided from neighboring tissues during migration of neural crest cells. For instance, grafting and ablation experiments in chick embryos demonstrate that the notochord is necessary but not sufficient to induce adrenergic phenotypes of neural crest-derived sympathetic ganglia [6,7]. A possible candidate for notochord-derived molecule(s) is sonic hedgehog [7]. In addition, a series of elegant experiments established that BMP family members, expressed from the dorsal aorta, play a crucial role in the differentiation and fate determination of the sympathetic nervous system [8 and references therein]. It is of note that these extracellular signaling molecules seem to work in concert with intracellular signals such as cAMP. Consistent with this, using neural crest cell culture, induction of differentiation and neurotransmitter phenotypes by BMP is enhanced by cAMP-elevating agents [4,9]. Interestingly, it appears that cAMP signaling acts as a bimodal regulator of sympathoadrenal (SA) cell development in neural crest cultures because its moderate activation promotes SA cell

Various transcription factors are thought to trigger a regulatory cascade by inducing the expression of downstream transcription factors, which eventually activate or repress the final target genes [4,12]. The regulatory cascade controlling the ANS’s noradrenergic neurotransmitter phenotype has been extensively studied, leading to the identification and functional characterization of critical transcription factors (Fig. 1.2). For example, the basic helix-loop-helix (bHLH) factor, Mammalian achaete-scute homolog-1 (Mash1, and the chicken homolog Cash1) induced by BMPs is the first transcription factor shown to be essential for noradrenergic neuron development. Downstream of Mash1 lies the homeodomain transcription factor Phox2a which is a critical regulator of noradrenergic cell lineage development. A closely related transcription factor Phox2b is also induced by BMPs independently of Mash1 and is another essential regulator of noradrenergic neuron development. In addition, GATA2/3 and dHand play critical roles for noradrenergic neuron development. Recent work from our laboratory showed that AP2β may critically regulate NA neuron development in the ANS. Specific functional roles of these key transcription factors in the development of the ANS have been identified as shown below (Fig. 1.2).

Mash1 (also called Cash1) Mash1, a basic helix-loop-helix (bHLH) protein, is the first transcription factor shown to be essential for development of the ANS. In Mash1/ mice, virtually all noradrenaline (NA) neurons of the nervous system are affected, suggesting that Mash1 is a critical factor for determining the NA fate. Mash1 appears to relay BMP molecules’ signals for sympathetic development. Consistent with this idea, Mash1 expression was induced by BMPs in neural crest cultures and it was largely diminished in sympathetic ganglia following inhibition of BMPs function [13]. In Mash1-inactivated mouse embryos, neural crest cells migrated to the vicinity of the dorsal aorta, but did not develop into mature sympathetic neurons, as evidenced by the lack of expression of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), as well as the absence of pan-neuronal markers [14]. In addition,

I. INTRODUCTION

TRANsCRIPTIONAl REgUlATORy CODE UNDERlyINg THE DEvElOPmENT AND PHENOTyPIC sPECIfICATION Of THE ANs

cAMP

whereas Phox2a expression is regulated by both Mash1 and Phox2b (Fig. 1.2). Recent promoter studies showed that Phox2a and 2b are able to directly activate the DBH promoter by interacting with multiple sequence motifs residing in the 5 flanking region (see below). Collectively, Phox2a and 2b appear to regulate both the development and neurotransmitter identity of sympathetic neurons and of other noradrenergic neurons.

BMPs

? Phox2b MASH1/CASH1

PKA

AP2 β

PNMT

CREB

GATA3

TH Neurotransmitter identity

Phox2a

DBH

5

c-Ret

GATA3

Survival

FIGURE 1.2 Diagrams depicting the regulatory network of the NA phenotype determination and maintenance. The diagram shows the possible regulatory interactions in the cascade of development and NA phenotype expression of ANS neurons. Thick arrows indicate likely direct regulation and thin arrows indicate direct or indirect regulation. BMPs are secreted from the dorsal aorta and activate the expression of MASH1/CASH1 and Phox2b. Phox2b then activates the expression of Phox2a. Phox2a and/or Phox2b in turn activate neurotransmitterspecifying genes TH and DBH. Direct action of Phox2a/2b on DBH transcription has been demonstrated. The transcription factor CREB has been shown to interact with the 5 promoter and directly activates transcription of the TH and DBH genes. The cAMP signaling pathway appears to regulate NA phenotype determination in concert with Phox2a/2b. The zinc finger factor GATA3, presumably downstream of Phox2b, also appears to be required for expression of NA-specific genes. Phox2a and 2b also regulate the expression of pan-neuronal genes including the GDNF receptor c-Ret, which may relate to their role in formation and/or survival of ANS neurons.

Mash1 directly or indirectly affects the expression of Phox2a, another key transcription factor important for DBH gene expression in a noradrenergic neuron-specific manner [15–17]. Thus, Mash1 is the first transcription factor known to control both differentiation and maintenance of noradrenergic neurons [18].

Phox2 Genes Phox2a and Phox2b are two closely related homeodomain transcription factors that are expressed in virtually all neurons that transiently or permanently express the noradrenergic neurotransmitter phenotype [19]. Gene inactivation studies have demonstrated that Phox2a and/or 2b are essential for proper development of all three divisions of the ANS and some noradrenergic containing structures of the CNS. For instance, in both Phox2a/ and 2b/ mouse brain, the major noradrenergic population in the locus coeruleus does not form, strongly suggesting that both genes are required for its development [20,21]. In contrast, only Phox2b seems to be required for the development of sympathetic neurons. In Phox2a/ mice, sympathetic neuron development is largely normal. Interestingly, however, both genes are able to induce sympathetic neuron-like phenotype when ectopically expressed in chick embryos [22]. During sympathetic development, Phox2b expression is induced by BMP molecules independently of Mash1,

The zinc finger transcription factor GATA-3 is a master regulator of type 2 T helper cell development. Interestingly, in GATA-3/ mice, noradrenaline deficiency is a proximal cause of embryonic lethality suggesting that GATA-3 is involved in the specification of noradrenergic neurotransmitter phenotype during noradrenergic neuron development [23]. Forced expression of GATA-3 in primary neural crest stem cell (NCSC) culture and developing chick embryos demonstrated that GATA-3 is able to increase the number of sympathoadrenergic neurons among NCSC culture, and induce ectopic expression of noradrenergic marker genes (TH and DBH) in developing chick embryos [24]. Furthermore, both TH and DBH promoters are robustly transactivated by GATA-3 via specific upstream subdomains encompassing binding motifs for transcription factors CREB, Sp1, and AP4. Protein–protein interaction assays showed that GATA-3 is able to physically interact with these transcription factors in vitro as well as in vivo [25]. Taken together, it is likely that GATA3 plays a critical role for specification of the NA phenotype via novel and distinct protein-protein interactions in both CNS and ANS development.

AP2(Activator Protein 2)β AP2 is a retinoic acid-inducible and developmentally regulated transcription factor with the basic helix-span-helix domain recognizing the palindromic 5-GCCNNNGGC-3 motif or its related GC-rich sequences. AP-2 family has 5 members, AP-2α, AP-2β, AP-2γ, AP-2δ, and AP-2, which show different spatiotemporal expression patterns during development. Based on its expression pattern in the neural crest, it was assumed that AP-2α regulates differentiation of neural crest-derived cells. However, our recent works demonstrated that AP2β is expressed in sympathetic ganglia of developing chick and mouse embryos and facilitates sympathoadrenergic differentiation in neural crest stem cells, while AP2α dramatically increases melanocytes at the expense of sympathoadrenergic cells [26,27]. Lossof-function studies of AP2β/ mouse revealed that DBH expression was significantly reduced in sympathetic ganglia as well as LC, indicating distinctive roles in neural crest differentiation and noradrenergic neurotransmitter specification in both central and peripheral nervous systems. Furthermore, these studies showed that AP2β directly controls the epinephrine phenotype by activating the

I. INTRODUCTION

6


phenylethanolamine-N-methyl-transferase (PNMT) gene expression (Fig. 1.2).

Other Transcription Factors Although the above transcription factors are the most promising candidates, additional factors are emerging as being important for the ANS’ development and phenotype specification. For instance, dHand is another bHLH transcription factor whose expression is induced by BMPs and is dependent on Mash1. Based on its specific expression in sympathetic neurons, dHand may directly contribute to the ANS development. However, analysis of dHand function was hampered because knockout mice die before sympathetic neuron development. It is worthwhile to note that transcriptional regulation of the ANS and phenotype identity may require the combinatorial action of cell type-specific factors (e.g., Phox2a and 2b) and general transcription factors. Such examples may include cAMP response element binding protein and Sp1, which are required for transcriptional activity of both the TH and DBH genes (see below).

NEUROTRANSMITTER PHENOTYPES OF THE ANS Among the various phenotypes of a particular neuron, neurotransmitter identity is an essential feature because it determines the nature of the chemical neurotransmission a given neuron will mediate, and influences its specific connectivity with target cells. For specification of neurotransmitter identity, given neurons should express relevant genes encoding the biosynthetic enzymes and cofactors, as well as the specific reuptake protein(s). In addition, expression of these genes needs to be matched with the appropriate receptors of the target tissues. Therefore, expression of particular neurotransmitter phenotypes should be coordinated with the differentiation and phenotype specification of the target tissues. Recently, molecular mechanisms underlying the specification of neurotransmitters in the nervous system have been investigated extensively and key signaling molecules and transcriptional factors have been identified. Among these, specification of the noradrenaline (NA) phenotype of the sympathetic nervous system and the CNS is well characterized. Therefore, we will focus our discussion on the molecular characterization of the NA phenotype determination and its phenotypic switch to the cholinergic phenotype.

NA Phenotype NA is a major neurotransmitter of the ANS, especially in sympathetic neurons, and fundamentally mediates the function of the ANS. Consistent with this, a rare human disease called the dopamine β-hydroxylase deficient disease, in which NA is undetectable, was identified to be associated with severe autonomic function failure [28].

NA is one of the catecholamine neurotransmitters that are synthesized from tyrosine by three consecutive enzymatic steps. While tyrosine hydroxylase is responsible for the first step of catecholamine biosynthesis, converting tyrosine to L-dopa, and is expressed in all catecholamine neurons, dopamine β-hydroxylase (DBH) is responsible for conversion of dopamine to NA and is specifically expressed in NA neurons. Thus, DBH is a hallmark protein of NA neurons and the control mechanism of its expression is an essential feature of the development of NA neurons.

Control Mechanism of DBH Gene Expression is Closely Related to the ANS Development Numerous investigators using both in vivo transgenic mouse approaches and in vitro cell culture systems have studied DBH gene regulation. As schematically summarized in Figure 1.3, the 5 1.1 kb region upstream of the DBH gene promoter has three functional domains that can drive reporter gene expression in a NA cell type-specific manner. More detailed deletional and sitedirected mutational analyses indicate that as little as 486 bp of the upstream sequence of the human DBH gene can direct expression of a reporter gene in a cell-specific manner [29]. While the distal region spanning 486 to 263 bp appears to have a cell-specific silencer function, the proximal part spanning 262 to 1 bp is essential for high-level and cell-specific DBH promoter activity. In this 262 bp proximal area, four protein-binding regions (domains I to IV), initially identified by DNase I footprinting analysis, were found to encompass functionally important, multiple cis-regulatory elements [29], including the cAMP response element (CRE), YY1, AP2, Sp1, and core motifs of homeodomain (HD) binding sites. Site-directed mutagenesis of each sequence motif has revealed that these multiple cis-acting elements synergistically and/ or co-operatively regulate the transcriptional activity of the DBH gene [16]. Among these, two ATTA-containing motifs in domain IV and another motif in domain II were identified to be NA-specific cis-acting motifs in that their mutation diminished the DBH promoter function only in NA cell lines. More recently, another NA-specific cisregulatory element was identified between domain II and III (Fig. 1.3). Interestingly, analysis of DNA-protein interactions on the DBH promoter demonstrated that all of these four NA-specific cis-regulatory elements are Phox2binding sites [15]. Taken together, this experimental evidence establishes that the DBH gene is an immediate downstream target of Phox2 proteins.

Mutations of DBH Gene are Closely Associated with the Autonomic Disorder, Orthostatic Hypotension DBH deficiency is a rare congenital disorder, first described in 1986 [28,30]. DBH deficiency is a severe autonomic disorder exhibiting sympathetic noradrenergic

I. INTRODUCTION

NEUROTRANsmITTER PHENOTyPEs Of THE ANs DBH deficiency

Normal

nucleus

nucleus

ER X

L-tyrosine

L-tyrosine

L-DOPA

L-DOPA

dopamine

dopamine

Golgi

LDCV

FIGURE 1.3 Effects of pathogenic mutations on DBH synthesis and trafficking. In normal, DBH enzymes (circle) are translated in the ER, transported to the Golgi, and then packaged into the LDCVs. In LDCV, DBH converts dopamine (triangle) to noradrenaline (rectangle). Noradrenaline are released by exocytosis upon stimulation of the nerve terminal and readily detectable in plasma. In DBH deficiency, DBH proteins (S-shape) encoded by missense mutations are retained in the ER and fail to be targeted to the LDCV due to their misfolding. As the result, noradrenaline is not synthesized in LDCV, leading to absence of noradrenaline in plasma. Instead, dopamine is dramatically increased.

failure and adrenomedullary failure but intact vagal and sympathetic cholinergic function [31]. DBH deficient patients exhibit severe deficits in autonomic regulation of cardiovascular function predisposing them to orthostatic hypotension. These patients display characteristic perturbations in the level of catecholamines: undetectable noradrenaline and its metabolites, and highly elevated dopamine and its metabolites. Given the fundamental role of noradrenaline in the nervous system, the report of adult patients with undetectable noradrenaline is both surprising and interesting. The report of frequent miscarriages and spontaneous abortions in mothers of known DBH deficiency cases suggests the interesting possibility that there could be many more undiagnosed fetal and neonatal deaths resulting from DBH deficiency and that those adult patients are lucky survivors [30]. In line with this, a DBH knock out mouse study showed less than 5% live births [32]. Mortality appeared to be due to cardiovascular failure caused by DBH deficiency in utero, which is reminiscent of tyrosine hydroxylase null mice. We, and others, have recently reported a mutation in the splice donor site of the first exon-intron junction and several missense mutations associated with the DBH deficiency syndrome in the DBH gene of these patients [33–35]. The mutation in the splice donor site (IVS1  2T→C) of DBH resulted in abnormal mRNA splicing and generated a transcript containing a premature stop codon as well as

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a normal transcript. Interestingly, all missense mutations so far identified in DBH deficient patients are subtle amino acid substitutions resulting in defective protein trafficking and mislocation, probably due to protein misfolding (Fig. 1.3) [35]. One of the well-known chemical chaperones, glycerol, could partially rescue defective secretion of DBH mutant proteins, suggesting the possibility that DBH deficiency disease could be more fundamentally treated with pharmacological chaperones [35].

Cholinergic Phenotype and the Switch of Neurotransmitter Phenotypes by Target Cell Interactions Another major neurotransmitter of the ANS is acetylcholine, and neurons generating this neurotransmitter are designated cholinergic. Among sympathetic neurons, the number of cholinergic neurons is much less than NA neurons. While differentiation and cholinergic specification are extensively investigated in central motor neurons [4], development of cholinergic ANS neurons is not well characterized. Therefore, it is of great interest to understand if key transcription factors such as HB9 and MNR2, likewise play key roles in determining the cholinergic phenotype during development of the ANS. Interestingly, it is well described that upon contacting developing sweat glands, the NA phenotype of sympathetic axons switches to the cholinergic phenotype [10]. The putative cholinergic-inducing factor secreted from sweat glands remains to be defined although leukemia inhibitory factor and ciliary neurotrophic factor are candidates. Consistent with the observation that TH and DBH expression remains even after neurotransmitter switch from the NA to the cholinergic phenotype, a recent study reported that levels of the TH cofactor, tetrahydrobiopterin (BH4), dropped significantly during the switch [36]. Immunoreactivity for the BH4-synthesizing GTP cyclohydrolase became undetectable in sweat gland neurons during this phenotypic switch, suggesting that suppression of cofactor expression underlies the neurotransmitter switch during development.

Acknowledgements This work was supported by NIH grants (MH048866, MH087903, and NS070577).

References [1] Liem Jr KF, Tremml G, Roelink H, Jessell TM. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 1995;82:969–79. [2] Sauka-Spengler T, Bronner-Fraser M. A gene regulatory network orchestrates neural crest formation. Nat Rev Mol Cell Biol 2008;9:557–68. [3] Christiansen JH, Coles EG, Wilkinson DG. Molecular control of neural crest formation, migration and differentiation. Curr Opin Cell Biol 2000;12:719–24.

I. INTRODUCTION

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[4] Edlund T, Jessell TM. Progression from extrinsic to intrinsic signaling in cell fate specification: a view from the nervous system. Cell 1999;96:211–24. [5] Wilson SI, Rydstrom A, Trimborn T, Willert K, Nusse R, et al. The status of Wnt signalling regulates neural and epidermal fates in the chick embryo. Nature 2001;411:325–30. [6] Stern CD, Artinger KB, Bronner-Fraser M. Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo. Development 1991;113:207–16. [7] Ernsberger U, Rohrer H. The development of the noradrenergic transmitter phenotype in postganglionic sympathetic neurons. Neurochem Res 1996;21:823–9. [8] Schneider C, Wicht H, Enderich J, Wegner M, Rohrer H. Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons. Neuron 1999;24:861–70. [9] Bilodeau ML, Boulineau T, Hullinger RL, Andrisani OM. Cyclic AMP signaling functions as a bimodal switch in sympathoadrenal cell development in cultured primary neural crest cells. Mol Cell Biol 2000;20:3004–14. [10] Francis NJ, Landis SC. Cellular and molecular determinants of sympathetic neuron development. Annu Rev Neurosci 1999;22:541–66. [11] Taraviras S, Pachnis V. Development of the mammalian enteric nervous system. Curr Opin Genet Dev 1999;9:321–7. [12] Goridis C, Brunet JF. Transcriptional control of neurotransmitter phenotype. Curr Opin Neurobiol 1999;9:47–53. [13] Goridis C, Rohrer H. Specification of catecholaminergic and serotonergic neurons. Nat Rev Neurosci 2002;3:531–41. [14] Guillemot F, Lo LC, Johnson JE, Auerbach A, Anderson DJ, et al. Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 1993;75:463–76. [15] Seo H, Hong SJ, Guo S, Kim HS, Kim CH, et al. A direct role of the homeodomain proteins Phox2a/2b in noradrenaline neurotransmitter identity determination. J Neurochem 2002;80:905–16. [16] Kim HS, Seo H, Yang C, Brunet JF, Kim KS. Noradrenergic-specific transcription of the dopamine beta-hydroxylase gene requires synergy of multiple cis-acting elements including at least two Phox2abinding sites. J Neurosci 1998;18:8247–60. [17] Yang C, Kim HS, Seo H, Kim CH, Brunet JF, et al. Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine betahydroxylase gene. J Neurochem 1998;71:1813–26. [18] Hirsch MR, Tiveron MC, Guillemot F, Brunet JF, Goridis C. Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system. Development 1998;125:599–608. [19] Brunet JF, Pattyn A. Phox2 genes - from patterning to connectivity. Curr Opin Genet Dev 2002;12:435–40. [20] Morin X, Cremer H, Hirsch MR, Kapur RP, Goridis C, et al. Defects in sensory and autonomic ganglia and absence of locus coeruleus in mice deficient for the homeobox gene Phox2a. Neuron 1997;18:411–23. [21] Pattyn A, Goridis C, Brunet JF. Specification of the central noradrenergic phenotype by the homeobox gene Phox2b. Mol Cell Neurosci 2000;15:235–43.

[22] Stanke M, Junghans D, Geissen M, Goridis C, Ernsberger U, et al. The Phox2 homeodomain proteins are sufficient to promote the development of sympathetic neurons. Development 1999;126:4087–94. [23] Lim KC, Lakshmanan G, Crawford SE, Gu Y, Grosveld F, et al. Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat Genet 2000;25:209–12. [24] Hong SJ, Huh Y, Chae H, Hong S, Lardaro T, et al. GATA-3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB. J Neurochem 2006;98:773–81. [25] Hong SJ, Choi HJ, Hong S, Huh Y, Chae H, et al. Transcription factor GATA-3 regulates the transcriptional activity of dopamine betahydroxylase by interacting with Sp1 and AP4. Neurochem Res 2008;33:1821–31. [26] Hong SJ, Lardaro T, Oh MS, Huh Y, Ding Y, et al. Regulation of the noradrenaline neurotransmitter phenotype by the transcription factor AP-2beta. J Biol Chem 2008;283:16860–16867. [27] Hong SJ, Huh YH, Leung A, Choi HJ, Ding Y, et al. Transcription factor AP-2b regulates the neurotransmitter phenotype and maturation of chromaffin cells. Mol Cell Neurosci 2011;46:245–51. [28] Robertson D, Goldberg MR, Onrot J, Hollister AS, Wiley R, et al. Isolated failure of autonomic noradrenergic neurotransmission. Evidence for impaired beta-hydroxylation of dopamine. N Engl J Med 1986;314:1494–7. [29] Seo H, Yang C, Kim HS, Kim KS. Multiple protein factors interact with the cis-regulatory elements of the proximal promoter in a cellspecific manner and regulate transcription of the dopamine betahydroxylase gene. J Neurosci 1996;16:4102–12. [30] Man in’t Veld AJ,, Boomsma F, Moleman P, Schalekamp MA. Congenital dopamine-beta-hydroxylase deficiency. A novel orthostatic syndrome. Lancet 1987;1:183–8. [31] Biaggioni I, Goldstein DS, Atkinson T, Robertson D. Dopaminebeta-hydroxylase deficiency in humans. Neurology 1990;40:370–3. [32] Thomas SA, Matsumoto AM, Palmiter RD. Noradrenaline is essential for mouse fetal development. Nature 1995;374:643–6. [33] Kim CH, Zabetian CP, Cubells JF, Cho S, Biaggioni I, et al. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency. Am J Med Genet 2002;108:140–7. [34] Deinum J, Steenbergen-Spanjers GC, Jansen M, Boomsma F, Lenders JW, et al. DBH gene variants that cause low plasma dopamine beta hydroxylase with or without a severe orthostatic syndrome. J Med Genet 2004;41:e38. [35] Kim CH, Leung A, Huh YH, Yang E, Kim DJ, et al. Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine b-hydroxylase. J Biol Chem 2011;286:9196–9204. [36] Habecker BA, Klein MG, Sundgren NC, Li W, Woodward WR. Developmental regulation of neurotransmitter phenotype through tetrahydrobiopterin. J Neurosci 2002;22:9445–52.

I. INTRODUCTION

C H A P T E R

2 Central Autonomic Control Eduardo E. Benarroch Central control of the sympathetic and parasympathetic outputs involves several interconnected areas distributed throughout the neuraxis. This central autonomic network has a critical role in moment-to-moment control of visceral function, homeostasis, and adaptation to internal or external challenges. The functions of the central autonomic network are organized in four hierarchical levels that are closely interconnected: spinal, bulbopontine, pontomesencephalic and forebrain levels (Fig. 2.1). The spinal level mediates segmental sympathetic or sacral parasympathetic reflexes and is engaged in stimulus-specific patterned responses under the influence of the other levels. The bulbopontine (lower brainstem) level is involved in reflex control of circulation, respiration, gastrointestinal function, and micturition. The pontomesencephalic (upper brainstem) level integrates autonomic control with pain modulation and integrated behavioral responses to stress. The forebrain level includes the hypothalamus, which is involved in integrated control of autonomic and endocrine responses for homeostasis and adaptation, and components of the anterior limbic circuit, including the insula, anterior cingulate cortex, and amygdala, which are involved in integration of bodily sensation with emotional and goal-related autonomic responses.

sympathetic and parasympathetic outputs, primarily via a relay in the lateral hypothalamus [4,5].

Anterior Cingulate Cortex The anterior cingulate cortex is interconnected with the anterior insula and is subdivided into ventral (affective) and dorsal (cognitive) regions [6]. The ventral anterior cingulate is part of the brain “default mode network” whereas the dorsal anterior cingulate is a component of the frontoparietal attention networks. The ventral anterior cingulate cortex includes subcallosal and precallosal portions that have extensive connections with the insula, prefrontal cortex, amygdala, hypothalamus, and brain stem. Via these projections, the anterior cingulate cortex controls sympathetic and parasympathetic functions [7].

Amygdala The amygdala provides affective or emotional value to incoming sensory information [8] and has multiple downstream targets that participate in the autonomic and neuroendocrine response to stress [9]. The central nucleus of the amygdala (CeA), both directly and via the bed nucleus of the stria terminalis, has a major role in integration of the stress responses, particularly fear responses, via its widespread connections with the hypothalamus and brainstem, particularly the periaqueductal gray and the medullary reticular formation [10].

FOREBRAIN COMPONENTS The forebrain regions involved in the control of autonomic functions include the insular cortex, anterior cingulate cortex, amygdala, and several areas of the hypothalamus.

Hypothalamus The hypothalamus integrates autonomic and endocrine responses necessary for homeostasis and adaptation. It acts as a visceromotor pattern generator that initiates specific patterns of autonomic and endocrine responses according to the stimulus, such as hypoglycemia, changes in blood temperature or osmolarity, or external stressors [2,11]. The preoptic-hypothalamic area is functionally subdivided into three functional zones, periventricular, medial, and lateral [11]: The periventricular zone includes the suprachiasmatic nucleus (the circadian pacemaker), and several areas involved in neuroendocrine control via the pituitary gland. The medial zone includes the medial

Insular Cortex The insular cortex is the primary interoceptive cortex and integrates visceral, pain and temperature sensations [1,2]. The dorsal insula has a viscerotropic organization [3] and receives inputs from gustatory, visceral, muscle, and skin receptors via the thalamus. The dorsal insula projects to the right anterior insula, which, via its connections with neocortical association and limbic areas, integrates these interoceptive inputs with emotional and cognitive processing to conveys the conscious experience of bodily sensation [1]. The insula is also a visceromotor area controlling both the


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10

2. CEnTRAl AuTonomIC ConTRol

FIGURE 2.1 Central autonomic control areas and levels of interaction of autonomic control.

preoptic area, paraventricular nucleus (PVN) and dorsomedial nucleus (DMH), which orchestrate coordinated autonomic and endocrine outputs for thermoregulation, osmoregulation, and stress responses [11–14]. The lateral zone includes nuclei that control sleep, arousal and motivated behavior [2]. The main autonomic outputs of the hypothalamus originate from the PVN, DMH, and lateral hypothalamic area [2]. The PVN contains different neuronal populations that are differentially activated during stress responses [12]. They include magnocellular neurons that release arginine-vasopressin (AVP) to the general circulation; neurons that release corticotropin releasing hormone and activate the adrenocortical axis, and neurons that project to autonomic nuclei of the brainstem and spinal cord [12]. Via these outputs, the PVN modulates stress responses, food and sodium intake, glucose metabolism, and cardiovascular, renal, gastrointestinal, and respiratory functions. The DMH participates in stress responses [13]; thermoregulation [14,15] and cardiovascular control [16].

Hypocretin/orexin neurons of the posterior lateral hypothalamus contribute to control autonomic output in the setting of arousal, feeding and reward driven behaviors [2].

BRAINSTEM COMPONENTS The brainstem areas controlling autonomic output include the periaqueductal gray matter of the midbrain (PAG), the parabrachial nucleus (PBN) and several medullary regions, including the nucleus of the solitary tract (NTS), ventrolateral reticular formation of the medulla, and medullary raphe (Fig. 2.1).

Periaqueductal Gray The PAG is the interface between the forebrain and the lower brain stem and has a major role in integrated autonomic and somatic responses to stress, pain modulation,

I. INTRODUCTION

AuTonomIC ouTPuT of THE CEnTRAl nERvous sysTEm

and other adaptive functions. It consists of different longitudinal columns that, via their different spinal, brainstem, and cortical connections, participate in cardiovascular responses associated with pain modulation [17]; coordination of the micturition reflex [18]; and control of respiration.

Parabrachial Complex and Adjacent Regions of the Pons The PBN is a major relay center that receives converging visceral, nociceptive and thermoreceptive inputs from the spinal cord and conveys this information to the hypothalamus, amygdala, and thalamus [2]. The PBN also participates in the control of respiratory, cardiovascular, and gastrointestinal functions. The dorsal pontine tegmentum also contains the pontine micturition center (PMC), also referred to as Barrington nucleus or M- (for medial) region, which is critical for the coordination of the micturition reflex and participates in the control of the function of the lower gastrointestinal tract and sexual organs [18].

Nucleus of the Solitary Tract The NTS is the first relay station of taste and visceral afferent information and includes several subnuclei with a viscerotropic organization. The rostral portion of the NTS receives taste inputs; the intermediate portion receives gastrointestinal afferents; and the caudal portion receives baroreceptor, cardiac, chemoreceptor, and pulmonary afferents [19]. The NTS relays this information, either directly or via the PBN, to rostral brainstem and forebrain areas [2,3]. The NTS is also the first central relay for all medullary reflexes controlling cardiovascular function (baroreflex and cardiac reflexes) [20], respiration (carotid chemoreflex and pulmonary mechanoreflexes), and gastrointestinal motility [21].

Rostral Ventrolateral Medulla The rostral ventrolateral medulla (RVLM), including the C1 group of epinephrine-containing neurons, is a key area for regulation of arterial blood pressure [20,21]. Glutamatergic neurons of the RVLM project directly and provide tonic excitation to sympathetic preganglionic neurons controlling cardiac output and total peripheral resistance [15]. The RVLM mediates all reflexes controlling arterial blood pressure, including the baroreflex, cardiopulmonary reflexes, and chemoreflexes [20,22]. The sympathoexcitatory RVLM neurons receive and integrate a large variety of inputs from the brainstem and forebrain [22]. These include inhibitory signals from baroreceptorsensitive neurons of the NTS and mediated via inhibitory gamma-aminobutyric acid (GABA) ergic neurons of the caudal ventrolateral medulla [22]. The RVLM also receives several inputs from the hypothalamus, including the PVN.

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Caudal Ventrolateral Medulla The caudal ventrolateral medulla contains GABAergic neurons that maintain a tonic inhibitory control on the RVLM and relay the inhibitory inputs from the NTS mediating the sympathoinhibitory component of the arterial baroreflex. Electrical stimulation studies indicate that the caudal medulla also contains pressor regions [23]. The caudal ventrolateral medulla also contains the A1 group of neurons that provide noradrenergic innervation to the hypothalamus and are a component of a reflex pathway that triggers AVP release in response to hypovolemia or hypotension [24].

Ventromedial Medulla and Caudal Raphe The rostral ventromedial medulla including the caudal raphe nuclei, has an important role in thermoregulation [15] pain modulation [17], and control of automatic ventilation [25]. One group of medullary raphe neurons initiate sympathetic responses to cold via input to preganglionic sympathetic neurons that activate skin vasoconstriction and non-shivering thermogenesis in the brown adipose tissue [26].

AUTONOMIC OUTPUT OF THE CENTRAL NERVOUS SYSTEM Sympathetic Preganglionic Units The sympathetic output is critical for maintenance of arterial pressure, thermoregulation, and redistribution of regional blood flow during stress and exercise. The sympathetic output originates from sympathetic preganglionic neurons located in the thoracolumbar spinal cord at the T1 to -L2 segments, primarily in the intermediolateral cell column. These neurons are organized into functionally separate units that innervate selective subpopulations of sympathetic ganglion neurons and receive distinct segmental afferent inputs triggering segmental somato- and viscerosympathetic reflexes [27]. Different preganglionic sympathetic units are recruited in a coordinated fashion by premotor neurons in the brainstem and hypothalamus to initiate different patterns of responses to specific internal or external stressors, such as postural changes, exercise, hypoglycemia, dehydration, exposure to heat or cold, or stress [2]. The main sources of premotor sympathetic innervation are the RVLM, medullary raphe, A5 noradrenergic group of the pons; PVN, and lateral hypothalamic area [4,28].

Parasympathetic Outputs In contrast to the sympathetic system, which mediates stimulus-specific patterns of responses affecting multiple effectors; the parasympathetic system mediates reflexes activated in an organ-specific fashion.

I. INTRODUCTION

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Vagal Output The vagal output originates from preganglionic neurons located in the dorsal motor nucleus of the vagus (DMV) and in the ventrolateral portion of the nucleus ambiguus (NAmb) in the medulla. The DMV contains most of the vagal preganglionic parasympathetic neurons that are organized in a viscerotropic fashion [29] and innervate the local ganglia in the respiratory tract, enteric nervous system (ENS), liver and pancreas. The DMV receives inputs from the NTS and mediates all vago-vagal reflexes controlling gastrointestinal motility and secretion [21]. Vagal preganglionic neurons located in the ventrolateral portion of the NAmb provide the primary control of the heart via the cardiac ganglia [30]. These cardiovagal outputs inhibit the automatism of the sinoatrial node exerting a beat-to-beat control of the heart rate. The cardiovagal NAMb neurons are activated by the NTS during the baroreflex and are inhibited during inspiration. Sacral Parasympathetic Output The sacral preganglionic output originates in neurons located in the lateral gray matter at the S2-S4 segments of the sacral spinal cord [31]. These neurons are critical for normal micturition, defecation, and sexual organs; this involves their coordinated interactions with both lumbar sympathetic neurons located at T12-L2 levels and somatic motor neurons of the Onuf nucleus at the S2-S4 levels innervating the external urinary sphincter and pelvic floor.

References [1] Craig AD. Interoception: the sense of the physiological condition of the body. Curr Opin Neurobiol 2003;13:500–5. [2] Saper CB. The central autonomic nervous system: conscious visceral perception and autonomic pattern generation. Annu Rev Neurosci 2002;25:433–69. [3] Cechetto DF. Central representation of visceral function. Fed Proc 1987;46:17–23. [4] Loewy AD. Descending pathways to the sympathetic preganglionic neurons. Prog Brain Res 1982;57:267–77. [5] Westerhaus MJ, Loewy AD. Central representation of the sympathetic nervous system. 2001 [6] Vogt BA, Vogt L, Farber NB, Bash E, et al. Architecture and neurocytology of monkey cingulate gyrus. J Comp Neurol 2005;485:218–39. [7] Verberne AJ, Owens NC. Cortical modulation of the cardiovascular system. Prog Neurobiol 1998;54:149–68. [8] LeDoux J. The amygdala. Curr Biol 2007;17:R868–874. [9] Ulrich-Lai YM, Herman JP. Neural regulation of endocrine and autonomic stress responses. Nat Rev Neurosci 2009;10:397–409. [10] Davis M. The role of the amygdala in fear and anxiety. Annu Rev Neurosci 1992;15:353–75. [11] Thompson RH, Swanson LW. Structural characterization of a hypothalamic visceromotor pattern generator network. Brain Res Brain Res Rev 2003;41:153–202.

[12] Sawchenko PE, Li HY, Ericsson A. Circuits and mechanisms governing hypothalamic responses to stress: a tale of two paradigms. Prog Brain Res 2000;122:61–78. [13] Dimicco JA, Zaretsky DV. The dorsomedial hypothalamus: a new player in thermoregulation. Am. J Physiol Regul Integr Comp Physiol 2007;292:R47–63. [14] Yoshida K, Li X, Cano G, et al. Parallel preoptic pathways for thermoregulation. J Neurosci 2009;29:11954–11964. [15] Morrison SF. RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue. Am J Physiol 1999;276:R962–973. [16] Dampney RA, Horiuchi J, McDowall LM. Hypothalamic mechanisms coordinating cardiorespiratory function during exercise and defensive behavior. Auton Neurosci 2008. [17] Bandler R, Keay KA, Floyd N, et al. Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Res Bull 2000;53:95–104. [18] Holstege G. Micturition and the soul. J Comp Neurol 2005;493:15–20. [19] Jean A. The nucleus tractus solitarius: neuroanatomic, neurochemical and functional aspects. Arch Int Physiol Biochim Biophys 1991;99:A3–52. [20] Dampney RA, Horiuchi J. Functional organisation of central cardiovascular pathways: studies using c-fos gene expression. Prog Neurobiol 2003;71:359–84. [21] Travagli RA, Hermann GE, Browning KN, Rogers C, et al. Brainstem circuits regulating gastric function. Annu Rev Physiol 2006;68:279–305. [22] Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci 2006;7:335–46. [23] Goodchild AK, Moon EA. Maps of cardiovascular and respiratory regions of rat ventral medulla: focus on the caudal medulla. J Chem Neuroanat 2009;38:209–21. [24] Lightman SL, Todd K, Everitt BJ. Ascending noradrenergic projections from the brainstem: evidence for a major role in the regulation of blood pressure and vasopressin secretion. Exp Brain Res 1984;55:145–51. [25] Corcoran AE, Hodges MR, Wu Y, Wang W, Wylie CJ, Deneris ES, Richerson S, et al. Medullary serotonin neurons and central CO2 chemoreception. Respir. Physiol Neurobiol 2009;168:49–58. [26] Morrison SF, Nakamura K, Madden CJ. Central control of thermogenesis in mammals. Exp Physiol 2008;93:773–97. [27] Janig W, Habler HJ. Neurophysiological analysis of target-related sympathetic pathways – from animal to human: similarities and differences. Acta Physiol Scand 2003;177:255–74. [28] Strack AM, Sawyer WB, Hughes JH, Platt KB, Lavey AD, et al. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res. 1989;491:156–62. [29] Huang XF, Tork I, Paxinos G. Dorsal motor nucleus of the vagus nerve: a cyto- and chemoarchitectonic study in the human. J Comp Neurol 1993;330:158–82. [30] Hopkins DA, Armour JA. Brainstem cells of origin of physiologically identified cardiopulmonary nerves in the rhesus monkey (Macaca mulatta). J Auton Nerv Syst 1998;68:21–32. [31] Birder L, de Groat W, Mills I, Morrison J, Thor K, Drake M, et al. Neural control of the lower urinary tract: peripheral and spinal mechanisms. Neurourol Urodyn 29:128–39.

I. INTRODUCTION

C H A P T E R

3 Imaging of Brainstem Sites Involved in Cardiovascular Control Vaughan G. Macefield, Luke A. Henderson The medulla is phylogenetically the oldest structure of the brain, containing dense clusters of neurons (nuclei) that coordinate many homeostatically critical functions, such as respiration, heart rate and blood pressure. Much of what we know of the functions of the medulla we have learnt from experiments in anesthetized or decerebrate animals. And, although its small size makes it difficult to study its function in awake humans, it is possible to use non-invasive neuroimaging approaches to delineate some of its nuclei. We shall limit our discussion to the control of arterial pressure, in which most of the work has been carried out. The maintenance of blood pressure within a relatively narrow physiological range is essential to ensure adequate perfusion of vital organs such as the brain. A primary determinant of arterial pressure is the level of sympathetically-mediated vasoconstriction within skeletal muscle. Muscle sympathetic nerve activity (MSNA) occurs as pulse-synchronous bursts, the incidence and magnitude of which are inversely correlated to diastolic pressure. The beat-to-beat control of arterial pressure is governed by the baroreflex – a classic negative-feedback loop in which increases in blood pressure, detected by the arterial baroreceptors (located in the carotid sinus and aortic arch), are corrected by withdrawal of muscle vasoconstrictor drive as well as decreases in heart rate and stroke volume. This short-term regulation of blood pressure survives removal of structures rostral to the medulla yet is abolished by transection of the spinal cord immediately below the medulla, indicating that the medulla contains structures critical for the generation and maintenance of vasomotor tone.

produce increases in activity in cutaneous sympathetic neurons yet no increase in blood pressure [1]. It is known from intracellular recordings from RVLM neurons that they exhibit an irregular tonic firing that decreases with increases in blood pressure and increases with decreases in blood pressure; it is also known that this tonic activity is modified by excitatory and inhibitory inputs, but that in the absence of such inputs this tonic activity continues [2]. The current understanding of the baroreflex circuitry defined primarily in anesthetized animal preparations – is that primary afferent axons from the baroreceptors project to the caudal region of the nucleus tractus solitarius (NTS), where they synapse onto second-order neurons, which in turn send excitatory (glutamatergic) projections onto GABAregic neurons within the region of the caudal ventrolateral medulla (CVLM). These CVLM GABAergic neurons synapse directly onto excitatory neurons within the RVLM and serve to inhibit the spontaneous activity of RVLM premotor sympathetic neurons. Nucleus ambiguus and the dorsal motor nucleus of the vagus also receive glutamatergic projections from NTS [3], activating vagal cardiac efferents and slowing the heart. In addition to these components of the basic baroreflex arc, it is known that other brainstem regions, such as the caudal pressor area (CPA), located in the most caudal part of the ventrolateral medulla, can also influence baroreflex activity by altering the activity in these baroreflex medullary nuclei [2]. Studies in conscious animals, using c-fos expression as a marker of neuronal activation, have confirmed the operation of the NTS-CVLM-RVLM serial pathway during maneuvers that increase or decrease arterial pressure [4,5].

IDENTIFICATION OF MEDULLARY CARDIOVASCULAR NUCLEI IN EXPERIMENTAL ANIMALS

IDENTIFICATION OF MEDULLARY CARDIOVASCULAR NUCLEI IN HUMANS

The circuitry responsible for the baroreflex has been examined in detail in anesthetized experimental animals. Early work had shown that excitation within discrete regions of the rostral ventrolateral medulla (RVLM) could activate muscle vasoconstrictor neurons and increase blood pressure, while activation within other areas could

Non-invasive neuroimaging has become a powerful tool in defining the functions of various regions in the human brain, but relatively little work has been done in cardiovascular control (for review see [6]). Most of the recent studies in this area use functional magnetic resonance imaging (fMRI), in which changes in blood-oxygen


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3. ImAgIng of BRAInSTEm SITES InvolvEd In CARdIovASCulAR ConTRol

FIGURE 3.1 Significant BOLD signal intensity changes in the medulla and cerebellum, obtained from the brainstem-specific scans (n  15) overlaid onto an average T2-weighted anatomical image set. Ventral is at the base of each image. The hot and cool color scales (coded for t-value) indicate regional signal increases and decreases, respectively. Slice positions (MNI space) are indicted in the top right of each image. To the right are three graphs showing the mean (SEM) percent changes in signal intensity (SI) over time for three significant clusters. Vertical grey boxes indicate each of the three inspiratory-capacity apnea periods. From Macefield et al. (2006) [8].

level dependent (BOLD) signal intensity are used as a proxy marker of neuronal activity: increases in signal intensity reflect increases in neuronal activity, while decreases reflect deactivations. Such approaches have been used in the anesthetized cat to identify changes in signal intensity within the medullary regions described above [7], but relatively few studies have applied fMRI to investigate the role of the brainstem in human cardiovascular control. One of the first investigations to do so used the Valsalva maneuver – a forced expiratory effort which evokes a stereotypical pattern of autonomic changes. The virtue in using this maneuver is that the changes in heart rate and arterial pressure are well known, with the increase in arterial pressure being brought about by an increase in muscle sympathetic nerve activity (MSNA). Significant changes in BOLD signal intensity occurred in the amygdala, hippocampus, insular and lateral frontal cortices, as well as in the dorsal pons, dorsal medulla, lentiform nucleus, and fastigial and dentate nuclei of the cerebellum [7]. This was one of the first studies to have attempted to identify the functional roles of the human brainstem in autonomic control, but the fact that the Valsalva maneuver is a complex, volitionally generated task makes it difficult to tease out elements related to motor planning, execution, sensory feedback and autonomic changes. A simpler maneuver, the inspiratory-capacity apnea – a maximal inspiratory

FIGURE 3.2 Left panel, axial sections of the brainstem during a series of inspiratory-capacity apneas, showing bilateral activation of the dorsolateral medulla. Data from Macefield et al. (2006) [8]. Right panel, axial sections of the human brainstem showing a high density of AngII binding in RVLM. Data reproduced with permission from Allen et al. (1988) [9]. See color plate at back of the book.

breath-hold – evokes a sustained increase in MSNA that is believed to be due primarily to unloading of the low-pressure baroreceptors. During the static phase of the maneuver, in which the inspiratory pump muscles are quiescent and only the laryngeal constrictors are active, intrathoracic pressure changes because of the elastic recoil of the lungs and chest wall against the closed glottis. This is different to the Valsalva maneuver, in which the diaphragm, abdominal and intercostal muscles are co-activated in a sustained effort that increases intra-abdominal and intrathoracic pressures. Like the Valsalva maneuver,

I. INTRODUCTION

IdEnTIfICATIon of mEdullARy CARdIovASCulAR nuClEI In HumAnS

15

FIGURE 3.3 Significant increases (warm color scale) and decreases (cool color scale) in BOL1D signal intensity within the brainstem correlated with MSNA total burst activity in the resting state; raw fMRI data from seven experiments. Equivalent histological sections are shown on the right. Figure from Macefield and Henderson (2010) [12].

the inspiratory-capacity apnea evokes discrete changes in BOLD signal intensity over multiple brain regions. Significant increases in BOLD signal intensity occur bilaterally in the lateral prefrontal cortex, anterior insular cortex, anterior cingulate cortex, dorsomedial hypothalamus and deep cerebellar nuclei; decreases in signal intensity occur in the hippocampus, posterior cingulate cortex and the cerebellar cortex [8]. As shown in the sagital and axial sections of the human brainstem in Figure 3.1, bilateral increases in activity also occur in the rostral lateral medulla, with decreases occurring in the dorsomedial and caudal lateral medulla. We believe the increases in the rostral lateral medulla reflect increases in activity of the RVLM. In humans, the RVLM and CVLM are displaced dorsally by the large inferior olivary nuclei [9]. The functional and anatomical localization of the human RVLM is illustrated in Figure 3.2: the left panel shows a bilateral increase in signal intensity during a maximal inspiratory breath-hold; the right panel shows the anatomical identification of the human RVLM, based on histochemical identification [9]. While McAllen and colleagues have functionally identified the medullary raphé nucleus during whole-body cooling [10], until recently no human fMRI studies had functionally identified NTS. As indicated in the right panel of Figure 3.2, the solitary tract nucleus is located dorsal and medial to the RVLM. We believe the decreases in signal intensity in the dorsomedial medulla reflect decreases in NTS, with the decreases in the caudal lateral medulla reflecting

decreases in neuronal activity in the CVLM. These responses can be explained by (i) the reduction of baroreceptor inputs to the NTS; (ii) a consequent reduction in activity of CVLM; and hence (iii) a reduction in inhibitory drive to the RVLM. The withdrawal of inhibitory drive to RVLM then allows this nucleus to increase its activity and thereby bring about an increase in MSNA. Sustained increases in MSNA can also be induced by static handgrip exercise and post-exercise ischemia: accumulation of the metabolites of contraction activates metaboreceptors within the muscle and induces a reflex increase in MSNA and arterial pressure. It is known that metaboreceptors project to the NTS, which sends excitatory projections to the RVLM without an intervening inhibitory synapse: increases in BOLD signal intensity occurred in discrete regions of the human medulla corresponding to the NTS and RVLM, but did not include CVLM [11]. A recent development has been concurrent microneurography and fMRI. By recording spontaneous bursts of MSNA and correlating these to spontaneous fluctuations in BOLD signal intensity it has been possible to functionally identify regions of the brainstem involved in the generation of spontaneous MSNA [12]. Analysis of the temporal coupling between BOLD signal intensity and nerve signal intensity revealed sites in which the two signals covaried, but these sites were limited to the medulla. The advantage of this approach is that maneuvers, with all of the associated confounds, need not be performed. As shown in Figure 3.3, increases in MSNA were associated

I. INTRODUCTION

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3. ImAgIng of BRAInSTEm SITES InvolvEd In CARdIovASCulAR ConTRol

with bilateral increases in signal intensity in the rostral lateral medulla that corresponds to the human equivalent of the RVLM. There was also a unilateral (left) increase in activity in the area that corresponds to the caudal pressor area (CPA). Reciprocal decreases in signal intensity occurred in the dorsomedial region of NTS and in CVLM, though on average the latter appeared to be limited to the right side. Although not illustrated here, increases in signal intensity also occurred in the medullary raphé and in the region that includes the dorsal motor nucleus of the vagus. The use of this approach promises to increase our understanding of the functional organization of the medulla and changes in sympathetic outflow that are associated with many pathophysiological states.

References [1] McAllen RM, May CN, Shafton AD. Functional anatomy of sympathetic premotor cell groups in the medulla. Clin Exp Hypertens 1995;17:209–21. [2] Dampney RA, Polson JW, Potts PD, Hirooka Y, Horiuchi J. Functional organization of brain pathways subserving the baroreceptor reflex: studies in conscious animals using immediate early gene expression. Cell Mol Neurobiol 2003;23:597–616. [3] Wang J, Irnaten M, Neff RA, Venkatesan P, Evans C, Loewy AD, et al. Synaptic and neurotransmitter activation of cardiac vagal neurons in the nucleus ambiguus. Ann N Y Acad Sci 2001;940:237–46. [4] Minson JB, Llewellyn-Smith IJ, Arnolda LF, Pilowsky PM, Chalmers JP. C-fos expression in central neurons mediating the arterial baroreceptor reflex. Clin Exp Hypertens 1997;19:631–43.

[5] Horiuchi J, Killinger S, Dampney RA. Contribution to sympathetic vasomotor tone of tonic glutamatergic inputs to neurons in the RVLM. Am J Physiol 2004;287:R1335–43. [6] Critchley HD, Nagai Y, Gray MA, Mathias CJ. Dissecting axes of autonomic control in humans: insights from neuroimaging. Auton Neurosci. 2011;161:34–42. [7] Henderson LA, Richard CA, Macey PM, Runquist ML, Yu PL, Galons JP, et al. Functional magnetic resonance signal changes in neural structures to baroreceptor reflex activation. J Appl Physiol 2004;96:693–703. [8] Macefield VG, Gandevia SC, Henderson LA. Neural sites involved in the sustained increase in muscle sympathetic nerve activity induced by inspiratory-capacity apnea – a fMRI study. J Appl Physiol 2006;100:266–73. [9] Allen AM, Moeller I, Jenkins TA, Zhuo J, Aldred GP, Chai SY, Mendelsohn FA. Angiotensin receptors in the nervous system. Brain Res Bull 1998;47:17–28. [10] McAllen RM, Farrell M, Johnson JM, Trevaks D, Cole L, McKinley MJ, et al. Human medullary responses to cooling and rewarming the skin: a functional MRI study. Proc Nat Acad Sci USA 2006;103:809–13. [11] Sander M, Macefield VG, Henderson LA. Cortical and brainstem changes in neural activity during static handgrip and post-exercise ischemia in humans. J Appl Physiol 2010;108:1691–700. [12] Macefield VG, Henderson LA. Real-time imaging of the medullary circuitry involved in the generation of spontaneous muscle sympathetic nerve activity in awake subjects. Human Brain Mapp 2010;31:539–59.

I. INTRODUCTION

C H A P T E R

4 Peripheral Autonomic Nervous System Robert W. Hamill, Robert E. Shapiro, Margaret A. Vizzard are paired structures that are located bilaterally along the vertebral column. They extend from the superior cervical ganglia (SCG), located rostrally at the bifurcation of the internal carotid arteries, to ganglia located in the sacral region. All told, there are three cervical ganglia (the SCG, middle cervical ganglion, and inferior cervical ganglion, which is usually termed the cervicothoracic or stellate ganglion because it is a fused structure combining the inferior cervical and first thoracic paravertebral ganglia), eleven thoracic ganglia, four lumbar ganglia, and four to five sacral ganglia. More caudally, two paravertebral ganglia join to become the ganglion impar. Prevertebral ganglia are midline structures located anterior to the aorta and vertebral column, and are represented by the celiac ganglia, aortico-renal ganglia, and the superior and inferior mesenteric ganglia. Previsceral, or terminal ganglia, are small collections of sympathetic ganglia located close to target structures; they are also referred to as short noradrenergic neurons since their axons cover limited distances. Generally, the preganglionic fibers are relatively short and the postganglionic fibers are quite long in the SNS. The axons of these postsynaptic neurons are generally unmyelinated and of small diameter (5 μm). The target organs of sympathetic neurons include smooth muscle and cardiac muscle, glandular structures, and parenchymal organs (e.g., liver, kidney, bladder, reproductive organs, muscles, etc. (see Fig. 4.1)) as well as other cutaneous structures. The spinal cells of origin for the presynaptic input to sympathetic peripheral ganglia are located from the first thoracic to the second lumbar level of the cord, although minor variations exist. The principal neurons generally have been viewed as located in the lateral horn of the spinal gray matter (intermediolateral cell column-IML), but four major groups of autonomic neurons exist: intermediolateralis pars principalis (ILP), intermediolateralis pars funicularis (ILF), nucleus intercalatus spinalis (IC) and the central autonomic nucleus (CAN) or dorsal commissural nucleus (DCN) (anatomical nomenclature is nucleus intercalatus pars paraependymalis (ICPE)). For paravertebral ganglia, 85–90% of the presynaptic fibers originate from cell bodies in ILP or ILF. Prevertebral ganglia and terminal ganglia receive a larger proportion of preganglionic terminals from the CAN/DCN. The nucleus intercalatus also contributes preganglionic fibers, but the exact

The autonomic nervous system (ANS) is structurally and functionally positioned to interface between the internal and external milieu, coordinating bodily functions to ensure homeostasis (cardiovascular and respiratory control, thermal regulation, gastrointestinal motility, urinary and bowel excretory functions, reproduction, and metabolic and endocrine physiology), and adaptive responses to stress (flight or fight response). Thus, the ANS has the daunting task of ensuring the survival as well as the procreation of the species. These complex roles require complex responses, and depend upon the integration of behavioral and physiological responses that are coordinated centrally and peripherally. In 1898, Langley, a Cambridge University physiologist coined the term “autonomic nervous system” and identified three separate components (sympathetic, parasympathetic, and enteric). The following section of the Primer will focus on the first two aspects of the peripheral ANS: sympathetic nervous system (SNS) including the adrenal medulla; and parasympathetic nervous system (PNS). The following précis will address the neuroanatomy of the SNS, adrenal medulla, and PNS, and then present a more detailed, albeit brief, review of the functional neuroanatomy, physiology and pharmacology of the peripheral autonomic nervous system. Importantly, the ANS’s role at multiple interfaces in normal and abnormal physiology is emerging as a key mediator of pathophysiology in a range of complex disorders (anxiety and panic, chronic fatigue syndrome, regional pain syndromes, autonomic failure) and a critical substrate underpinning the field of neurocardiology. The following information will serve as a framework from which to view the complexity of the ANS as revealed in the more detailed descriptions that follow.

SYMPATHETIC NERVOUS SYSTEM (SEE FIG. 4.1) The SNS is organized at a spinal and peripheral level such that cell bodies within the thoracolumbar segments of the spinal cord provide preganglionic efferent innervation to sympathetic neurons that reside in ganglia dispersed in three arrangements: paravertebral, prevertebral, and previsceral or terminal ganglia. Paravertebral ganglia


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4. PERIPHERAl AuToNomIC NERvouS SySTEm

Parasympathetic Division

Brainstem & Spinal Cord

Sympathetic Division

Target Organs

Trunk

Trunk

Target Organs

Oliary Ganglion

III

Nucleus Ambigours Superior Cervical Ganglion

Parotid Gland

Middle Cervical Ganglion Stellate

Heart

Ganglion

Respiratory Tract & Lungs

Small Intestine Adrenal Medulla Small Intestine

Celiac Ganglion

Greater Splanchnic Nerve Lesser Splanchnic Nerve Superior Mesenteric Ganglion

Colon Pelvin Colon & Rectum Genitourinary Tract

Interior Mesenteric Ganglion

C1 C2 C3 C4 C5 C6 C7 C8 T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 L1 L2 L3 L4 L5 S1 S2 S3 S4 S5

Submandibular Ganglion

Sweat Glands, Hair Follicles, Blood Vessels

Lacrimal & Submandibular Glands

Stomach

VII IX X X

Edinger-Westphal Nucleus Superior Salivatory Nucleus Inferior Salivatory Nucleus Dorsal Motor Nucleus

Eye

Pterygopalatine Ganglion

Otic Ganglion

Eye Lacrimal & Palatine Glands Submandibular & Sublingual Glands Parotid Gland

Respiratory Tract & Lungs Gastrointestinal Tract, Liver & Pancreas Heart Nasopharynx Esophagus

Pelvic Colon & Rectum Pelvic Nerve

Genitourinary Tract

Hypogastric Ganglion

FIGURE 4.1 Schematic diagram of the sympathetic and parasympathetic divisions of the peripheral autonomic nervous system. The paravertebral chain of the sympathetic division is illustrated on both sides of the spinal outflow in order to demonstrate the full range of target structures innervated. Although the innervation pattern is diagrammatically illustrated to be direct connects between preganglionic outflow and postganglionic neurons, there is overlap of innervation such that more than one spinal segment provides innervation to neurons within the ganglia.

extent of these is not fully understood and is probably limited. These spinal autonomic nuclei receive substantial supraspinal input from multiple transmitter systems located at multiple levels of the neuraxis; diencephalon (hypothalamus) and brainstem (raphe, locus coeruleus, reticular formation and ventral lateral medulla) provide the largest input and the pattern of innervation viewed in horizontal sections reveals a ladder like arrangement of the distribution of nerve terminals [1]. Without detailing the source or course of specific systems, it is important to point out that the following different neurotransmitter systems impinge on preganglionic neurons within this ladder like structure: monoamines – epinephrine, norepinephrine (NE), serotonin; neuropeptides – substance P, thyrotropin-release hormone (TRH), met-enkephalin, vasopressin, oxytocin, and neuropeptide Y (NPY); amino acids – glutamate, GABA, and glycine. Undoubtedly, others exist and more will be found. It is apparent that dysfunction of these supraspinal systems, or alterations of these

neurotransmitters by disease or pharmacological agents will alter the spinal control of peripheral ganglia and result in clinical dysfunction. The outflow from the spinal cord to peripheral ganglia is segmentally organized with some overlap. Retrograde tracing studies indicate that there is a rostral-caudal gradient: SCG receives innervation from spinal segments T1–T3; stellate ganglia – T1–T6; adrenal gland – T5–T11; celiac and superior mesenteric ganglia – T5–T12; inferior mesenteric and hypogastric ganglion from L1–L2. These presynaptic fibers, which are small in diameter (2–5 μm) and thinly myelinated, exit the ventral roots via the white rami communicantes to join the paravertebral chain either directly innervating their respective ganglion at the same level or traveling along the chain to innervate a target ganglion many levels away (Fig. 4.2). The distribution of postsynaptic fibers also follow a regional pattern with the head, face and neck receiving innervation from the cervical ganglia (spinal segments T1–4), the upper limb and

I. INTRODUCTION

19

SymPATHETIC NERvouS SySTEm

Parasympathetic Nervous System

Sympathetic Nervous System Thoracolumbar Spinal Cord

Sacral Spinal Cord

Spinal ganglion Afferent autonomic fiber Vasomotor Sudomotor Pilomotor fibers

Preganglionic fiber Postganglionic fiber

Gray communicating ramus (post ganglionic) White communicating ramus (preganglionic)

Paravertebral ganglion

Splanchnic nerve

Descending colon Rectum Urinary Bladder Sexual organs

Pelvic splanchnic nerve Prevertebral ganglion Pelvic ganglion

Postganglionic visceral nerves to smooth muscle of inner organs & visceral blood vessels

FIGURE 4.2 Schematic illustration of the segmental spinal arrangement of the sympathetic and parasympathetic nervous system. Although segmental interactions exist, they are polysynaptic operating via interneurons; the primary input to spinal preganglionic neurons is supraspinal originating from brainstem structures (not shown).

thorax from the stellate and upper thoracic ganglia (spinal segments T1–8); the lower trunk and abdomen from lower thoracic ganglia (spinal segments T4–12), and the pelvic region and lower limbs from lumbar and sacral ganglia (spinal segments T10–L2). More recently, with the introduction of trans-neuronal tracing techniques, using pseudorabies virus (PRV), it has been possible to inject ganglia in the periphery and examine the trans-synaptic passage of PRV. Thus, supraspinal neurons projecting to the specific sets of preganglionic neurons that innervate the peripheral ganglia injected may be examined. Interestingly, a surprisingly common set of central pathways influencing the thoracolumbar sympathetic outflow were labeled. For instance, following injections in either the SCG, stellate or celiac ganglia, or the adrenal gland, the following five brain areas are labeled: ventromedial and rostral ventrolateral medulla; caudal raphe nuclei; A5 noradrenergic cell group; and the paraventricular nucleus of the hypothalamus [2]. Apparently, these central loci must share regulatory functions that are coordinated through similar pathways of thoracolumbar outflow. These same studies indicate that other brain areas are only labeled from specific ganglia; thus, site specific central control exists as well. Of additional interest is that numerous, small interneurons in Rexed laminae VII and X of the spinal cord were labeled providing structural support

for the observation that spinal intersegmental and intrasegmental autonomic interactions (including autonomic reflexes) exist. It is apparent that the structural organizational of the sympathetic nervous system permits the integration and dissemination of responses depending on demand. Multiple supraspinal descending pathways provide a dense innervation of all four major autonomic cell groups in the spinal cord, but clearly specific topographic responses exist as well. In turn, each preganglionic neuron innervates anywhere from 4 to 20 postganglionic sites, and each spinal outflow level may reach multiple peripheral ganglia which in turn supply multiple targets, permitting additional dispersion of sympathetic responses when indicated. At each thoracic level there are an estimated 5000 preganglionic neurons (these counts have generally been limited to the cells located in the ILP). Since preganglionic output to prevertebral ganglia originates from more medially placed cell bodies, it is conceivable that a greater number of neurons at certain segmental levels contribute to the output. Thus, a given spinal segment has a powerful base to influence greater than 100,000 postganglionic neurons. Although original thinking suggested that responses were “all-or-none and widespread”, anatomical studies have continued to reveal subtleties of structural arrangements that indicate that the system is not only poised for

I. INTRODUCTION

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generalized activation of the peripheral sympathetic nervous system, but is also able to exert control of relatively specific sites and functions. The postganglionic fibers in the SNS travel quite lengthy paths to arrive at target organs. For instance, fibers from the SCG traverse the extracranial and intracranial vasculature to reach such targets as the lacrimal glands, parotid glands, pineal gland, and pupils. Fibers from the stellate ganglia course through the brachial plexus to reach vascular and cutaneous targets in the upper limb and hand. Within the abdomen, axons originating from the paravertebral ganglia supply the viscera as well as the mesenteric vasculature. Lumbar and sacral paravertebral ganglia course distally along peripheral nerves and blood vessels to reach the distal vasculature and cutaneous structures in the feet. In humans, the innervation to the leg requires a sympathetic axon to be 50 cm long and with an estimated overall diameter of 1.2 μm the axonal volume is approximately 565,000 μm3. This axonal cytoskeleton and its metabolic requirements are supported by a perikaryon of about 30 μm with a somal volume of 14,000 μm3. With this structural architecture to maintain, these neurons are vulnerable to various metabolic and structural insults. Although most preganglionic fibers have a relatively short course to their ganglion targets, the upper thoracic preganglionic fibers travel relatively longer distances to reach the stellate and SCG, and preganglionic fibers to the adrenal medulla and prevertebral ganglia course through the paravertebral chain reaching these visceral targets as the splanchnic nerves. Along the course, fiber systems may be interrupted, resulting in local autonomic dysfunction. For example, the Horner’s syndrome results from lesion of either preganglionic fibers to the SCG or the postganglionic axons which leave the SCG to innervate Müller’s muscle of the upper eyelid, pupillodilator muscles, facial vasculature, and sudomotor structures of the face (see Fig. 4.1). The autonomic neuroeffector junction is generally a poorly defined synaptic structure lacking the pre- and postjunctional specializations that are observed in the central nervous system or skeletal muscle motor end plates. The unmyelinated, highly branched postganglionic fibers become beaded with varicosities as they approach their targets. The varicosities are not static; they move along as structures with a diameter of 0.5 to 2 μm, and a length of approximately 1.0 μm. The number of varicosities varies from 10,000/mm3 to over 2 million per mm3 depending on the target being innervated. The varicosities are packed with mitochondria and vesicles containing various transmitters, and are at varying distances from their target organs. For instance, for smooth muscle targets, this distance varies from 20 nm in the vas deferens to 1–2 μm in large arteries. In a sense the release of transmitter is accomplished en passage as the impulse travels along an autonomic axon. The lack of a restrictive synaptic arrangement permits the released NT to diffuse various distances along a target organ and activate multiple receptors,

again expanding the overall effect of sympathetic activation. Between 100 and 1000 vesicles exist in each varicosity in noradrenergic fibers. Traditional teaching suggests that vesicle characteristics indicate the transmitter system: small granular vesicles are noradrenergic; small agranular vesicles are cholinergic; large granular vesicles are peptidergic. However, exceptions to these correlations exist. The principal neuronal phenotype in peripheral sympathetic ganglia is the noradrenergic neuron which is generally multipolar in character with synapses mainly located more on dendrites than somata. Depending on which ganglia are examined, studies indicate that from 80–95% of ganglion cells will stain positively for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, or have positive catecholamine fluorescence. The remaining cells have a mixture of transmitters, or are postganglionic cholinergic cells (the sudomotor and periosteal components of sympathetic function). Within sympathetic ganglia there is a small group of small intensely fluorescent (SIF) neurons. The transmitters identified in SIF cells include dopamine, epinephrine, or serotonin. As will be described later, the original concept that preganglionic neurons in the SNS are cholinergic and postganglionic neurons are noradrenergic has given way to new information that a whole array of molecules (cholinergic, catecholaminergic, monoaminergic, peptidergic, “non-cholinergic, non-adrenergic”, and gaseous) appear to be involved in neurotransmission either as agents themselves or as neuromodulators (vide infra).

SYMPATHOADRENAL AXIS AND THE ADRENAL GLAND Interactions between the adrenal cortex and adrenal medulla constitute a critical link between the autonomic and endocrine systems. The adrenal cortex is largely regulated by the hypothalamic-pituitary-adrenocortical axis, whereas the adrenal medulla is primarily under neural control. Both adrenal cortex and medulla, respond to stress and metabolic aberrations. The coordinated response of elevated plasma cortisol and catecholamines during stress indicate that central limbic and hypothalamic centers exert combined influences to ensure the needed neurohumoral adaptations. The interdependence of these two components of the adrenal gland arise early in development: migrating sympathoblasts destined for the adrenal medulla require the presence of the cortical tissue to change their developmental fate from neurons to that of chromaffin cells. These cells, named because they exhibit brown color when treated with “chrome salts”, do not develop neural processes but instead serve an endocrine function by releasing their neurohumors (epinephrine, norepinephrine, and neuropeptides) into the blood stream. During adulthood, the presence of the cortex is critical for maintaining the levels of epinephrine since the induction of the enzyme

I. INTRODUCTION

PARASymPATHETIC NERvouS SySTEm

phenylethanolamine-n-methyltransferase is dependent on local levels of cortisol. Although traditional teaching emphasizes the preganglionic cholinergic splanchnic innervation of the adrenal medulla, there is also evidence that postganglionic sympathetic fibers, vagal afferents, and other sensory afferents are present. Tracing studies indicate that dye placed within the adrenal medulla is transported retrogradely within spinal preganglionic sympathetic neurons in a somewhat bell shaped distribution from approximately T2–L1 with the predominant innervation originating from T7–T10. Neuronal cell bodies are primarily within the nucleus intermediolateralis pars principalis (ILP) with the pars funicularis and pars intercalatus providing a relatively small portion of the innervation. The exiting nerve roots pass through the sympathetic chain, join to form the greater splanchnic nerve, and distribute themselves beneath the adrenal capsule and within the medulla. A small number of nerve cells are labeled in ganglia within the sympathetic chain suggesting that postganglionic sympathetic fibers innervate the gland. Whether these terminals are labeled as they pass along blood vessels within the gland or whether they innervate medullary or cortical cells is not fully resolved. Also, at least in the guinea pig, tracing studies indicate that the parasympathetic system may contribute a small efferent innervation to the gland since neurons in the dorsal motor nucleus of the vagus are labeled after injections in the medulla. Also, cell bodies within the dorsal root ganglia and vagal sensory ganglia (nodose) are also labeled after tracer studies of the adrenal medulla indicating an afferent innervation as well. Lastly, although not a prominent innervation pattern, there appears to be an intrinsic innervation that arises from ganglion cells sparsely populating the subcapsular, cortical and medullary regions of the gland. The innervation pattern of the adrenal medulla is thus more complex than the traditionally listed thoracolumbar preganglionic cholinergic outflow, although the major adaptive responses depend on the preganglionic cholinergic innervation since surgical section of these nerves, or pharmacological blockade with cholinergic antagonists preclude the induction of tyrosine hydroxylase and appropriate release of catecholamines following various stress paradigms. Morphological studies of the adrenal medulla have revealed the presence of two basic types of granules in chromaffin cells. A diffuse spherical granule contains the predominant monoamine secreted by medullary cells, epinephrine, whereas eccentrically located dense core granules contain norepinephrine. As indicated above for ganglion neurons, chromaffin cells of the adrenal medulla also cocontain other molecules. For instance, the opioid molecules are well represented: enkephalin is co-contained in vesicles with the monoamines. The signaling cascade responsible for enhancing the synthesis and release of these neurohormonal agents is complicated and includes preganglionic innervation, steroid hormones (e.g., glucocorticoids), and growth factors (e.g., nerve growth factor, NGF).

21

PARASYMPATHETIC NERVOUS SYSTEM (SEE FIG. 4.1) The craniosacral outflow is the source of central neuronal pathways providing the efferent innervation of peripheral ganglia of the parasympathetic nervous system (PNS). The cranial nerves involved include cranial nerves III, VII, IX, and X, and the sacral outflow is largely restricted to sacral cord levels 2, 3, and 4. As indicated for the SNS, the preganglionic innervation is largely cholinergic with these terminals releasing acetylcholine (ACh) at the ganglion synapses. In contrast to the SNS, the major transmitter postsynaptically is also ACh. These cholinergic neurons also co-contain other transmitter substances: preganglionic neurons contain enkephalins, and ganglionic cholinergic neurons frequently contain vasoactive intestinal peptide (VIP) and/or NPY. The parasympathetic fibers in cranial nerve III originate in the Edinger–Westphal nuclei of the midbrain and travel in the periphery of the nerve (where they are subject to dysfunction secondary to nerve compression), exiting along with the nerve to the inferior oblique to supply the ciliary ganglion. Second order postganglionic fibers exit in the ciliary nerves and supply the pupiloconstrictor fibers of the iris and the ciliary muscle where their combined action permits the near response, including accommodation. The salivatory nuclei, located near the pontomedullary junction, provide the preganglionic parasympathetic innervation for cranial nerves VII and IX. The superior salivatory nucleus sends preganglionic fibers, which leave the facial nerve at the level of the geniculate ganglion (non-parasympathetic sensory ganglion), to form the greater superficial petrosal nerve to the pterygopalatine (sphenopalatine) ganglia that provides postganglionic secretomotor and vasodilator fibers to the lacrimal glands via the maxillary nerve. Other preganglionic fibers in the facial nerve continue and subsequently leave via the chorda tympani to join the lingual nerve, eventually synapsing in the submandibular ganglion. Postsynaptic cholinergic fibers supply the sublingual and submandibular glands. Postganglionic fibers from the pterygopalatine and submandibular ganglia also supply glands and vasculature in the mucosa of the sinuses, palate, and nasopharynx. The inferior salivatory nucleus sends preganglionic fibers via the glossopharyngeal nerve (cranial nerve IX) to the otic ganglion that in turn relays postganglionic fibers to the parotid gland via the auriculotemporal nerve. The preganglionic fibers in cranial nerve IX branch from the nerve at the jugular foramen and contribute to the tympanic plexus and form the lesser superficial petrosal nerve. This nerve exits the intracranial compartment via the foramen ovale along with the third division of the trigeminal nerve to reach the otic ganglion. The most caudal cranial nerve participating in the preganglionic parasympathetic system is the vagus nerve (cranial nerve X). The dorsal motor nucleus of the vagus is located in the medulla and sends preganglionic fibers to innervate essentially all organ systems within the chest

I. INTRODUCTION

22


and abdomen including the gastrointestinal tract as far as the left colonic flexure (splenic flexure). Also, the nucleus ambiguus supplies preganglionic fibers to the vagus and these fibers are believed to be involved mostly with regulating visceral smooth muscle whereas the dorsal motor vagus neurons may be secretomotor in nature. The glossopharyngeal and vagus nerves also contain a substantial number of afferent fibers (in the vagus, afferent fibers may exceed the efferent fiber system by a ratio of 9 to 1) so that a sensory component related to autonomic control exists within cranial nerve IX and X. These afferents provide a critical component of the baroreceptor reflex arc relaying information regarding the systemic blood pressure to central cardiovascular areas in the nucleus tractus solitarii and other medullary centers involved in blood pressure and heart rate control. The parasympathetic cell bodies in the spinal cord are located in the sacral parasympathetic nucleus of second, third, and fourth sacral segments (see Fig. 4.2). These neurons send preganglionic nerve fibers via the pelvic nerve to ganglia located close to or within the pelvic viscera. Postganglionic fibers are relatively short, in contradistinction to their length in the SNS, and supply cholinergic terminals to structures involved in excretory (bladder and bowel) and reproductive (fallopian tubes and uterus, prostate, seminal vesicles, vas deferens, and erectile tissue) functions. Of interest, the pelvic ganglia involved in some of these functions appear to be mixed ganglia (especially in rodents) where sympathetic and parasympathetic neurons are components of the same pelvic ganglion. They appear to receive their traditional preganglionic input, but may have local interconnections that are not fully revealed by current studies. As indicated below, there is clear evidence that the cholinergic postganglionic neurons in the pelvic ganglion in the rodent co-contain vasoactive intestinal peptide and nitric oxide as two other neuroactive compounds. These neurons are believed to be integrally involved in sexual functions in the male, permitting the development and maintenance of potency. The exact regulatory factors controlling the synthesis and release of these transmitter molecules, and the specific receptor systems involved remain to be fully explored.

THE CONCEPT OF PLURICHEMICAL TRANSMISSION AND CHEMICAL CODING The notion of the presence of multiple transmitters and a chemical coding system of autonomic neurons is now firmly established. Originally it was posited that principal neurons were only noradrenergic (contained NE), but over the last two decades it has become clear that within a single neuron multiple transmitter systems may exist, and that within a given ganglion the variety and pattern of neurotransmitters may be quite extensive (see Table 4.1). Also, the composition of neurotransmitters (NT) may change depending on the location of the ganglia: paravertebral

TABLE 4.1 Neurotransmitter Phenotypes in Autonomic Neurons Autonomic Neurons Sympathetic neurons Paravertebral ganglia Prevertebral ganglia Terminal ganglia (previsceral ganglia) Parasympathetic neurons Major parasympathetic ganglia Ciliary Sphenopalatine Otic Submandibular/sublingual Pelvic ganglia Terminal parasympathetic ganglia (previsceral ganglia) Enteric neurons Myenteric plexus (Auerbach’s) Submucosal plexus (Meisner’s)

Transmitter Characteristics (not all inclusive) NE, CCK, somatostatin, SP, Enk, Ach VIP, 5-HT, NPY, DYN1-8, DYN1-17

ACh, VIP, SP, CAs-SIF, NPY, NO

GABA, ACh, VIP, 5-HT SP, Enk, SRIF, motilinlike peptide, bombesinlike peptide

Enteric ganglia Chromaffin cells of adrenal medulla Paraganglia-chromaffin

E, NE, Enk, NPY, APUD

SIF cells, ganglia

ganglia tend to have fewer transmitters whereas prevertebral and terminal ganglia may have various NT, although as noted for the guinea pig SGC (Fig. 4.3), some paravertebral ganglia have a broad array of transmitters. The exact co-location and functions of these multiple transmitters are not fully understood, but some general principles exist. Neuropeptide NPY is probably the most prominent peptide in sympathetic ganglia and is highly co-localized with NE. The sudomotor component of ganglia is dependent upon a population of cells, which are cholinergic in character [contain ACh as NT], and the most frequent peptide colocalized with ACh is vasoactive intestinal peptide (VIP). The distribution of these cholinergic cells varies: in paravertebral ganglia they may represent 10–15% of the neuronal population whereas they represent 1% of the neurons in prevertebral ganglia. NE  NPY, and ACh  VIP are believed to be released together, but some degree of activity associated-chemical coding exists. That is, at lower levels of activation NE is preferentially released, whereas higher levels of stimulation result in NPY being released. Both agents have vasoconstrictor properties and are integral in cardiovascular control, especially in the maintenance of blood pressure. Of course, the eventual action and effect of a transmitter rests with the receptor system that is activated (vide infra). Purinergic neurotransmission expands the co-transmission motif. Presynaptic and postsynaptic mechanisms exist: the purine nucleotide adenosine triphosphate (ATP) is in high concentration in sympathetic synaptic vesicles and following release ATP is catabolized to adenosine

I. INTRODUCTION

vISCERAl AffERENT NEuRoNS ANd AuToNomIC NERvouS SySTEm

23

FIGURE 4.3 Chemical coding and target organization. Chemical coding of sympathetic neurons projecting from the superior cervical ganglion to various targets in the head of guinea-pigs. Each population of neurons has a specific combination of neuropeptides. Note that all neurons containing a form of dynorphin A (DYN1-8 or DYN1-17) also contain dynorphin B and neo-endorphin. No neuropeptides have been found in neurons projecting to secretory tissue in the salivary or lacrimal glands. Neurons with similar peptide combinations also occur in other paravertebral ganglia of guinea-pigs, except that the salivary secretomotor neurons are absent. Conversely, the paravertebral ganglia have many non-noradrenergic vasodilatory neurons containing prodynorphin-derived peptides, VIP and NPY. AVAs, arterio-venous anastomoses; NA, noradrenaline; NPY, neuropeptide Y; ACh, acetylcholine; CGRP, calcitonin gene-related peptide; SP, substance P; VIP, vasoactive intestinal polypeptide (see Elfvin, Lindh and Hokfelt, 1993 [11]; Jänig, 2006 [12]).

moieties. There are at least eight receptors (four purinergic and four adenosinergic) that serve to translate purinergic and adenosinergic effects (vasoconstriction and vasodilatation) to vascular beds via endothelium-dependent and endothelium-independent mechanisms. Chemical coding also reveals that ganglion neurons with specific transmitter molecules innervate specific targets or receive specific afferent inputs. Apparently, anterograde and retrograde transsynaptic information appears to determine the transmitter phenotype of the neuron. Thus, studies of neuronal circuitry indicate that pathwayspecific combinations determine the presence and combinations of specific peptides within autonomic neurons. This is particularly so in the prevertebral ganglia, but studies in the guinea pig SCG demonstrate that the transmitter molecules will vary depending on the target organ supplied (see Fig. 4.3). Although all principal cells portrayed are noradrenergic in character (as indicated by NA), the neuropeptides co-contained in neurons vary depending on whether the targets are secretory (salivary and lacrimal glands), vascular (small vs. large arteries, arterioles vs. arterio-venous anastomoses), pupil or skin. Detailed pictures of these chemically coded circuits are beginning to emerge from studies of paravertebral (SCG), prevertebral (superior mesenteric), and previsceral (pelvic) ganglia. This phenomenon pertains to both the SNS and PNS. Preganglionic sympathetic neurons in the spinal cord have traditionally been viewed as cholinergic neurons.

More recently, it has been recognized that neuronal cell bodies in the cat ILP may contain a variety of transmitters including enkephalin, neurotensin, somatostatin, and substance P. In rodents, VIP and calcitonin gene-related pepitde (CGRP) containing neurons have also been localized to the ILP by immunocytochemistry. Also, preganglionic fibers in the sacral parasympathetic outflow co-contain enkephalin. It is apparent that neurotransmission is plurichemical in both preganglionic and postganglionic-fiber systems.

VISCERAL AFFERENT NEURONS AND AUTONOMIC NERVOUS SYSTEM Visceral afferent neurons are polymodal and are excited by physical (distention, contraction), chemical and thermal events. These stimuli are transmitted to the spinal cord and brainstem neurons resulting in target organ regulation, reflexes and sensation. In rats, vagal afferent neurons arising in the stomach may terminate within the medullary nucleus tractus solitarius in proximity to dendrites from dorsal motor nucleus efferent neurons sending axons back to the stomach [3]. Spinal primary afferent neurons that innervate viscera have multiple functions (afferent, efferent and trophic functions) [4] and contain a diverse assortment of neuropeptides including calcitonin generelated peptide (CGRP), substance P, vasoactive intestinal

I. INTRODUCTION

24


polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) [5]. For example, bladder afferent neurons travel in the hypogastric and pelvic nerves, and their cell bodies are located in dorsal root ganglia (DRG) at spinal segments T11–L2 and S2–S4 in humans and L1–L2 and L6–S1 in rats [5,6]. Bladder afferent fibers consist of lightly myelinated Aδ fibers and unmyelinated C-fibers. Sensation of bladder filling is conveyed by Aδ fibers, the most important mechanoreceptors of the bladder. C-fibers are normally “silent”, but they do respond to chemical or noxious stimuli, including extreme bladder pressure [5,6].

FUNCTIONAL NEUROANATOMY AND BIOCHEMICAL PHARMACOLOGY The peripheral ANS is well structured to provide the physiological responses critical for homeostasis and acute adaptations to stressful, perhaps life-threatening, circumstances. As outlined in Figure 4.1 and Table 4.2, multiple organ systems respond to neurotransmitters released from autonomic endings, and circulating catecholamines released from the adrenal medulla. Traditional teaching is that the effects of activation of the SNS and PNS are generally antagonistic; this is still largely the case. However, viewed more specifically, the relationships between these two major components of the ANS are far from simple. For instance, not all organs receive an equal number of both sets of fibers, and in some situations, both SNS and PNS effects are similar. It is important to remember that receptor systems, including the signal transduction components, on the target organs are the critical molecular proteins that determine the actual effects of ligand-receptor interactions on the cell membrane. Thus, when the SNS is stimulated with its capability to produce a widespread response, a host of receptor systems are activated to effect the necessary and desired change. An example of these responses includes the following aspects of SNS activation: dilatation of the pupil; slight increase in glandular secretions; bronchodilatation; increased heart rate and force of contraction; decreased gastrointestinal tract motility; decreased function of the reproductive organs; and mobilization of energy substrates to meet demands. The receptor systems mediating these responses include α1A, α1B,, α1D,, α2A, α2B, α2C, β1, β2 and β3 receptors. α1 receptors have subtypeselective distributions, second messenger systems and functions. Activation of these receptors occurs following interaction with NE and variously results in contraction of smooth muscle in the vasculature and iris, and relaxation of smooth muscle in the gut. α1 receptors exert a limited positive inotropic effect on the heart, as well as mediate salivary gland secretion and contraction of the prostate gland. α2 receptors also have discrete subtype-specific localizations and functions in brain, peripheral nerve, and target tissues. α2 receptors serve as autoreceptors on sympathetic nerve terminals and inhibit the release of NE as

TABLE 4.2 Autonomic Nervous System functions Organ

Sympathetic Nervous System


Eye Pupil Ciliary muscle

Dilatation Relax (far vision)

Constriction Constrict (near vision)

Lacrimal gland

Slight secretion

Secretion

Parotid gland

Slight secretion

Secretion

Submandibular gland

Slight secretion

Secretion

Heart

Increased rate Positive inotropism

Slowed rate Negative inotropism

Lungs

Bronchodilation

Bronchodilation

Gastrointestinal tract

Decreased motility

Increased motility

Kidney

Decreased output

None

Bladder

Relax detrusor Contract sphincter

Contract detrusor Relax sphincter

Penis

Ejaculation

Erection

Sweat glands

Secretion

Palmar sweating

Piloerection muscles

Contraction

None

Blood vessels Arterioles

Constriction

None

Constriction or dilatation Glycogenolysis

None

Muscle Arterioles Metabolism

None

part of a negative feed back loop. α2 receptors also can act as constrictors of both arterial and venous vascular smooth muscle. Furthermore, these receptors play important roles in nociception and also mediate metabolic and endocrine changes such as inhibition of lipolysis in adipose tissue and reduction of insulin release from the pancreas. β receptors (β1) provide positive inotropic and chronotropic effects on the heart and stimulate renin release from the kidney. β2 receptors relax smooth muscle of the bronchi and pelvic organs as well as the vascular structures of the gut and skeletal muscle. β2 receptors located in liver and skeletal muscle elicit activation of glycogenolysis and gluconeogenesis. This receptor system is particularly activated by epinephrine rather than norepinephrine. The parasympathetic nervous system is poised for more focal responses, but some effects may be quite broad, particularly with the wide-ranging innervation of the vagus nerve. Activation of parasympathetic pathways lead to pupillary constriction, substantial secretion from lacrimal and salivary glands, slowed cardiac rate and negative inotropism, bronchoconstriction, enhanced gastrointestinal motility and contraction of the detrusor muscle of the bladder. In contrast to the SNS, the PNS does not appear to influence metabolic or endocrine processes in any major way. However, recent evidence indicates that preganglionic vagal fibers originating in the dorsal motor nucleus innervate postganglionic parasympathetic ganglia in the

I. INTRODUCTION

STRESS ANd AuToNomIC dySfuNCTIoN

pancreas, and appear to influence exocrine and endocrine function. Thus, as new data emerge, the integrated roles of the SNS and PNS will continue to expand. An understanding of the receptor systems mediating the responses to PNS activation is incomplete. Preganglionic cholinergic receptors, which exist in the SNS as well as PNS, are nicotinic in character whereas postganglionic cholinergic receptors are muscarinic. Recently, molecular cloning studies have revealed multiple subtypes of both sets of receptors. The subtypes of muscarinic receptors (mAChRs), M(1)-M(5), are more fully understood (at least for the first three subtypes): M1 receptors are excitatory to neurons in ganglia and lead to noradrenaline release in sympathetic neurons; M2 receptors mediate the bradycardia and decreased contractility of the heart following vagal activation; M3 stimulation leads to contraction of smooth muscle and enhanced secretion from glandular tissues. Nicotinic acetylcholine receptor subtypes (nAChRs) are widely expressed in the mammalian CNS and PNS, playing a central role in autonomic transmission. Neuronal nicotinic acetylcholine receptors (nAChR) are composed of 12 subunits (α2–α10, β2–β4) but very little was known, until recently, about their physiological roles. The repertoire of nicotinic subunits in autonomic ganglia includes α3, α5, α7, β2 and β4 subunits. In the periphery, nicotinic receptors mediate vital excitatory fast synaptic cholinergic transmission at both the neuromuscular junction and ganglia. However, there is only a limited understanding of subunit actions in the presynaptic and postsynaptic components of the peripheral ANS. Functional deletions of subunit by gene knockout in animals are beginning to overcome these limitations.

STRESS AND AUTONOMIC DYSFUNCTION A prominent role for stress in the pathophysiology of cardiovascular disease and presentation of clinical pain states, including functional gastrointestinal disorders have been well documented. In addition, anxiety and stress may generate and worsen urinary symptoms and functional urinary disorders [7], such as interstitial cystitis (IC)/bladder pain syndrome (BPS). The majority of IC/BPS patients report symptom exacerbation by stress, and clinical studies have shown that acute stress increases bladder pain and urgency in IC/BPS patients [7,8]. Clinical populations afflicted with bladder disorders have a greater incidence of comorbid anxiety disorders than populations without bladder dysfunction. Both anxiety and bladder dysfunction can be caused and/or exacerbated by exposure to environmental stressors in human clinical populations and animal models. A number of ongoing studies from various laboratories are evaluating whether autonomic nervous system pathology and stress/anxiety share overlapping neuronal circuitry. The complexity of autonomic control and the range of mechanisms available to peripheral sympathetic and

25

parasympathetic neurons and their targets are expanded by presynaptic gaseous molecules (nitric oxide [NO]) and postsynaptic endothelium released peptides. Also, central nervous system centers involved in autonomic control contain nitric oxide synthase and evidence suggests that NO may mediate sympatho-inhibition or sympathoexcitation depending on the nuclear groups involved. Peripherally, NO exerts tonic vasodilation and mediates acetylcholine-induced vasodilation, as well as mediating catecholamine release and action. Endothelins, released abluminally by endothelial cells, bind to endothelial and adrenoceptors on vascular smooth muscle and endothelial cells and appear to regulate sympathetic terminals as well. The recent discoveries that a number of neurotrophic factors (neurotrophins) and their receptors are part and parcel of the development and integrity of the SNS and PNS expand the horizons regarding how anatomical pathways are established and maintained, and adapt to intrinsic and extrinsic demands. Because of its relatively simple anatomical architecture, the peripheral ANS continues to serve as a model system to understand neuronal development, structural and functional linkages among neurons and their targets, and the integrative role(s) these “little brains” (6000–30,000 neurons) serve. More recently, observations of the clinical phenotype and neuropathology of Parkinson Disease (PD) suggest that the peripheral ANS may serve as a model to study human neurodegenerative disease since peripheral and preganglionic, including medullary preganglionic vagal (DMV) neurons, may be involved at the earliest stages of the disease [9], before the onset of the well-known motor system dysfunctions related to the basal ganglia. A neuropathological cascade that originates in peripheral autonomic nerves may gradually and progressively ‘spread’ to involve central nervous system structures with the eventual involvement of the classically described neurons of the locus, coeruleus, substantia nigra and more rostral structures. Recent investigations in animal models of PD have taken advantage of the relative simplicity of the peripheral ANS to explore pathophysiological mechanism of how α-synuclein might initiate the premotor clinical dysfunction and presumed underlying alteration is neurotransmitter biochemistry and neurophysiology of peripheral autonomic neurons [10]. As the structure-function relationships and molecular pharmacology of the peripheral ANS are clarified, and more understandings accrue regarding their involvement in disease processes, new approaches to understanding ANS functions in health and disease and treatments of its maladies will undoubtedly emerge. The following sections of this Primer will expand on these issues.

References [1] Romagnano MA, Hamill RW. Spinal sympathetic pathway: an enkephalin ladder. Science 1984;225:737–9. [2] Strack AM, Sawyer WG, Hughes JH, Platt KB, Loewy AD. A general pattern of CNS innervation of the sympathetic outflow

I. INTRODUCTION

26

[3] [4]

[5]

[6] [7]

[8] [9] [10]

[11] [12]


demonstrated by transneuronal pseudorabies viral infections. Brain Res 1989;491:156–62. Shapiro RE, Miselis RR. The central organization of the vagus nerve innervating the stomach of the rat. J Comp Neurol 1985;238:473–88. Jänig W, Habler HJ. Specificity in the organization of the autonomic nervous system: a basis for precise neural regulation of homeostatic and protective body functions. Prog Brain Res 2000;122:351–67. Arms L, Vizzard MA. Neuropeptides in lower urinary tract function. In: Urinary tract, handbook of experimental pharmacology 202. Berlin Heidelberg: Springer-Verlag; 2011. Fowler CJ, Griffiths D, de Groat WC. The neural control of micturition. Nat Rev Neurosci 2008;9(6):453–66. Baldoni F, Ercolani M, Baldaro B, Trombini G. Stressful events and psychological symptoms in patients with functional urinary disorders. Percept Mot Skills 1995;80:605–6. Koziol JA, Clark DC, Gittes RF, Tan EM. The natural history of interstitial cystitis: a survey of 374 patients. J Urol 1993;149:465–9. Braak H, Del Tredici K. Invited Article: Nervous system pathology in sporadic Parkinson disease. Neurology 2008;70:1916–25. Hamill RW, Girard B, Tompkins JD, Galli JR, Parsons RL, Kershen RT, et al. Synucleinopathy model: Autonomic plasticity in the major pelvic ganglion (MPG) in mice with human alpha-synuclein overexpression. Developmental Neurobiol, in revision. Elfvin L-G, Lindh B, Hökfelt TJ. The chemical neuroanatomy of sympathetic ganglia. Ann Rev Neurosci 1993;16:471–507. Jänig W. The Integrative Action of the Autonomic Nervous System Neurobiology of homeostasis. Cambridge: Cambridge University Press; 2006.

Further Reading Baloh RH, Enomoto H, Johnson Jr. EM, Milbrandt J. The GDNF family ligands and receptors-implications for neural development. Curr Opin Neurobiol 2000;10:103–10. Burnstock G, Milner P. Structural and chemical organization of the autonomic nervous system with special reference to non-adrenergic, non-cholinergic transmission. In: Banister R, Mathias CJ, editors. Autonomic failure (4th edn.). New York: Oxford University Press; 1999. p. 63. Chowdhary S, Townend JN. Role of nitric oxide in the regulation of cardiovascular autonomic control. Clin Sci (Lond) 1999;97(July):5–17. Dinner DS, editor. The autonomic nervous system. (Review articles) J Clin Neurophysiol, 10; 1993 1–82 Gibbins I. Chemical neuroanatomy of sympathetic ganglia. In: McLachlan EM, editor. Autonomic ganglia. Luxembourg: Harwood Academic Publishers; 1995. p. 73–121. Goldstein DS. The autonomic nervous system in health and disease. New York: Marcel Dekker, Inc; 2001. 23–135 Schober A, Unsicker K. Growth and neurotrophic factors regulating development and maintenance of sympathetic preganglionic neurons. Int Rev Cytol 2001;205:37–76. Zansinger J. Role of nitric oxide in the neural control of cardiovascular function. Cardiovasc Res 1999;43:639–49.

I. INTRODUCTION

C H A P T E R

5 Cotransmission Geoffrey Burnstock

EARLY STUDIES

TABLE 5.1 Cotransmitters in the Peripheral and Central Nervous System

For many years, understanding of neurotransmission incorporated the concept that one neuron releases only a single transmitter, known as “Dale’s Principle”. This idea arose from a widely adopted misinterpretation of Dale’s suggestion in 1935 that the same neurotransmitter was stored in and released from all terminals of a single neuron, a suggestion which did not specifically preclude the possibility that more than one transmitter may be associated with the same neuron. Early hints that nerves might release more than one transmitter began in the 1950s with evidence for the involvement of both noradrenaline (NA) and acetylcholine (ACh) in sympathetic transmission. Koelle identified acetylcholinesterase in some adrenergic neurons in 1955, while Burn and Rand introduced the concept of a “cholinergic” link in adrenergic transmission in 1959. Another line of evidence provided by Hillarp concerned the coexistence of adenosine 5-triphosphate (ATP) with catecholamines, first in adrenal chromaffin cells and later in sympathetic nerves. Inconsistencies in the single transmitter hypothesis provided by these and other studies, including those concerned with invertebrate neurotransmission, from the early literature were rationalized in an article by Burnstock in 1976 [1] with the provocative title: “Do some nerve cells release more than one transmitter?” Today, it is widely accepted that cotransmission is an integral feature of neurotransmission. A role for ATP as a cotransmitter in sympathetic, parasympathetic, sensory-motor and enteric non-adrenergic, non-cholinergic (NANC) inhibitory nerves was supported by research from Burnstock and colleagues, while Hökfelt and colleagues focused on the colocalization, vesicular storage and release of peptides from both peripheral and central nerves (see [2–4]). Furness and Costa introduced the concept of “chemical coding” to describe the combination of potential neurotransmitters found in enteric nerves and this concept has since been applied to other nerve types, in both peripheral and central nervous systems (CNS) [5]. Colocalized substances are not necessarily cotransmitters, they can (especially peptides) act as pre- and/or postjunctional neuromodulators of the release and actions of the principal cotransmitters. The proportions of cotransmitters vary considerably between species and organs, and show


PERIPHERAL NERVOUS SYSTEM Sympathetic nerves

ATP  NA  NPY

Parasympathetic nerves

ATP  ACh  VIP

Sensory-motor

ATP  CGRP  SP

NANC enteric nerves

ATP  NO  VIP

Motor nerves (in early development)

ATP  Ach

CENTRAL NERVOUS SYSTEM Cortex, caudate nucleus

ATP  Ach

Hypothalamus, locus ceruleus

ATP  NA

Hypothalamus, dorsal horn, retina

ATP  GABA

Mesolimbic system

ATP  DA

Hippocampus, dorsal horn

ATP  glutamate

Compiled from [4]. ACh, acetylcholine; ATP, adenosine 5-triphosphate; CGRP, calcitonin gene-related peptide; DA, dopamine; GABA, γ-amino butyric acid; NA, noradrenaline; NO, nitric oxide; NPY, neuropeptide Y; SP, substance P; VIP, vasoactive intestinal peptide.

plasticity of expression during development and in pathological conditions. In general, classical transmitters are contained in small synaptic vesicles, whereas peptides are stored in large granular (dense-cored) vesicles (LGVs), although small molecule transmitters are sometimes stored together with peptides in LGVs. Pharmacological studies of pre- and postjunctional neuromodulation provide evidence, which is complementary to the concept of cotransmission. Evidence for ATP being a cotransmitter with established neurotransmitters in the CNS as well as in the periphery has been reported (see Table 5.1).

SYMPATHETIC NERVES There is substantial evidence to show that NA, ATP and neuropeptide Y (NPY) are cotransmitters in sympathetic nerves, having differentially important roles as transmitters and neuromodulators depending on the tissue, the species, and on the parameters of stimulation.

27


28

5. CoTRANSmISSIoN

The first indication that ATP might be released from sympathetic nerves was the demonstration that stimulation of sympathetic nerves by Su et al. in 1971 [6] led to release of tritium from taenia coli preincubated in [3H]adenosine (which is taken up and converted to [3H]ATP). Later Sol Langer and colleagues suggested that the substantial residual NANC response of the cat nictitating membrane observed after depletion of NA by reserpine was due to the release of ATP remaining in the sympathetic nerves. Most of the early studies establishing the model of cotransmission of NA and ATP were made by Dave Westfall and colleagues on the vas deferens, a tissue with a high density of sympathetic nerves. In retrospect, there was a good indication that the excitatory junction potentials (EJPs) recorded in the guinea pig vas deferens when the electrophysiology of sympathetic nerve smooth muscle transmission was first described by Burnstock and Holman in 1960, were due to ATP released as a cotransmitter from sympathetic nerves, rather than to NA. It was puzzling at the time that adrenoceptor antagonists failed to block the EJPs, although guanethidine, a drug that prevents the release of sympathetic transmitters, was effective. It was over 20 years later that NANC EJPs were shown to be blocked by desensitization of the ATP (P2) receptors by α,β-methylene ATP (α,β-meATP) and mimicked by ATP [7]. After destruction of sympathetic nerves with 6-hydroxydopamine, purinergic nerve-mediated responses were abolished. ATP is co-stored with NA in small and large vesicles. Differential prejunctional modulation of the release of NA and ATP by various agents has been shown in the vas deferens, perhaps indicating that NA and ATP are stored in different vesicles. Cotransmission of NA and ATP in perivascular sympathetic nerves supplying the rabbit aorta, portal vein and saphenous, pulmonary and mesenteric arteries, and the dog basilar artery has been demonstrated (see [8]). Electrophysiological studies have shown that in a number of vessels the electrical response to stimulation of perivascular sympathetic nerves is biphasic; an initial fast, transient depolarization or EJP of the vascular smooth muscle is followed by a slow, prolonged depolarization. The EJP and slow depolarization are mimicked by the effects of ATP and NA, respectively. Considerable variation exists in the proportions of NA and ATP utilized by sympathetic nerves. For example, in guinea pig submucosal arterioles both vasoconstriction and EJPs, evoked in response to electrical stimulation of sympathetic nerves, are mediated exclusively by ATP, with NA assuming the role of a neuromodulator, by acting through prejunctional α2-adrenoceptors to depress transmitter release. At the other extreme, in rat renal arteries the purinergic component is relatively small. It has also been noted that the purinergic component is optimal with short bursts of low frequency stimulation, whereas longer durations of higher frequency favor adrenergic transmission. NPY has been found to be present in LGV in most sympathetic nerves. The release of NPY, as well as NA and ATP, in response to electrical stimulation of sympathetic nerve terminals is prevented by guanethidine. The major role of

FIGURE 5.1 Schematic of sympathetic cotransmission. ATP and NA released from small granular vesicles (SGV) act on P2X and α1 receptors on smooth muscle, respectively. ATP acting on inotropic P2X receptors evokes excitatory junction potentials (EJPs), increase in intracellular calcium ([Ca2]i) and fast contraction; while occupation of metabotropic α1 adrenoceptors leads to production of inositol triphosphate (InsP3), increase in [Ca2]i and slow contraction. Neuropeptide Y (NPY) stored in large granular vesicles (LGV) acts on release both as a prejunctional inhibitory modulator of release of ATP and NA and as a postjunctional modulatory potentiator of the actions of ATP and NA. Nucleotidases are released from nerve varicosities, and are also present as ectonucleotidases. (Reproduced from [14] with permission from Elsevier.)

NPY in the vasculature, and in the vas deferens, appears to be that of a pre- and/or postjunctional modulator of sympathetic transmission, since it has little direct postjunctional action or causes contraction only at high concentrations (see Fig. 5.1). Direct vasoconstrictor actions of NPY have, however, been demonstrated in some vessels. At the prejunctional level, NPY has potent inhibitory effects, reducing the release of NA and ATP from sympathetic nerves. Postjunctionally, NPY generally acts to enhance the actions of sympathetic nerve stimulation, NA and ATP. Although 5-hydroxytryptamine (5-HT) immunofluorescent nerves have been localized in a number of vessels, for the most part 5-HT is not synthesized and stored in separate nerves, but is taken up, stored in, and released as a “false transmitter” from sympathetic nerves. Enkephalins have been shown to coexist with NA in cell bodies and fibers of some postganglionic sympathetic neurons. The functional significance of sympathetic coexistence of opioids is likely to be related to their prejunctional inhibitory effects on sympathetic transmission.

PARASYMPATHETIC NERVES The classical evidence for cotransmission of ACh and vasoactive intestinal polypeptide (VIP) in certain

I. INTRODUCTION

ENTERIC ANd CARdIAC NERvES

postganglionic parasympathetic neurons comes from pharmacological studies performed by Lundberg in 1981 on cat salivary glands (see [9]). ACh and VIP are released from the same parasympathetic nerve terminals in response to transmural nerve stimulation. During low frequency stimulation ACh is released to cause an increase in salivary secretion from acinar cells and also to elicit some minor dilatation of blood vessels in the gland. VIP is preferentially released at high frequencies to cause marked vasodilatation of blood vessels and, while it has no direct effect on acinar cells, it acts as a neuromodulator to enhance both the postjunctional effect of ACh on acinar cell secretion and the release of ACh from nerve varicosities via prejunctional receptors. Vasodilator nerves to the uterine arteries in the guinea pig contain immunoreactivity to VIP, which coexists with dynorphin, NPY and somatostatin. NPY-like immunoreactivity has been reported in some of the choline acetyltransferase-/VIP-containing neurons of the parasympathetic ciliary, sphenopalatine, otic and pterygopalatine ganglia with targets including the iris and cerebral vessels. Autonomic control of penile erection, involving relaxation of the smooth muscle of the corpus cavernosum as well as dilatation of other penile vascular beds, has traditionally been attributed to the vasodilator effects of ACh and VIP released from parasympathetic nerves. More recently, nitric oxide (NO) released from nerves arising from nerves in the pelvic ganglia, have been claimed to play a role in smooth muscle relaxation leading to penile erection. NO synthase (NOS)-containing fibers, shown by lesion studies to arise from parasympathetic cell bodies in the sphenopalatine ganglia, have been localized in the adventitia of cerebral arteries and many of these also contain VIP. A functional role for perivascular neuronal NO in cerebral arteries has been identified in studies showing that stimulation of adventitial nerve fibers causes vascular relaxation, which is attenuated by inhibitors of NOS. Parasympathetic nerves supplying the urinary bladder utilize ACh and ATP as cotransmitters, in variable proportions in different species, and by analogy with sympathetic nerves, ATP again acts through P2X ionotropic receptors to produce EJPs and fast contraction, while the slow component of the response is mediated by a metabotropic receptor, in this case muscarinic (see [10]). There is also evidence for parasympathetic, purinergic cotransmission to resistance vessels in the heart and airways.

SENSORY-MOTOR NERVES The neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) are the principal transmitters of primary afferent nerves and have been shown to coexist in the same terminals. Furthermore, with the use of colloidal gold particles of different sizes, they have been shown to coexist in the same large granular vesicles. The motor (efferent) function of sensory nerves has been

29

demonstrated in rat mesenteric arteries where evidence exists for a role for CGRP as the mediator of vasodilatation following release from sensory motor nerves. In contrast, SP is not co-released with CGRP by electrical stimulation and SP has little or no vasodilator action on rat mesenteric arteries. While it is possible that SP released from nerves supplying the microvasculature could produce vasodilatation via SP receptors on endothelial cells, it is most unlikely to reach the endothelium without degradation in larger blood vessels. It may be that the role of the coexisting SP is either trophic or sensory (and not motor). Other peptide and non-peptide substances including neurokinin A, somatostatin, VIP and ATP have been described in capsaicin-sensitive sensory neurons. Unmyelinated sensory neurons containing cholecystokinin (CCK)/CGRP/dynorphin/SP have been shown to project to cutaneous arterioles in guinea pig skin. Neurons from the same ganglia which contain CCK/CGRP/SP, innovate arterioles of skeletal muscle, CGRP/dynorphin/ SP nerve fibers mostly supply the pelvic viscera, and CGRP/SP fibers run mainly to the heart, large arteries, and veins. There is also evidence for a sensory role for ATP and it has been proposed that ATP may coexist in sensory nerve terminals with SP and CGRP (Fig. 5.2).

ENTERIC AND CARDIAC NERVES Intrinsic neurons exist in most of the major organs of the body. Many of these are part of the parasympathetic nervous system, but certainly in the gut and perhaps also in the heart and airways, some of these intrinsic neurons are derived from neural crest tissue, which differs from that which forms the sympathetic and parasympathetic systems and appear to participate in local reflex actions independent of the CNS. The enteric nervous system contains several hundred million neurons located in the myenteric plexus between muscle coats and the submucous plexus. The chemical coding of these nerves has been examined in detail. A subpopulation of these intramural enteric nerves provides NANC inhibitory innervation of the gastrointestinal smooth muscle. It seems likely that three major cotransmitters are released from these nerves. ATP produces fast inhibitory junction potentials, NO also produces inhibitory potentials but with a slower time course, while VIP produces slow tonic relaxations. The proportions of these three transmitters varies considerably in different regions of the gut and in different species; for example, in some sphincters the NANC inhibitory nerves utilize largely VIP, in others largely NO, while in non-sphincteric regions of the intestine ATP is more prominent. In recent papers, evidence has suggested that ACh and ATP are fast excitatory cotransmitters to myenteric neurons and that there may be colocalization of ACh, ATP and 5-HT in enteric Dogiel Type I/S neurons. Detailed studies have allowed a very complete mapping of the complex neuronal markers and

I. INTRODUCTION

30

5. CoTRANSmISSIoN

FIGURE 5.2 Schematic hypotheses of the neurogenic basis of reflex vasodilatation of skin vessels. One suggestion is that substance P (a putative transmitter in sensory nerve endings in the spinal cord) is released from sensory nerve collaterals in the skin; another that ATP is the transmitter released from these collaterals. Both substances are powerful vasodilators and can release histamine from mast cells. ATP is also a potent inducer of prostaglandin synthesis. Another alternative, although less likely, is that a peripherally placed purinergic neuron is interposed between the sensory nerve collateral and the effector system. (This hypothesis incorporates early histologic reports of neurons in the skin and provides an alternative or additional explanation for the initiation of the vasodilator reflex by nicotine and its block by hexamethonium.) (Reproduced from [15], with permission.)

projections of enteric neurons. Several peptidergic substances, including NPY, VIP, enkephalin, somatostatin, peptide histidine isoleucine, galanin, SP and CGRP have been identified in enteric neurons, often coexisting (up to five peptides in the same neuron) with the neurotransmitters NA, ACh, 5-HT, NO and ATP (see Table 5.2). Studies of intrinsic cardiac neurons in culture have shown that subpopulations of intrinsic nerves in the atrial and intra-atrial septum contain and/or release cotransmitters, including ATP, NO, NPY, ACh and 5-HT. Many of these nerves project to the coronary microvasculature and produce potent vasomotor actions. NO together with ATP have been shown to be the mediators of NANC vasodilatation of the rabbit portal vein.

PHYSIOLOGICAL SIGNIFICANCE OF COTRANSMISSION In general, cotransmission offers greater physiological events by peripheral mechanisms rather than the all-ornone control by messages coming from the CNS, that has been the dominant view for many years (see [11]).

Cotransmitters with Different Firing Patterns Although single presynaptic action potentials release small molecule neurotransmitters, trains of impulses are

needed to release neuropeptides. For sympathetic and parasympathetic cotransmission, release of ATP is favored at low frequency stimulation, whereas NA and ACh are released at higher frequencies. There are instances where more than one fast cotransmitter is released (e.g. glutamate and ATP) together with one or more peptides.

Different Cotransmitters act on Different Postjunctional Cells Neurons using multiple transmitters may project to two or more targets. For example, ACh released at low frequency stimulation from parasympathetic nerves supplying salivary glands acts on acinar cells to produce secretion and a minor dilatation of vessels, whereas, at higher frequency stimulation, its cotransmitter VIP causes powerful vasodilatation of vessels in the glands and postjunctional enhancement of ACh-induced saliva secretion.

Neuromodulation A cotransmitter can feed back on presynaptic receptors that increase or decrease its own release or that of its cotransmitter(s). For example, ATP released as a cotransmitter with glutamate from primary afferent fibers in lamina II of the spinal cord can act on prejunctional P2X3 receptors to facilitate the release of its cotransmitter, glutamate,

I. INTRODUCTION

31

PHySIologICAl SIgNIfICANCE of CoTRANSmISSIoN

TABLE 5.2 Types of Neurons in the Enteric Nervous System Proportion

Chemical Coding

Function/Comments

Excitatory circular muscle motor neurons

12%

Short: ChAT/TK/ENK/GABA Long: ChAT/TK/ENK/NFP

To all regions, primary transmitter ACh, cotransmitter TK

Inhibitory circular muscle motor neurons

16%

Short: NOS/VIP/PACAP/ENK/ NPY/GABA Long: NOS/VIP/PACAP/ Dynorphin/BN/NFP

Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP

Excitatory longitudinal muscle motor neurons

25%

ChAT/Calretinin/TK

Primary transmitter ACh, cotransmitter TK

Inhibitory longitudinal muscle motor neurons

~2%

NOS/VIP/GABA

Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP

Ascending interneurons (local reflex)

5%

ChAT/Calretinin/TK

Primary transmitter ACh

Descending interneurons (local reflex)

5%

ChAT/NOS/VIP  BN  NPY

Primary transmitter ACh, ATP may be a cotransmitter

Descending interneurons (secretomotor reflex)

2%

ChAT/5-HT

Primary transmitter ACh, 5-HT (at NK3 receptors)

Descending interneurons (migrating myoelectric complex)

4%

ChAT/SOM


Myenteric intrinsic primary afferent (primary sensory) neurons

26%

ChAT/Calbindin/TK/NK3 receptor

Primary transmitter TK

Intestinofugal neurons

1%

ChAT/BN/VIP/CCK/ENK


Motor neurons to gut endocrine cells

N/A

N/A

For example, myenteric neurons innervating gastrin cells. Neurons of this type may be in submucosal ganglia

Non-cholinergic secretomotor/vasodilator neurons

45%

VIP/GAL

Primary transmitter VIP. A small proportion of these have cell bodies in myenteric ganglia

Cholinergic secretomotor/vasodilator neurons

15%

ChAT/Calretinin/Dynorphin


MYENTERIC NEURONS

SUBMUCOSAL NEURONS

*Also listed are three types of motor neuron that are found in other parts of the tubular digestive tract, marked by asterisks. Reproduced from [13], with permission from Elsevier. ACh, acetylcholine; ATP, adenosine 5-triphosphate; BN, bombesin; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; ChAT, choline acetyltransferase; DA, dopamine; ENK, enkephalin; GABA, γ-amino butyric acid; GAL, galanin; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; NFP, neurofilament protein; NK, neurokinin; NOS, nitric oxide synthase; NPY, neuropeptide Y; PACAP, pituitary adenylyl cyclase activating peptide; SOM, somatostatin; SP, substance P; TK, tachykinin; VIP, vasoactive intestinal peptide.

whereas adenosine resulting from ectoenzymatic breakdown of ATP acts on presynaptic P1 receptors to inhibit glutamate release. Both NA and ATP can prejunctionally modulate sympathetic transmission, NA via prejunctional α2-adrenoceptors and ATP via P1 receptors following breakdown to adenosine. Modulation of cotransmitter release and presynaptic action by other agents also occurs and might provide a new level of synaptic flexibility, in which individual neurons utilize more than one transmitter but retain independent control over their synaptic activity.

Synergism There are an increasing number of reports of the synergistic actions of cotransmitters. ATP and NA released from sympathetic nerves have synergistic actions on

smooth muscle of vas deferens and blood vessels, and ATP released with ACh from motoneurons facilitates the nicotinic actions of ACh at the skeletal neuromuscular junction. Co-operativity between receptors for ATP and N-methyl-D-aspartate (NMDA) in induction of long-term potentiation in hippocampal CA1 neurons has also been demonstrated. Thyrotropin-releasing hormone and 5-HT have been reported to have synergistic actions in spinal cord neurons. The mechanisms underlying cotransmitter synergism are not well understood. However, it has been suggested that postjunctional synergism between the responses of vas deferens to NA and ATP is caused by the ability of NA to potentiate the contractile responses to ATP by sensitizing smooth muscle cells to Ca2 via an inhibitory action on myosin light chain phosphatase, an action mediated by protein kinase C.

I. INTRODUCTION

32

5. CoTRANSmISSIoN

Negative Cross-Talk Co-application of nicotinic and P2X receptor agonists produces less than the additive responses predicted by independent receptor activation. Inhibitory interactions between 5-HT3 and P2X receptors have been described in submucosal and myenteric neurons.

Trophic Factors Some co-stored and co-released substances can act as long-term (trophic) factors, as well as neurotransmitters. For example, ATP can act on P2 receptors, or P1 (adenosine) receptors after ectoenzymatic breakdown, to promote vascular cell proliferation, motility, differentiation or death. NPY released from sympathetic nerves has cardiovascular trophic effects in end-stage renal disease. There is growing evidence that neurotrophic factors might be synthesized, stored and released from nerve terminals together with fast neurotransmitters.

Excitatory and Inhibitory Cotransmitters Although cotransmitters generally have similar actions on postjunctional cells, there are some examples of cotransmitters having opposite actions. For example, in the mammalian uterus, one or other cotransmitter dominates depending on the hormonal and/or tonic status of the postjunctional muscle cells. Brain-derived neurotrophic factor (BDNF) increases the release of ACh and reduces NA release from sympathetic nerves to cause a rapid shift from excitatory to inhibitory transmission.

False Cotransmitters For example, it has been known for some time that sympathetic nerves take up 5-HT, which can then be released as a “false transmitter”, rather than a genuine “cotransmitter”. A “false transmitter” is a substance actively taken up and subsequently released by a neuron that does not synthesize it.

Coexisting Peptides Acting as Neuromodulators In general, most neuropeptides act as neuromodulators rather than neurotransmitters. For example, NPY released from sympathetic nerves acts as a pre- and postjunctional modulator of ATP and NA release and postjunctional actions.

COTRANSMITTER PLASTICITY Cotransmitter plasticity occurs during development and ageing, following trauma or surgery and after chronic exposure to drugs, as well as in disease (see [12]). There were some outstanding early studies of the factors

influencing cotransmitter expression in sympathetic nerves. Evidence was presented that cholinergic differentiation in sympathetic neurons is promoted by neurotrophic factors from three different protein families (glial cell line-derived neurotrophic factor, neurotrophin 3 and ciliary neurotrophic factor), whereas noradrenergic differentiation is promoted by nerve growth factor. In another study, BDNF was claimed to switch sympathetic neurotransmission to the heart from an adrenergic excitation to cholinergic inhibition; it was also shown that the action of BDNF was mediated by the P75 neurotrophic receptor. Histamine, galanin and γ-aminobutyric acid (GABA) acting as cotransmitters in neurons of the tuberomammillary nucleus (hypothalamus) have independent control mechanisms. CGRP-like immunoreactivity was found earlier than SP-like immunoreactivity in cerebrovascular nerves during development, and increased in old age, while the density of SP-like immunoreactive fibers did not change. NA and NPY also show different expression in cerebrovascular nerves during development. Changes in chemical coding of myenteric neurons in ulcerative colitis have been reported, with a shift from cholinergic to more SP-mediated cotransmission. In interstitial cystitis and outflow obstruction, there is a substantial increase in ATP as the cotransmitter with ACh in parasympathetic nerves supplying the human bladder, and there is increase in ATP as a cotransmitter with NA in sympathetic nerves in spontaneous hypertensive rats (see [4]).

CONCLUDING COMMENTS It has been particularly difficult to establish cotransmitter roles for the many peptides found in nerves, partly because specific receptors and physiological roles have not been established for some of these and partly because of the lack of selective antagonists. In some enteric neurons, up to five neuropeptides have been identified. However, it is important to distinguish between neuromodulator, neurotransmitter and neurotrophic roles for released peptides or indeed as yet unrecognized roles. It is becoming clear that ATP is a primitive signalling molecule that has been retained as a cotransmitter in every nerve type in both the peripheral and central nervous systems, although the relative role of ATP varies considerably in different species and pathophysiological conditions. ATP appears to become a more prominent cotransmitter in stress and inflammatory conditions. Most nerves contain and release ATP as a fast cotransmitter together with classical transmitters such as ACh, NA, glutamate, GABA and one or more peptides. Now that cotransmission is recognized as a universal mechanism, it is recommended that the terms “adrenergic”, “cholinergic”, “peptidergic”, “purinergic”, “aminergic” and “nitrergic” should not be used when nerves are described, although adrenergic, cholinergic, peptidergic, purinergic, aminergic or nitrergic transmission is still meaningful.

I. INTRODUCTION

CoNCludINg CommENTS

References [1] Burnstock G. Do some nerve cells release more than one transmitter? Neuroscience 1976;1:239–48. [2] Kupfermann I. Functional studies of cotransmission. Physiol Rev 1991;71:683–732. [3] Merighi A. Costorage and coexistence of neuropeptides in the mammalian CNS. Prog Neurobiol 2002;66:161–90. [4] Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 2007;87:659–797. [5] Furness JB, Morris JL, Gibbins JL, Costa M. Chemical coding of neurons and plurichemical transmission. Ann Rev Pharmacol Toxicol 1989;29:289–306. [6] Su C, Bevan JA, Burnstock G. [3H]adenosine triphosphate: release during stimulation of enteric nerves. Science 1971;173:337–9. [7] Sneddon P, Burnstock G. Inhibition of excitatory junction potentials in guinea-pig vas deferens by α,β-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur J Pharmacol 1984;100:85–90. [8] Burnstock G. Noradrenaline and ATP as cotransmitters in sympathetic nerves. Neurochem Int 1990;17:357–68.

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[9] Lundberg JM. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol Rev 1996;48:113–78. [10] Burnstock G. Cotransmission with particular emphasis on the involvement of ATP. In: Fuxe K, Hökfelt T, Olson L, Ottoson D, Dahlström A, Björklund A, editors. Molecular Mechanisms of Neuronal Communication. A Tribute to Nils-Ake Hillarp. Oxford: Pergamon Press; 1996. p. 67–87. [11] Burnstock G. Cotransmission. Curr Opin Pharmacol 2004;4:47–52. [12] Burnstock G. Co-transmission. The fifth Heymans memorial lecture – Ghent, February 17, 1990. Arch Int Pharmacodyn Ther 1990;304:7–33. [13] Furness JB. Types of neurons in the enteric nervous system. J Auton Nerv Syst 2000;81:87–96. [14] Burnstock G, Verkhratsky A. Vas deferens - a model used to establish sympathetic cotransmission. Trends Pharmacol Sci 2010;31:131–9. [15] Burnstock G. Autonomic neuroeffector junctions - reflex vasodilatation of the skin. J Invest Dermatol 1977;69:47–57.

I. INTRODUCTION

P A R T II

BIOCHEMICAL AND PHARMACOLOGICAL MECHANISMS



C H A P T E R

6 Noradrenergic Neurotransmission David S. Goldstein The main chemical transmitter of the sympathetic nervous system mediating regulation of the circulation is the catecholamine norepinephrine (NE). Sympathetic stimulation releases NE, and binding of NE to adrenoceptors on cardiovascular smooth muscle cells causes the cells to contract. Sympathoneural NE therefore satisfies the main criteria defining a neurotransmitter – a chemical released from nerve terminals by electrical action potentials that interacts with specific receptors on nearby structures to produce specific physiological responses. Unlike most other neurotransmitters, NE can be measured in human plasma. This capability, coupled with the ability to assay simultaneously levels of NE and other compounds related to synthesis and disposition of NE, provides the bases for clinical neurochemical biomarkers of sympathetic innervation and function and for metabolomic approaches to diagnose several disorders. Such neurochemical measures often are more sensitive than physiological measures in clinical laboratory assessments of patients with autonomic dysfunction.

fibers in the ansae subclaviae pass along the dorsal surface of the pulmonary artery into the plexus that supplies the left main coronary artery. In primates, cardiac sympathetic nerves originate about equally from the superior, middle, and inferior cervical (stellate) ganglia. The right sympathetic chain generally projects to the anterior left ventricle and the left to the posterior left ventricle. Sympathetic innervation of the sinus and atrioventricular nodes also has a degree of sidedness, the right sympathetics projecting more to the sinus node and the left to the atrioventricular node. This sidedness means that left stellate stimulation produces relatively little sinus tachycardia. Epicardial sympathetic nerves provide the main source of noradrenergic terminals in the myocardium. Sympathetic nerves travel with the coronary arteries in the epicardium before penetrating into the myocardium, whereas vagal nerves penetrate the myocardium after crossing the atrioventricular groove and then course distally in the subendocardium. Post-ganglionic noradrenergic fibers reach all parts of the heart. The sinus and atrioventricular nodes and the atria receive the densest innervation, the ventricles less dense innervation, and the Purkinje fibers the least. Sympathetic and vagal afferents follow similar intracardiac routes to those of the efferents. The heart contains relatively high NE concentrations compared to other organs. In humans, the left ventricular myocardial NE concentration is roughly 10 pmoles per mg wet weight, with substantial inter-individual variability, corresponding to about 10 times the plasma concentration. The coronary arteries possess sympathetic noradrenergic innervation, but assessing the physiological role of this innervation has proven difficult, because several interacting factors complicate neural control of the coronary vasculature. Alterations in myocardial metabolism and systemic hemodynamics change coronary blood flow, coronary vasomotion in response to sympathetic stimulation depends on the functional integrity of the endothelium, and coronary arteries appear to possess less dense innervation than do other arteries. The body’s myriad arterioles largely determine total resistance to blood flow in the body and therefore contribute importantly to blood pressure. Sympathetic nerves enmesh blood vessels in lattice-like networks in the adventitial outer surface that extend inward to the adventitial-medial border. The concentration of sympathetic

CATECHOLAMINES, AND CATECHOLS The endogenous catecholamines are NE, dopamine (DA), and epinephrine (EPI, synonymous with adrenaline). Catecholamines are catechols, which are chemicals that have adjacent hydroxyl groups on a benzene ring. Catechol itself does not exist in the human body, but compounds that contain catechol as part of their molecular structure are called catechols. Human plasma normally contains six catechols – the catecholamines, L-DOPA (the amino acid precursor of the catecholamines), and two metabolites of the catecholamines, dihydroxyphenylglycol (DHPG, the main neuronal metabolite of NE), and dihydroxyphenylacetic acid (DOPAC, the main neuronal metabolite of DA). As will be seen, particular patterns of levels of catechols characterize different disorders of catecholamine systems.

NORADRENERGIC INNERVATION OF THE CARDIOVASCULAR SYSTEM Sympathetic nerves to the heart travel via the ansae subclaviae, branches of the left and right stellate ganglia. The


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6. NORADRENERGIC NEuROTRANSMISSION

nerves increases as the arterial caliber decreases, so that small arteries and arterioles, the smallest nutrient vessels possessing smooth muscle cells, have the most intense innervation. The architectural association between sympathetic nerves and the vessels that determine peripheral resistance has enticed cardiovascular researchers particularly in the area of autonomic regulation for many years. Sympathetic vascular innervation varies widely among vascular beds, with dense innervation of resistance vessels in the gut, kidney, skeletal muscle, and skin. Sympathetic stimulation in these beds produces profound vasoconstriction, whereas stimulation in the coronary, cerebral, and bronchial beds elicits weaker constrictor responses, consistent teleologically with the “goal” of preserving blood flow to vital organs in emergencies.

NOREPINEPHRINE: THE MAIN SYMPATHETIC CARDIOVASCULAR NEUROTRANSMITTER Norepinephrine Synthesis Catecholamine biosynthesis begins with uptake of the amino acid tyrosine (TYR) into the cytoplasm of sympathetic neurons, adrenomedullary cells, possibly para-aortic enterochromaffin cells, and specific centers in the brain. Tyrosine hydroxylase (TH) catalyzes the conversion of TYR to DOPA. This is the enzymatic rate-limiting step in catecholamine synthesis. The enzyme is stereospecific, and tetrahydrobiopterin, ionized iron, and molecular oxygen regulate TH activity. Dihydropteridine reductase (DHPR) catalyzes the reduction of dihydropterin, produced during the hydroxylation of TYR. Since the reduced pteridine, tetrahydrobiopterin, is a key co-factor for TH, DHPR deficiency decreases the amount of TYR hydroxylation for a given amount of TH enzyme. Both phenylalanine hydroxylase and TH require tetrahydrobiopterin as a co-factor. DHPR deficiency therefore also inhibits phenylalanine metabolism and presents clinically as an atypical form of phenylketonuria. Multiple and complex mechanisms contribute to TH activation. Short-term mechanisms include feedback inhibition by DOPA and catecholamines and phosphorylation of the enzyme, the latter depending on membrane depolarization, contractile elements, and receptors. Long-term mechanisms include changes in TH synthesis. During stress-induced sympathetic stimulation, acceleration of catecholamine synthesis in sympathetic nerves helps to maintain tissue stores of NE. Even with diminished stores after prolonged sympathoneural activation, increased nerve traffic can maintain extracellular fluid levels of the transmitter. L-aromatic-amino-acid decarboxylase (LAAAD, also called DOPA decarboxylase) catalyzes conversion of DOPA to DA. Many types of tissues contain this enzyme – especially the kidneys, gut, liver, and brain. LAAAD activity depends on pyridoxal phosphate (vitamin B6).

Although LAAAD metabolizes most of the DOPA formed in catecholamine-synthesizing tissues, some of the DOPA enters the circulation unchanged. This provides the basis for using plasma DOPA levels to examine catecholamine synthesis. LAAAD inhibitors include carbidopa and benserazide. Both are catechols that do not readily penetrate the blood–brain barrier. By inhibiting conversion of DOPA to DA in the periphery, carbidopa and benserazide enhance the efficacy of L-DOPA and reduce its side effects in the treatment for Parkinson disease. LAAAD blockade increases DOPA levels and decreases levels of DOPAC, the deaminated metabolite of DA. Rates of increase in extracellular fluid DOPA levels and of decrease in DOPAC levels after acute LAAAD inhibition provide in vivo indices of TH activity. Dopamine-β-hydroxylase (DBH) catalyzes conversion of DA to NE. DBH is localized to vesicles in cells that synthesize catecholamines, such as noradrenergic neurons and chromaffin cells. Treatment with reserpine, which blocks translocation of amines from the axonal cytoplasm into vesicles, prevents conversion of DA to NE in sympathetic nerves. DBH contains – and its activity depends on – copper. Because of this dependence, children with Menkes disease, a rare, X-linked recessive inherited disorder of copper metabolism, have neurochemical evidence of concurrently increased catecholamine biosynthesis and decreased conversion of DA to NE, with high plasma ratios of DOPA:DHPG. Patients with congenital absence of DBH have virtually undetectable levels of both NE and DHPG and high levels of DA and DOPAC. DBH activity also requires ascorbic acid (vitamin C), which provides electrons for the hydroxylation. Phenylethanolamine-N-methyltransferase (PNMT) catalyzes the conversion of NE to EPI in the cytoplasm of chromaffin cells and some brainstem neurons.

Norepinephrine Storage Vesicles generated near the Golgi apparatus in cell bodies travel by axonal transport to the nerve terminals. Noradrenergic vesicles may also form by endocytosis within the axons. Since reserpine eliminates the electrondense cores of the small but not the large vesicles, the cores of the small vesicles may represent NE, whereas the electron-dense cores of the large vesicles may represent additional components. Cores of both types of vesicle contain adenosine triphosphate (ATP). The vesicles also contain at least three types of polypeptides: chromogranin A, an acidic glycoprotein; enkephalins; and neuropeptide Y (NPY). Extracellular fluid levels of each of these compounds have been considered as indices of exocytosis. Vesicles in sympathetic nerves actively remove and trap axoplasmic amines via the type 2 vesicular monoamine transporter (VMAT). Adrenomedullary chromaffin cells express both types of VMAT. Vesicular uptake favors l- over d-NE, Mg and ATP accelerate the uptake, and reserpine effectively and irreversibly blocks it. VMAT

II. BIOCHEMICAL AND PHARMACOLOGICAL MECHANISMS

NOREPINEPHRINE: THE MAIN SyMPATHETIC CARDIOvASCuLAR NEuROTRANSMITTER

proteins resemble the neuronal uptake carriers of the cell membrane. Neurotransmitter specificity appears to depend on different transporters in the cell membrane rather than on different vesicular transporters. Tissue NE stores are maintained by a balance of synthesis and loss. Under resting conditions, the main determinant of loss of NE from the tissue is net leakage of NE from the vesicles into the axoplasm, with subsequent enzymatic breakdown of the axoplasmic NE. This loss is balanced by ongoing synthesis of DA in the neuronal cytosol, vesicular uptake of cytosolic DA, and conversion of DA to NE catalyzed by DBH. Sympathetic neuroimaging using radio-iodinated metaiodobenzylguanidine, positron-emitting analogs of sympathomimetic amines, and 6-[18F]fluorodopamine depends on uptake of the imaging agents into the axoplasm via the cell membrane norepinephrine transporter (NET) and subsequent translocation into vesicles via the VMAT. Visualization of sympathetic innervation in organs such as the heart, therefore, results in radiolabeling of the vesicles in noradrenergic nerves.

Norepinephrine Release Adrenomedullary chromaffin cells, much easier to study than sympathetic nerves, have provided the most commonly used model for studying mechanisms of catecholamine release. Agonist occupation of nicotinic acetylcholine receptors releases catecholamines from the cells. Since nicotinic receptors mediate ganglionic neurotransmission, researchers have presumed that findings in adrenomedullary cells apply to post-ganglionic sympathetic noradrenergic neurons. Low plasma levels of NE in patients with autoimmune autonomic ganglionopathy from circulating antibodies to the neuronal nicotinic receptor support this view. According to the exocytotic theory of NE release, acetylcholine depolarizes terminal membranes by increasing membrane permeability to sodium. The increased intracellular sodium levels directly or indirectly enhance transmembrane influx of calcium, via voltage-gated calcium channels. The increased cytoplasmic calcium concentration evokes a cascade of biomechanical events resulting in fusion of the vesicular and axoplasmic membranes. Vesicle poration causes the interior of the vesicle to exchange with the extracellular compartment, and the soluble contents of the vesicles diffuse into the extracellular space. As predicted from this model, manipulations besides application of acetylcholine that depolarize the cell, such as electrical stimulation or increased K concentrations in the extracellular fluid, also activate voltage-gated calcium channels and trigger exocytosis. During cellular activation, simultaneous, stoichiometric release of soluble vesicular contents – ATP, enkephalins, chromogranins, and DBH – without similar release of cytoplasmic macromolecules, has provided biochemical support for the exocytosis theory.

39

At least two storage pools of NE seem to exist in sympathetic nerve terminals – a small, readily releasable pool of newly-synthesized NE and a large reserve pool in longterm storage. Because of conversion of DA to NE within vesicles, plasma DA responses during sympathetic stimulation may reflect release from the readily releasable pool. Sympathetic nerve endings can also release NE by calcium-independent, non-exocytotic mechanisms. One such mechanism is reverse transport through the neuronal uptake carrier. The indirectly acting sympathomimetic amine, tyramine, releases NE non-exocytotically, since tyramine releases NE independently of calcium and does not release DBH. Myocardial ischemic hypoxia also evokes calcium-independent release of NE. As noted above, sympathetic stimulation releases other compounds besides NE. Some of these compounds may function as co-transmitters. ATP, adenosine, NPY, acetylcholine, DA, and EPI have received the most attention. Pharmacological stimulation of a large variety of receptors on noradrenergic terminals affects the amount of NE released during cellular activation. Compounds inhibiting NE release include acetylcholine, gamma-aminobutyric acid (GABA), prostaglandins of the E series, opioids, adenosine, and NE itself. Compounds enhancing NE release include angiotensin II, acetylcholine (at nicotinic receptors), ACTH, GABA (at GABAA receptors), and EPI (via stimulation of pre-synaptic β2-adrenoceptors). In general, whether at physiological concentrations these compounds exert modulatory effects on endogenous NE release remains unproven, especially in humans. An exception, however, is inhibitory presynaptic modulation by NE itself, via autoreceptors on sympathetic nerves. This modulatory action appears to vary with the vascular bed under study, being prominent in skeletal muscle beds such as the forearm, relatively weak in the kidneys, and virtually absent in the adrenals. In addition to local feedback control of NE release, reflexive “long-distance” feedback pathways via high- and low-pressure baroreceptors elicit reflexive changes in sympathoneural impulse activity. Alterations in receptor numbers or of intracellular biomechanical events after receptor activation also affect responses to agonists. These factors may, therefore, regulate NE release by trans-synaptic local and reflexive long-distance mechanisms. NE released from sympathetic nerve terminals acts mainly locally, with only a small proportion of released NE reaching the bloodstream. One must, therefore, keep in mind the indirect and distant relationship between plasma NE levels and sympathetic nerve activity in interpreting plasma NE levels in response to stressors, pathophysiologic situations, and drugs.

Norepinephrine Disposition NE is inactivated mainly by uptake into cells, with subsequent intracellular metabolism or storage (Fig. 6.1). Reuptake into nerve terminals – Uptake-1 – via the cell


40

6. NORADRENERGIC NEuROTRANSMISSION Exo

VMA

Exo

DAv

DBH

LIVER

NEv

3-MT

VMA NET

3-MT

NET Leak

VMAT

COMT

ADH, ALDH

VMAT

DA DOPA TYR

TH

DOPA

LAAAD

NE

NEc

DAc MAO

COMT

COMT

3-OMeTyramine

NMN DOPAL

3-OMeTyramine

AR

HVA

DOPET

DOPEGAL

ALDH

DOPAC

AR

DHPG

COMT

NMN

ALDH

MHPG

DHMA COMT

HVA DOPAC

MHPG

DHPG

FIGURE 6.1 Diagram of steps in norepinephrine biosynthesis, release, cellular uptake, and metabolism. Abbreviations: ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; AR, aldose/aldehyde reductase; COMT, catechol-O-methyltransferase; DA, dopamine; DBH, dopamine-β-hydroxylase; DHMA, dihydroxymandelic acid; DHPG, dihydroxyphenylglycol; DOPA, dihydroxyphenylalanine; DOPAC, dihydroxyphenylacetic acid; DOPEGAL, dihydroxyphenylglycolaldehdye; Exo, exocytosis; HVA, homovanillic acid; LAAAD, L-aromatic-amino-acid decarboxylase; MAO, monoamine oxidase; MHPG, methoxyhydroxyphenylglycol; 3-MT, 3-methoxytyrosine; 3-OMdTyramine, 3-methoxytyramine; NEc, cytoplasmic norepinephrine; NEv, vesicular norepinephrine; NET, cell membrane norepinephrine transporter; NMN, normetanephrine; TH, tyrosine hydroxylase; TYR, tyrosine; VMA, vanillylmandelic acid; VMAT, vesicular monoamine transporter.

membrane NET is the predominant means of terminating the actions of released NE. Uptake-1 is energy-requiring and can transport catecholamines against large concentration gradients. The only common structural feature of all known substrates for Uptake-1 is an aromatic amine, with the ionizable nitrogen moiety not incorporated in the aromatic system. Uptake-1 does not require a catechol nucleus. Alkylation of the primary amino group decreases the effectiveness of the transport, explaining why sympathetic nerves take up NE more efficiently than they do EPI and why they do not take up isoproterenol, an extensively alkylated catecholamine, at all. Methylation of the phenolic hydroxyl groups also markedly decreases susceptibility to Uptake-1, and so sympathetic nerves do not take up O-methylated catecholamine metabolites such as normetanephrine. Many drugs or in vitro conditions inhibit Uptake-1, including cocaine, tricyclics such as desipramine, NET blockers such as reboxetine, low extracellular Na concentrations, and Li(Fig. 6.2). Neuronal uptake absolutely requires intracellular K and extracellular Na. Transport does not directly require ATP; however, maintaining ionic gradients across cell membranes depends on ATP, and the carrier uses the energy expended in maintaining the transmembrane Na gradient to co-transport amines with Na.

There are distinct cell membrane transporters for NE and DA. The human NET protein includes 12–13 hydrophobic and therefore membrane-spanning domains, a structure that differs substantially from that of adrenoceptors and other receptors coupled with G-proteins but resembles that of transporters for DA, GABA, serotonin, and vesicular monoamines. Non-neuronal cells remove NE actively by a process called Uptake-2, which is characterized by the ability to transport isoproterenol, susceptibility to blockade by O-methylated catecholamines, corticosteroids, and β-haloalkylamines, and absence of susceptibility to blockade by cocaine and tricyclics. In contrast with Uptake-1, Uptake-2 functions independently of extracellular Na. The Uptake-2 carrier has little if any stereoselectivity and has low affinity and specificity for catecholamines. For instance, extraneuronal cells remove imidazolines such as clonidine by Uptake-2. Whereas reverse transport via the Uptake-1 carrier requires special experimental conditions, one can readily demonstrate reverse transport via the Uptake-2 carrier. Because of reverse transport, during infusion of a catecholamine at a high rate, the catecholamine can accumulate in extraneuronal cells, with re-entry of the catecholamine into the extracellular fluid via the Uptake-2 carrier after the infusion ends.


NOREPINEPHRINE: THE MAIN SyMPATHETIC CARDIOvASCuLAR NEuROTRANSMITTER

SNS

Uptake-1

PLASMA

NE

FIGURE 6.2 Diagram of sympathoneural, hepatic, adrenomedullary, and gut contributions to plasma levels of norepinephrine and its metabolites. Additional abbreviations: DHPG-S, DHPG sulfate; MHPG-S, MHPG sulfate; MN-S, MN sulfate; mPST, monoamine-preferring phenolsulfotransferase; NMN-S, NMN sulfate.

NE NE

MAO

41

DHPG

MHPG

GUT CELL

DHPG

LIVER CELL

COMT MAO AD

VMA

VMA DHPG-S MHPG-S

COMT

EPI, NE NMN

DHPG-S MHPG-S

NMN-S

NMN-S

ADRENOMEDULLARY CELL

mPST

mPST

NMN

Norepinephrine Metabolism NE taken up into the axoplasm by the Uptake-1 transporter is subject to two fates, translocation into storage vesicles and deamination by monoamine oxidase (MAO). The combination of enzymatic breakdown and vesicular uptake constitute an intraneuronal “sink,” which keeps axoplasmic NE concentrations very low. Reserpine prevents the conservative recycling of NE and depletes NE stores. After reserpine injection, plasma DHPG levels increase rapidly, reflecting marked net leakage of NE from vesicular stores, and then decline to very low levels, reflecting decreased vesicular uptake of DA and decreased conversion of DA to NE. MAO catalyzes the oxidative deamination of DA to form DOPAC and of NE to form DHPG. Because of the efficient uptake and reuptake of catecholamines into the axoplasm of catecholamine neurons, and the rapid exchange of amines between the vesicles and axoplasm, the neuronal pool of MAO, located in the outer mitochondrial membrane, figures prominently in the overall functioning of catecholamine systems. Two isozymes of MAO, MAO-A and MAO-B, have been described. Clorgyline blocks MAO-A, and deprenyl and rasagiline block MAOB. MAO-A predominates in neural tissue, whereas both subtypes exist in non-neuronal tissue. Inhibitors of MAO-A potentiate pressor effects of tyramine, whereas inhibitors of MAO-B do not. NE and EPI are substrates for MAO-A, and DA is a substrate for both MAO-A and MAO-B. The immediate products of the deamination are short-lived aldehydes. For DA, the aldehyde intermediate,

dihydroxyphenylacetaldehyde (DOPAL) is converted rapidly to DOPAC by aldehyde dehydrogenase; for NE, the aldehyde intermediate is converted mainly to DHPG by an aldehyde/aldose reductase. The formation of the aldehydes reduces a flavine component of the enzyme. The reduced enzyme reacts with molecular oxygen, regenerating the enzyme but also producing hydrogen peroxide. Catechol-O-methyltransferase (COMT) in non-neuronal cells catalyzes the O-methylation of DHPG to form methoxyhydroxyphenylglycol (MHPG) and of DOPAC to form homovanillic acid (HVA). In the liver MHPG is converted vanillylmandelic acid (VMA), and in splanchnic organs catecholamines (especially DA) and their glycol metabolites are extensively sulfoconjugated. The main end-products of NE metabolism in plasma, therefore, are MHPG, conjugated MHPG, and VMA. Although MAO inhibitors are effective anti-depressants, a phenomenon known as the “cheese effect” limits their clinical use. In patients taking MAO inhibitors, administration of sympathomimetic amines such as in many nonprescription decongestants or ingestion of foods such as aged cheese, wine, or meat, which contain tyramine, can produce paroxysmal hypertension. Since tyramine and other sympathomimetic amines displace NE from sympathetic vesicles into the axoplasm, blockade of MAO in this setting causes non-exocytotic NE release, stimulating cardiovascular smooth muscle cells and producing intense vasoconstriction and hypertension. COMT catalyzes the conversion of NE to normetanephrine (NMN) and EPI to metanephrine (MN). Uptake-2 and COMT probably act in series to remove and degrade


42

6. NORADRENERGIC NEuROTRANSMISSION

circulating catecholamines. The methyl group donor for the reaction is S-adenosyl methionine. COMT is expressed by non-neuronal cells and adrenomedullary chromaffin cells but not by sympathetic neurons. Ongoing production of NMN and MN in such cells explains the high sensitivity of plasma levels of free (unconjugated) metanephrines in the diagnosis of pheochromocytomas, which are chromaffin cell tumors.

BIOMARKERS OF SYMPATHETIC NORADRENERGIC INNERVATION AND FUNCTION Simultaneous measurement of plasma levels of NE and its metabolites provides valuable information about sympathetic noradrenergic innervation and function. In general, sympathetic noradrenergic stimulation produces similar absolute increments in plasma NE and DHPG levels. In contrast, decreased Uptake-1 activity increases plasma levels of plasma NE more than of DHPG. Under resting conditions plasma DHPG is determined mainly by net leakage of NE from vesicles into the cytosol – an ongoing passive process. Thus, plasma DHPG is related indirectly to NE stores in sympathetic nerves. Because loss of sympathetic noradrenergic terminals can compensatorily increase nerve traffic, NE release and plasma NE levels may be maintained until denervation is advanced. Accordingly, plasma DHPG provides a more sensitive index of sympathetic noradrenergic denervation than does plasma NE. Assessments of effects of neuropharmacologic probes such as tyramine, desipramine, atomoxetine, yohimbine, clonidine, trimethaphan, and isoproterenol are often included in clinical studies of sympathetic noradrenergic function. In general, physiological measurements such as of blood pressure, heart rate, or local vascular resistances are not as sensitive as neurochemical measurements in detecting and characterizing sympathetic noradrenergic dysfunction. This is because patients with autonomic failure typically also have baroreflex failure, and patients with sympathetic noradrenergic denervation typically also have augmented cardiovascular responses to agonists at adrenoceptors, complicating the relationship between NE release and physiological dependent measures.

NE AND METABOLOMICS Patterns of plasma levels of catechols yield metabolomic information reflecting abnormal enzymatic activities that can be relevant to the diagnosis of several disorders. Because of the dependence of DBH on copper, all patients with Menkes disease, an X-linked recessive disorder of a copper-ATPase, have high plasma DOPAC:DHPG and DA:NE ratios. Familial dysautonomia and DBH deficiency also entail elevated ratios of DA metabolites to NE

metabolites. Due to the vesicular localization of DBH, the same abnormal pattern characterizes low VMAT-2 activity. Decreased TH activity, from reduced synthesis either of the enzyme itself or of the required co-factor tetrahydrobiopterin, results in low levels of all the endogenous catechols. LAAAD deficiency is associated with high DOPA:NE, DOPA:DHPG, and DOPA:DOPAC ratios. Decreased Uptake-1 activity, such as by hypofunctional mutation of the gene encoding the NET, results in relatively larger increments in plasma NE than in plasma DHPG during sympathetic noradrenergic stimulation. Decreased MAO-A activity from treatment with an MAO inhibitor or from mutation of the X-linked gene encoding MAO-A manifests as high NE:DHPG and DA:DOPAC ratios. Since DHPG exiting sympathetic noradrenergic neurons is extensively O-methylated by COMT, low COMT activity is associated with increased DHPG:MHPG ratios. Theoretically, decreased alcohol dehydrogenase activity should be associated with elevated MHPG:VMA ratios.

DIFFERENTIAL NORADRENERGIC VS. ADRENERGIC ACTIVATION IN STRESS Different stressors can elicit different patterns of sympathoneural outflows and therefore differential NE release in the various vascular beds. This patterning redistributes blood flows. Local sympathoneural release of NE also markedly affects cardiac function and glandular activity. The adjustments usually are not sensed, and the organism usually does not feel distressed. Examples of situations involving prominent changes in sympathoneural outflows include orthostasis, mild exercise, post-prandial hemodynamic changes, mild changes in environmental temperature, and performance of non-distressing locomotor tasks. In contrast, in response to perceived global threats whether from external physical or internal psychological or metabolic stimuli – especially when the organism senses an inability to cope with those stimuli – increased neural outflow to the adrenal medulla elicits catecholamine secretion into the adrenal venous drainage. In humans, the predominant catecholamine in the adrenal venous drainage is EPI. EPI rapidly reaches all cells of the body (with the exception of most of the brain), producing a wide variety of hormonal effects at low blood concentrations. One can comprehend all of the many effects of EPI in terms of countering acute threats to survival that mammals have faced throughout their evolution, such as sudden lack of metabolic fuels, trauma with hemorrhage, and antagonistic confrontations. Even mild hypoglycemia elicits marked increases in plasma levels of EPI, in contrast with relatively small increases in levels of NE. Distress accompanies all these situations, the experience undoubtedly fostering the long-term survival of the individual and the species by motivating avoidance learning and producing signs that are understood instinctively by other members of the species.


DIffERENTIAL NORADRENERGIC vS. ADRENERGIC ACTIvATION IN STRESS

Across a variety of stressors, increases in adrenomedullary activity as indicated by elevated plasma EPI levels correlate more closely with increases in pituitaryadrenocortical activity as indicated by elevated plasma levels of corticotropin than with increases in sympathoneural activity as indicated by elevated plasma levels of NE. Thus, insulin-induced hypoglycemia produces large increases in plasma EPI and ACTH levels and rather mild NE responses, whereas cold exposure produces increases in plasma NE levels, with little or no increases in EPI or ACTH. These findings call into question the long held concept of a unitary sympathoadrenal system activated in a stereotypical manner during stress.

Further reading Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: A contemporary view with implications for physiology and medicine. Pharmacol Rev 2004;56:331–49.

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Goldstein DS. The autonomic nervous system in health and disease. New York: Marcel Dekker, Inc. 2001. Goldstein DS. Adrenaline and the inner world: An introduction to scientific integrative medicine. Baltimore: The Johns Hopkins University Press; 2006. Goldstein DS. Catecholamines 101. Clin Autonomic Res 2010;20:331–52. Goldstein D.S. Neurocardiology: Therapeutic implications for cardiovascular disease. Cardiovasc Ther 2010 Nov 25. doi: 10.1111/j.17555922.2010.00244.x. [Epub ahead of print] Goldstein DS, Holmes C, Kaler SG. Relative efficiencies of plasma catechol levels and ratios for neonatal diagnosis of Menkes disease. Neurochem Res 2009;34:1464–8. Goldstein DS, Holmes C. Neuronal source of plasma dopamine. Clin Chem 2008;54:1864–71. Goldstein DS, Kopin IJ. Adrenomedullary, adrenocortical, and sympathoneural responses to stressors: A meta-analysis. Endocr Regul 2008;42:111–9. Lenders JWM, Pacak K, Walther MM, Linehan WM, Mannelli M, Friberg P, et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 2002;287:1427–34.


C H A P T E R

7 Tyrosine Hydroxylase Kazuto Kobayashi, Toshiharu Nagatsu INTRODUCTION

approximately 8.5 kb (GenBank Accession Number: BC104967). Alternative splicing from a single gene produces four kinds of mRNAs encoding TH isoforms (hTH1-4) that are different in amino acid sequences in the N-terminal region (see Fig. 7.1A) [see ref. 1 for detail]. The hTH1-4 isoforms are distinguishable by the combination of insertion/deletion of the 12-bp sequence (4 amino acids) in the 3-terminal portion of exon 1 and the 81-bp sequence (27 amino acids) corresponding to exon 2. The hTH1 isoform contains the shortest amino acid sequence (497 amino acids), and the hTH2, hTH3, and hTH4 isoforms contain additional sequences of 4, 27, and 31 amino acids, respectively, between the 30th and 31st amino acids in the hTH1 isoform. TH activity is regulated by phosphorylation mainly at serines 19, 31, and 40 in the N-terminal region (hTH1). In particular, the TH isoforms appear to be differentially modified by extracellular signal-regulated protein kinase (ERK) [3]. ERK phosphorylates serine 31 in the hTH1 isoform, whereas ERK phosphorylation of the corresponding site in the hTH2-4 isoforms is less efficient. The phosphorylation of serine 19 increases the rate of phosphorylation on serine 44 in the hTH2 isoform (corresponding to serine 40 in the hTH1) more greatly than phosphorylation of the site in the hTH1 isoform. Dumas et al. [4] subsequently demonstrated the generation of more isoforms encoding human TH through skipping exon 3 in the adrenal medulla (Fig. 7.1B). These isoform are overexpressed in the tissue of patients with progressive supranuclear palsy, a neurodegenerative disease mostly affecting the basal ganglia, cerebral cortex, and brain stem. The isoforms lack 74 amino acids containing the two major phosphorylation sites (serines 31 and 40). In addition, Ohye et al. [5] report a new splicing variant in the adrenal medulla that encodes TH isoform lacking exon 4 sequence, resulting in truncation of the C-terminal region of the protein. This splicing seems to cause inactivation of TH function. These recent data support that alternative splicing produces the functional diversity of TH isoforms, which may be involved in the complex mechanisms that regulate catecholamine metabolism. Altered metabolism related to TH gene expression may underlie the pathophysiology of some neurological and neurodegenerative diseases.

Tyrosine hydroxylase (TH; tyrosine 3-monooxygenase) (EC 1.14.16.2) catalyzes the conversion of L-tyrosine to L-3,4dihydroxyphenylalanine (L-DOPA), which is the initial and rate-limiting step in the biosynthetic pathway of catecholamines including dopamine, noradrenaline, and adrenaline [reviewed in ref. 1]. These catecholamines play important roles in a variety of physiological and behavioral functions in the nervous and endocrine systems. TH activity is regulated through different mechanisms at the levels of transcription of the gene and posttranslational modification of the protein. Gene targeting technique of the mouse genome reveals that TH function is essential for animal development and survival as well as brain function [reviewed in ref. 2]. Disruption of the TH gene (homozygous mutation) causes the lethality at the embryonic and neonatal stages because of alterations in the cardiovascular system. Reduction of TH activity in the mice heterozygous for the mutation impairs retention of long-term memory at the adult stage. Recent advances in molecular biological study of the human TH gene have shown that a number of TH isoforms are generated by alternative splicing from a single gene, suggesting a complex mechanism that generates the diversity of TH isoforms in the normal and pathological conditions. Human genetic studies have found mutations that lead to TH deficiency, showing an infantile onset, progressive hypokinetic-rigidity with dystonia or a complex encephalopathy with neonatal onset. In addition, the use of TH gene function has provided a useful approach for gene therapy trials of Parkinson’s disease. In the present chapter, we describe recent advances in molecular biological and genetic studies related to human TH gene. We recapitulate the mechanism that generates the diversity of human TH isoforms, the implication of TH mutations in congenital diseases, and the update of gene therapy trials for neurological disease by the use of TH gene.

DIVERSITY OF HUMAN TH GENE EXPRESSION The human TH gene is encoded on chromosome 11p15.5 and composed of 14 exons, spanning

Primer on the Autonomic Nervous System. DOI: 10.1016/B978-0-12-386525-0.00007-X

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46

7. TyRosInE HydRoxylAsE

FIGURE 7.1 Schematic illustration of alternative splicing from a single TH gene that generates the diversity of isoforms. (A) Alternative splicing events producing four kinds of mRNAs encoding TH isoforms (hTH1-4). These mRNAs are distinguishable by the combination of insertion/deletion of the 12-bp sequence in the 3-terminal portion of exon 1 and the 81-bp sequence corresponding to exon 2. (The data are cited from ref. 1.) (B) Splicing events producing the mRNAs lacking exon 3. These mRNAs correspond to hTH1, hTH3 and hTH4 mRNAs deleting exon 3. (The data are cited from ref. 4.)

CONGENITAL DISEASE ASSOCIATED WITH TH MUTATION

GENE THERAPY WITH TH GENE FUNCTION

TH deficiency is an autosomal recessive disorder associated with the mutations in the TH gene (see Fig. 7.2). The clinical phenotype of the disease shows an infantile onset, progressive L-DOPA-responsive dystonia or a progressive encephalopathy with L-DOPA-nonresponsive dystonia. This deficiency can be diagnosed by measuring the levels of catecholamine metabolites such as homovanillic acid and 3-methoxy-4-hydroxyphenylethylene glycol in cerebrospinal fluid and direct sequencing of the TH gene. So far, the deficiency is reported in fewer than 40 patients worldwide. TH mutations in the deficiency have been extensively studied, and the clinical, biochemical, and genetic data are summarized in ref. [6]. The majority of TH mutations (~95%) were missense mutations that lead to amino acid substitutions in the protein-coding region. Some of these mutations are clarified to result in partial loss of enzymatic activity in the bacterial expression system. In addition, deletion of a single nucleotide in the coding region is reported that generates a truncated form of TH protein. Single nucleotide substitutions in the cyclic AMP response element (CRE) in the promoter region of TH gene are also found in some cases. Since the CRE in the TH promoter is known to be important for both basal and inducible transcriptional activity, the mutations in the CRE may down-regulate the transcription of TH gene, affecting the TH expression level. The relationship between the mutation and clinical phenotype does not appear to be fully understood.

Parkinson’s disease is an age-related neurodegenerative disease caused by the progressive loss of dopamine neurons in the ventral midbrain and the consequent reduction of the dopamine level. The patients exhibit a range of clinical phenotypes with the most common motor deficits such as the resting tremor, rigidity, akinesia, and postural instability. The standard procedure to treat the disease is pharmacotherapy by oral administration of L-DOPA, but many patients gradually develop L-DOPA-induced dyskinesia and motor fluctuation. Gene therapy trials offer alternative, complementary approaches for clinical application of Parkinson’s disease [7]. One potential strategy for gene therapy is to produce dopamine in striatal cells through gene transfer of enzymes related to dopamine biosynthesis. Muramatsu et al. [8] report gene therapy trial of a non-human primate model for Parkinson’s disease by adeno-associated viral vectors that carry the genes encoding TH, GTP cyclohydrolase I (GCH1), and aromatic L-amino acid decarboxylase (AADC). GCH1 is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, tetrahydrobiopterin (BH4), and AADC converts L-DOPA to dopamine. Ectopic expression of these three enzymes successfully achieves the increased production and release of dopamine, and thus improves behavioral deficits in the parkinsonian model. Gene therapy for continuous L-DOPA delivery is also a potentially useful therapeutic strategy for the disease. Björklund et al. [9] report that gene transfer of TH and GCH1 genes into striatal cells


ConClusIon

47

FIGURE 7.2 Overview of the mutations in TH deficiency. The exon/intron structure of the human TH gene is shown. The closed boxes indicate the protein-coding regions and the open boxes indicate 5- and 3-untranslated regions. Three mutations (71C  T, 70G  A, and 69T A) are located in the cyclic AMP response element in the TH promoter (the nucleotides 67 to 74 upstream of the initiation codon). The 295delC and 296delT mutations cause the frame shift in the protein-coding region. The 1198-24T  A mutation in the putative branchpoint sequence in intron 11 results in alternative splicing that produces aberrant mRNAs. Other mutations generate amino acid substitutions in the protein-coding region. (The data are cited from ref. 6.)

of the parkinsonian model rats with adeno-associated viral vectors results in ectopic L-DOPA production, showing the optimal ratio of the amount of TH and GCH1 enzymes for the in vivo DOPA production. The elevated level in BH4 by GCH1 expression appears to contribute to the stabilization of TH protein. A subsequent study with positron emission tomography imaging demonstrates that the straital transfer with TH and GCH1 genes enables behavioral recovery correlated with correction of dopamine transmission in the striatum [10]. These gene therapy trials aiming at the delivery of dopamine or L-DOPA provide a reasonable, promising strategy for clinical application in the disease.

CONCLUSION In the present chapter, we presented a short review of recent advances of molecular biological and genetic studies of human TH gene. The functional diversity of TH isoforms is generated by alternative splicing from a single gene. This diversity appears to mainly affect the regulatory mechanism of TH activity through phosphorylation of the protein. The different regulatory mechanism may be implicated in the control of catecholamine metabolism. Mutations in the coding or promoter region of the human TH gene exist in congenital TH deficiency with the infantile onset, progressive hypokinetic-rigidity with dystonia or a complex encephalopathy with neonatal onset. Future genetic study will address the question whether mutations in the TH gene are involved in the neuropsychiatric diseases. Gene therapy trials with the use of TH gene function aiming at the in vivo delivery of dopamine or L-DOPA indeed show the recovery of behavior and brain dopamine metabolism in animal models for Parkinson’s disease. This strategy will generate a viable procedure for clinical application in the disease.

The progress on the TH gene studies provides a clearer understanding of catecholamine function in the pathophysiology in neurological and neurodegenerative diseases and a novel strategy for the management of these diseases.

References [1] Nagatsu T. The catecholamine system in health and diseases – Relation to tyrosine 3-monooxygenase and other catecholamine-synthesizing enzymes. Proc Jpn Acad Ser B Phys Biol Sci 2006;82:388–415. [2] Kobayashi K, Nagatsu T. Molecular genetics of tyrosine 3-monooxygenase and inherited diseases. Biochem Biophys Res Commun 2005;338:267–70. [3] Lehman IT, Bobrovskaya L, Gordon SL, Dunkley PR, Dickson PW. Differential regulation of the human tyrosine hydroxylase isoforms via hierarchical phosphorylation. J Biol Chem 2006;281:17644–17651. [4] Dumas S, Hir HL, Bodeau-Péan S, Hirsch E, Thermes C, Mallet J. New species of human tyrosine hydroxylase mRNA are produced in variable amounts in adrenal medulla and are overexpressed in progressive supranuclear palsy. J Neurochem 1996;67:19–25. [5] Ohye T, Ichinose H, Yoshizawa T, Kanazawa I, Nagatsu T. A new splicing variant for human tyrosine hydroxylase in the adrenal medulla. Neurosci Lett 2001;312:157–60. [6] Willemsen MA, Verbeek MM, Kamsteeg E-J, de Rijk-van Andel JF, et al. Tyrosine hydroxylase deficiency: a treatable disorder of brain catecholamine biosynthesis. Brain 2010;133:1810–22. [7] Feng LR, Maguire-Zeiss KA. Gene therapy in Parkinson’s disease: rationale and current status. CNS Drugs 2010;24:177–92. [8] Muramatsu S, Fujimoto K, Ikeguchi K, Shizuma N, et al. Behavioral recovery in a primate model of Parkinson’s disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Hum Gene Ther 2002;13:345–54. [9] Björklund T, Hall H, Breysse N, Soneson C, Carlsson T, Mandel RJ, et al. Optimization of continuous in vivo DOPA production and studies on ectopic DA synthesis using rAAV5 vectors in Parkinsonian rats. J Neurochem 2009;111:355–67. [10] Leriche L, Björklund T, Breysse N, Besret L, Grégoire M-C, Carlsson T, et al. Positron emission tomography imaging demonstrates correlation between behavioral recovery and correction of dopamine neurotransmission after gene therapy. J Neurosci 2009;29:1544–53.


C H A P T E R

8 Antidepressant-Sensitive Norepinephrine Transporters: Structure and Regulation Maureen K. Hahn Chemical signaling at central and peripheral noradrenergic synapses is terminated through reuptake of released norepinephrine (NE) [1]. The protein executing this activity, the antidepressant-sensitive NE transporter (NET), has been cloned from multiple species including man [2]. The human NET (hNET) gene encodes a 617 amino acid polypeptide. A high resolution structure of a SLC6 bacterial family member, the leucine transporter (LeuTAa), from Aquifex aeolicus, was recently achieved, confirming the structure of this family as twelve transmembrane domain proteins with intracellular NH2 and COOH termini (Fig. 8.1) [3]. Expression of the original hNET isolate is sufficient to confer antidepressant-sensitive NE transport [4]. During biosynthesis, hNET protein is N-glycosylated at sites on a large extracellular loop between transmembrane domains (TMDs) 3 and 4, and the protein is subsequently trafficked to the plasma membrane [5]. Although hNET glycosylation is not known to be regulated, this modification enhances protein stability and transport activity [6]. Once inserted in the plasma membrane, hNET is exposed to the transmembrane sodium gradient that the transporter utilizes to bring about NE uptake. Extracellular chloride also facilitates uptake of NE

and intracellular potassium may offer further stimulation, though evidence that potassium is countertransported is lacking. Additionally, hNET supports NE-gated channel states that may allow NE to translocate hNET at high rates when synaptic concentrations are elevated [7]. To take advantage of this possibility, NETs would need to be enriched at synaptic sites of release. Consistent with this, studies with NET-specific antibodies reveal NET localized to the plasma membrane of synaptic boutons [8]. Additional hNET splice variants have been identified at the mRNA level, predicting additional NET species with altered COOH termini [9]. Whereas related, though not identical, mRNA variants have been identified in rat brain and bovine adrenal, evidence of splice variant protein expression is currently lacking [10,11]. Recent heterologous expression studies incorporating these variants reveal significantly disrupted maturation and surface trafficking, raising doubt as to their functional relevance, at least as monomers [12]. In this regard, although a single hNET cDNA can confer NE transport activity, multiple hNET proteins may assemble together as an oligomeric complex. Indeed, dimer or higher order complexes have been documented for the closely related dopamine transporter (DAT) and serotonin transporter (SERT) and the presence of oligomers of hNET would offer an explanation for genetic NET deficiency exhibited by subjects with a mutation in one of two hNET alleles [13–15]. Mouse models of genetic NET deficiency support a contribution of transporter expression to presynaptic NE homeostasis, extracellular NE clearance and psychostimulant action [16]. Studies conducted over the past few years have revealed that NET and related transporters are subject to rapid regulation including changes in transporter surface expression as well as intrinsic transport activity [17,18]. This regulatory potential is believed to involve both transporter phosphorylation and the regulated associations of accessory proteins. Thus, NET has been found to complex with the catalytic subunit of protein phosphatase 2A (PP2Ac), the SNARE protein syntaxin 1A as well as the scaffolding protein PICK1 [18–20]. The NH2 terminus of hNET supports syntaxin 1A associations whereas the transporter’s COOH terminal PDZ domain is required for PICK1 binding. Protein kinase C activators disrupt Syntaxin and PP2Ac associations,

NE, Na+, Cl–

Extracellular

Intracellular P P

FIGURE 8.1 Norepinephrine transporters. Depicted is the 12transmembrane topology predicted for the NET protein, bearing three N-glysosylated residues on the second extracellular loop. The cytoplasmic NH2 and COOH termini bear multiple consensus sites for Ser/Thr phosphorylation. Norepinephrine (NE) is depicted as cotransported with Na and Cl which provide the energy for uptake of catecholamine into the presynaptic terminal.


49


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8. ANTIdEPRESSANT-SENSITIvE NoREPINEPHRINE TRANSPoRTERS: STRuCTuRE ANd REgulATIoN

NE

[3] TAPs dissociate

[4]

Phosphorylation NT

[5]

Recycling

Internalization

[6]

Dephosphorylation

[7]

FIGURE 8.2 Norepinephrine transporter regulation. Illustrated is a cycle for the regulated trafficking of NET proteins. NETs are depicted in complex with multiple associated proteins that may stabilize the transporter at the plasma membrane. Stimuli linked to receptor activation by other neurotransmitters, hormones or cytosolic second messengers influence the stability of the NET associated protein complex and enhance NET phosphorylation. In parallel with, or as a result of such stimulation, NET proteins internalize leaving reduced presynaptic NE transport capacity. NETs can recycle to the plasma membrane, a process that likely occurs at basal rates but which may be linked to regulatory stimuli. TAP, transporter-associated proteins.

stimulation that also leads to NET internalization. PP2Ac also appears to be required for alterations in hNET intrinsic activity triggered by insulin and MAP-kinase linked pathways [17]. Possibly, trafficking and intrinsic function are coregulated through kinase-dependent mechanisms. Insulin signaling to promote NET internalization, an effect mediated through protein kinase B (Akt), occurs in both brain and peripheral neurons in vivo, thus providing a potential link between diseases of metabolism and neurobehavioral disorders [2]. Although these are early days in molecular studies of NET regulation, it is already clear that NE clearance capacity is likely to involve multiple regulatory proteins that localize, stabilize and activate NET (Fig. 8.2). They may also influence the tendency of NET to support catecholamine efflux when ion gradients are perturbed as they can be in ischemic insults. Finally, successful assimilation of these proteins into a regulatory model of NE inactivation should offer new opportunities to manipulate NET in autonomic and mental disorders, such as depression, where altered noradrenergic signaling has been recognized.

Acknowledgements

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

M.K. Hahn gratefully acknowledges the NIMH for their support of studies on NET genetics and regulation. [19]

References [1] Iversen LL. Uptake processes for biogenic amines. In: Iversen LL, editor. Handbook of Psychopharmacology (third ed.). New York: Prenum Press; 1978. p. 381–442. [2] Robertson SD, Matthies HJ, Owens WA, Sathananthan V, Christianson NS, Kennedy JP, et al. Insulin reveals Akt signaling as

[20]

a novel regulator of norepinephrine transporter trafficking and norepinephrine homeostasis. J Neurosci 2010;30(34):11305–11316. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E, et al. Crystal structure of a bacterial homologue of Na/Cl -dependent neurotransmitter transporters. Nature 2005;437(7056):215–23. Pacholczyk T, Blakely RD, Amara SG. Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 1991;350(6316):350–4. Melikian HE, McDonald JK, Gu H, Rudnick G, Moore KR, Blakely RD, et al. Human norepinephrine transporter. Biosynthetic studies using a site-directed polyclonal antibody. J Biol Chem 1994;269(16):12290–12297. Melikian HE, Ramamoorthy S, Tate CG, Blakely RD. Inability to N-glycosylate the human norepinephrine transporter reduces protein stability, surface trafficking, and transport activity but not ligand recognition. Mol Pharmacol 1996;50(2):266–76. Galli A, Blakely RD, DeFelice LJ. Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake [see comments]. Proc Natl Acad Sci USA 1998;95(22):13260–13265. Matthies HJ, Han Q, Shields A, Wright J, Moore JL, Winder DG, et al. Subcellular Localization of the antidepressant-sensitive norepinephrine transporter. BMC Neurosci 2009;10(1):65. Pörzgen P, Bönisch H, Hammermann R, Brüss M. The human noradrenaline transporter gene contains multiple polyadenylation sites and two alternatively spliced C-terminal exons. Biochim Biophys Acta 1998;1398(3):365–70. Burton LD, Kippenberger AG, Lingen B, Bruss M, Bonisch H, Christie DL, et al. A variant of the bovine noradrenaline transporter reveals the importance of the C-terminal region for correct targeting to the membrane and functional expression. Biochem J 1998; 330(Pt 2):909–14. Kitayama S, Ikeda T, Mitsuhata C, Sato T, Morita K, Dohi T, et al. Dominant negative isoform of rat norepinephrine transporter produced by alternative RNA splicing. J Biol Chem 1999; 274(16):10731–10736. Bauman PA, Blakely RD. Determinants within the C-terminus of the human norepinephrine transporter dictate transporter trafficking, stability, and activity. Arch. Biochem Biophys 2002;404(1):80–91. Hastrup H, Karlin A, Javitch JA. Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment. Proc Natl Acad Sci U S A 2001;98(18):10055–10060. Kilic F, Rudnick G. Oligomerization of serotonin transporter and its functional consequences. Proc Natl Acad Sci USA 2000;97(7):3106–11. Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, et al. Clues to the origin of orthostatic intolerance: a genetic defect in the cocaine- and anti-depressant sensitive norepinephrine transporter. N Eng J Med 2000;342(8):541–9. Xu F, Gainetdinov RR, Wetsel WC, Jones SR, Bohn LM, Miller GW. et al. Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat Neurosci 2000;3(5):465–71. Apparsundaram S, Sung U, Price RD, Blakely RD. Traffickingdependent and -independent pathways of neurotransmitter transporter regulation differentially involving p38 mitogen-activated protein kinase revealed in studies of insulin modulation of norepinephrine transport in SK-N-SH Cells. J Pharmacol Exp Ther 2001;299(2):666–77. Bauman HJ, Apparsundaram S, Ramamoorthy S, Wadzinski BE, Vaughan RA, Blakely RD, et al. Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A. J Neurosci 2000;20(20):7571–8. Sung U, Apparsundaram S, Galli A, Kahlig K, Savchenko V, Schroeter S, et al. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J Neurosci 2003;23(5):1697–709. Torres GE, Yao W, Mohn AR, Quan H, Kim K, Levey AI, et al. Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 2001;30(1):121–34.


C H A P T E R

9 α1-Adrenergic Receptors Marion C. Mohl, Robert M. Graham

a1-ADRENORECEPTOR SUBTYPES

gene family in the human genome – α1-ARs are long, single chain, integral membrane proteins. Like rhodopsin and β-ARs, α1-ARs contain seven transmembrane (TM)spanning α-helical domains linked by three intracellular and three extracellular loops [1]. In addition, the juxtamembranous portion of the C-terminal tail likely forms an eighth α-helix that lies parallel to the plain of the membrane and is importantly involved in receptor signaling – a domain structure found in all class A GPCRs for which highresolutions structures are available, but not in the recently determined structure of the CXCR4 chemokine receptor [2]. As with all GPCRs, the N-terminus of α1-AR subtypes is located extracellularly and the C-terminus, intracellularly (Fig. 9.1). The ligand-binding pocket is formed by the clustering of the seven α-helical domains to form a water accessible region for agonist to bind, and is located in the outer (extracellular) one third of the membranespanning domain. Residues of the intracellular (cytoplasmic) domains, particularly the third intracellular loop, mediate specific interactions with their cognate G proteins and, thus, are involved in receptor-engagement with its signaling and regulatory pathways.

Three subtypes have been identified by molecular cloning in several species including humans. They are classified as the α1A (previously the α1A/c)-, the α1B- and α1D (previously the α1a or α1a/d)-ARs (for a detailed consideration of α1-AR subtype-classification, see ref. 1). For the α1A-AR, four splice variants (α1A-1, α1A-2, α1A-3, α1A-4) have been identified, and are expressed at different levels in various tissues, including the liver, heart and prostate. However, the functional significance of these splice variants is unclear, since they all display similar ligandbinding and functional activity when expressed in heterologous cell systems. The characteristics of the various α1-AR subtypes are shown in Table 9.1. In addition to these subtypes, a putative fourth subtype, the α1L-AR, which displays low affinity for prazosin and other α1-antagonists, has been identified by functional but not by molecular studies. This subtype is postulated to mediate contraction of prostate smooth muscle, but it remains unclear if it is a distinct subtype or a merely a functional variant of one of the cloned α1-subtypes, such as the α1A splice variants, since the latter all display some of the characteristics of an α1L-subtype when expressed in heterologous cell systems. Another molecular variant of the α1A-AR is a coding region polymorphism that involves an arginine to cysteine (Arg492Cys) substitution in an arginine-rich region of the C-terminal tail. This polymorphic receptor is found with higher frequency in African-Americans, but it is not associated with essential hypertension. In addition, it is unclear if it alters receptor regulation or produces any other functional effects.

LIGAND BINDING AND ACTIVATION OF a1-ADRENORECEPTORS Binding of catecholamines to α1-AR involves an ionic interaction between the basic aliphatic nitrogen atom common to all sympathomimetic amines and an aspartate (Asp125 in the hamster α1B-AR) in the third transmembranespanning segment (TMIII) [3]. In the ground state, this TMIII aspartate forms a salt bridge with a lysine residue (Lys331 in the α1B-adrenergic receptor) in TMVII. Activation of α1-ARs, likely involves disruption of this ionic interaction. This is due to competition between the protonated amine of catecholamines and the TMVII lysine, which is just favored by the slightly more basic pKa of the protonated amine (pKa 11.0) versus the lysine (pKa 10.5). Agonistbinding to α1-ARs also involves an H-bond interactions between the meta hydroxyl group of catecholamines and a serine residue (Ser188, α1A-receptor numbering) in TMV,

STRUCTURE OF a1-ADRENORECEPTORS α1-ARs are integral membrane glycoproteins and members of the biogenic amine or class A family (also includes α2- and β-ARs, as well as the light-activated photoreceptor, rhodopsin) of G-protein-coupled receptors (GPCRs) [1]. Like other members of the GPCR-superfamily – the largest family of membrane receptors and possibly the largest


51


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9. α1-ADRENERGIC RECEPTORS

TABLE 9.1 Characteristics of the α1A-Adrenoreceptor Subtypes Receptor Subtype α1A

α1B

α1D

68

80

≈65

Amino acids

431–501

515–520

561–572

Gylcosylation sites (N-terminus)

3

4

2

Phosphorylation sites

PKA

PKA

–

Genomic organization Introns

1

1

2

2

2

8

5

20 p13

5-Methylurapidil, ()niguldipine, oxymetazoline, A-61603, SNAP5089, KMD-3213, RS17053

AH11110A, L-765,314

()Norepinephrine, BMY 7378, SKF105854

–

Prazosin, phentolamine, benoxathian, abanoquil, terazosin, doxazosin, tamulosin, phenylephrine, methoxamine, cirazoline

–

Prototypic tissues

Rat kidney and submaxillary gland, rabbit liver, human heart and liver

Rat spleen, liver, and heart

Rat aorta, lung, and cerebral cortex

Receptor-coupled signaling

–

Ca2 mobilization, PLC, PLA2, PLD

–

G-protein coupling

Gq/11/4

Gq/11/4/16, Gh

Gq11, Gh

Characteristics *

Mr

§

Exons †

Chromosomal localization

Pharmacological selectivity Subtype-selective agents

Nonselective agents‡

*Apparent molecular weights (Mr) determined by SDS-PAGE are shown (Table adapted from ref. 1). † Refers to the human genome. ‡ These characteristics are the same for all subtypes. § Differences due to species variability and/or to splice variants.

whereas an interaction between the para hydroxyl and another TMV serine (Ser192) contributes only minimally to receptor activation. A further important interaction, for both binding and activation, involves aromatic-aromatic bonding between the catecholamine ring and that of a phenylalanine (Phe310 for the α1B-adrenergic receptor) in TMVI [3]. This interaction is also important in receptor activation, which in addition to the TMIII–TMVII salt bridge disruption, mentioned above, involves movement of TMVI that is likely required to allow interaction between the intracellular third loop and the receptor’s cognate G-protein. Aromatic–aromatic interactions between two phenylalanines (Phe163 and Phe187) in TMIV and TMV, respectively, and the catecholamine ring, have also been suggested to be important for agonist binding [3], but not for activation, although that with Phe163 may be indirect, since this residue is replaced by a leucine in the α1B- and α1D-adrenergic receptors. Residues critical for subtype-selective agonist recognition have been evaluated and, importantly, just two of the approximately 172 residues in the transmembrane domains (Ala204 in TMV and Leu314 in TMVI, and Val185 in TMV and

Met293 in TMVI of the α1B- and α1A-receptors, respectively) have been shown to account entirely for the selective agonist-binding profiles of the α1A- and α1B-subtypes [1]. Interactions between antagonists and α1-ARs are less well-defined, although the selectivity of two α1Aantagonists, phentolamine and WB4101, involves interactions with three consecutive residues (Gly196, Val197 and Thr198) in the second extracellular loop [3].

FUNCTION OF a1-ADRENORECEPTORS The best characterized α1-AR-mediated function is that of smooth muscle contraction. As such, these receptors have a major role in the vascular system in controlling blood pressure, the baroreflex response to changes in blood pressure, and in temperature control. The importance of α1ARs in controlling sympathetically regulated arteriolar tone is evident, for example, from the fact that a component of the blood pressure lowering action of all currently available antihypertensive agents is attributable to an attenuation of α1-AR-responses. This is due either to a direct action at


FUNCTION OF α1-ADRENORECEPTORS

VI

VII

V

I

IV III

II

LIGAND

Extracellular

II

VII

IV

LIGAND

V

I Intracellular

III

FIGURE 9.1 Cut away 3D model of the hamster α1B-AR, showing the seven α-helical transmembrane domains indicated by Roman numerals and by dashed circles and backbone ribbons (corkscrews), with the catecholamine agonist, epinephrine (ball and stick model with surrounding dot surface) modeled in its binding pocket. Upper panel: top view (looking down onto the plane of the membrane). Lower panel: side view.

the level of post-junctional, vascular smooth muscle α1-ARactivation or signaling, or to an indirect effect via inhibition of sympathetic outflow and, thus, availability of the endogenous receptor-agonists, the catecholamines, epinephrine and norepinephrine. Therefore α1-ARs agonists or antagonists can be used in the treatment of hypotension or hypertension, respectively, although their use in hypertension may result in heart failure in susceptible patients, due possibly to blockade of important cardiomyocyte pro-survival effects [4,5]. Despite the critical role of α-ARs in mediating arteriolar vasoconstriction, the particular subtype(s) responsible remains unclear. Consistent with findings in rodents, clinical trials of α1A-selective blockers have implicated this subtype as a major regulator of blood pressure [11]. In contrast, studies of a patient with sympathotonic orthostatic hypotension indicate that the α1B-AR may be a major regulator of vascular resistance in humans. Moreover, expression of this subtype increases in the elderly at a time when hypertension is commonly manifested. Thus, there is evidence for both the α1A- and α1B-adrenergic receptors being involved in blood pressure control in humans. Studies in the rat and in mouse knockout-models indicate that the α1D-subtype

53

can also play a role in blood pressure regulation. Indeed, such studies of genetically engineered animal models [5] indicate that sympathetic regulation of vascular tone by the various α1-subtypes is complex and may involve crossregulation of their contractile effects and/or of their expression. For example, inactivation of the α1A-AR results in a small but significant decrease in basal blood pressure but no change in pressor responses to the α1-agonist, phenylephrine. However, inactivation of the α1B-AR results in no effect on basal blood pressure but the pressor responses to phenylephrine were significantly blunted or unchanged. Interestingly, with inactivation of both the α1A-AR and α1B-AR there was no significant fall in blood pressure. Inactivation of the α1D-AR, or both α1D-AR and α1B-AR resulted in a significant drop of the resting blood pressure and impaired vasoconstrictor responses to noradrenaline and phenylephrine. Given that overexpression of the α1Badrenergic receptor in transgenic mice does not increase systemic arterial blood pressure, and that inactivation of this subtype results in attenuation of phenylephrine pressorresponses, with no change in basal blood pressure, it could reasonably be suggested that the α1A- and α1D-AR are the mediators of vasoconstriction and that the α1B-AR has a modulatory rather than a direct contractile role. However, mice lacking both the α1A- and α1B-receptors, or both the α1B- and α1D-receptors, show profound attenuation of phenylephrine pressor-responses. Thus, under appropriate circumstances the α1B-subtype may contribute importantly to sympathetic regulation of arteriolar tone. Further studies are thus required to fully elucidate the mechanisms involved in this apparent latent contribution of the α1B-AR to peripheral resistance, and to determine if these findings in animal models are also germane to the regulation of vascular resistance and blood pressure in humans. Other functions mediated by α1-ARs, include bronchoconstriction, regulation of human lipid metabolism, uptake of glucose into adipocytes, and contractile effects in various tissues, such as the vas deferens and the myocardium. For example, inactivation of the α1A-AR in mice results in a 50% reduction in pregnancy rate, which is markedly enhanced to 10% in animals with inactivation of all three α1A/B/C-AR subtypes – an effect likely due to impaired contraction of the vas deferens and, thus, decreased sperm ejaculation. Cardiac specific overexpression of the wild type α1B-AR in mice results in depressed contractile responses to β-AR stimulation, whereas cardiac-specific overexpression of a constitutively active mutant α1B-AR results in hypertrophic responses to pressure overload, leading to heart failure and premature death. In contrast, α1A-AR overexpression leads to increased cardiac contractility but no hypertrophy and, furthermore, moderate overexpression (66–fold) improves outcomes after pressure overload and after myocardiac infarction. Marked overexpression (170–fold), however, leads to sudden cardiac death that is likely due to calcium overload rather than to arrhythmia development. α1-ARs, particularly, the α1Asubtype, also have important cardiomyocyte pro-survival and developmental growth effects [4,5].


54

9. α1-ADRENERGIC RECEPTORS

Other important smooth muscle constrictor effects mediated by α1-ARs, include those relating to the lower urinary tract (bladder and prostate). The role of the α1A-subtype, in particular, is well-documented with α1A-selective antagonists being useful to relax prostate smooth muscle, relieve bladder outlet obstruction and, thus, to enhance urine flow (reviewed in ref. 6). Moreover, urinary incontinence is an unfortunate side effect of therapy with α1-AR blockers in patients with impaired bladder neck function. α1-ARs also regulate prejunctional or pre-synaptic neurotransmitter release, including inhibition of norepinephrine, acetylcholine and vasopressin release, and stimulation of inhibitory GABAergic neurotransmission in neurons and glial cells [7].

SIGNALING OF a1-ADRENORECEPTORS All three α1-ARs subtypes couple to a variety of second messenger proteins via heterotrimeric G-proteins, particularly of the Gαq/11 family, to phospholipase C leading to increased Ca2 entry via voltage-dependent or TRPC3 and or TRPC6 channels, or to release of Ca2 from intracellular stores [8]. Activation of α1-ARs augments arachidonic acid release through stimulation of phospolipase A or D, which can increase cAMP levels [8]. Inotropic effects of α1-ARs in rat hearts involve stimulation of Gαs-protein and reduction of the K current via the cAMP/PKA-mediated pathway. In addition to signaling through hetertriomeric G-proteins, α1ARs also mediate responses through the RhoA/Rho-kinase signaling pathway leading to Ca2 sensitization through phosphorylation of the myosin light chain phosphatase (MLCP) and the α1B and α1D, but not the α1A-subtype may also couple to phospholipase δ1-activation via Gh [9]. There is also evidence that α1-ARs form hetero- and homodimers, which alter their ligand binding and signaling properties, as well as their trafficking [8].

REGULATION OF a1-ADRENORECEPTORS α1-ARs are subject to agonist-induced regulation that results in both short and long-term desensitization of signaling [8,10]. These regulatory responses are mediated by agonist-induced conformational changes that lead to C-terminal tail receptor-phosphorylation, both by

receptor-linked protein kinase C as well as by G-proteincoupled receptor kinases, followed by the binding of arrestins and internalization by the clathrin pathway. These responses have been studied in most detail for the α1Breceptors. With the α1A- and α1D-ARs, the carboxyl terminal tail appears not to be essential for signaling or desensitization, and receptor internalization is not associated with α1A-AR desensitization.

Acknowledgements We are most grateful to Dr J. Novotny for the α1B-AR 3D model. Work from the author’s laboratory is supported in part by a Program Grant from the National Health and Medical Research Council of Australia (#573732), and a Grant-in-Aid from the Heart Foundation of Australia (G09S4342).

References [1] Graham RM, Perez DM, Hwa J, Piascik MT. Alpha(1)-adrenergic receptor subtypes – molecular structure, function, and signaling. Circ Res 1996;78:737–49. [2] Wu B, Chien EYT, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R, Brooun A, Wells P, Bi FC, Hamel DJ, Kuhn P, Handel TM, Cherezov V, Stevens RC. Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists. Science 2010;330:1066–71. [3] Perez DM. Structure-function of α1-adrenergic receptors. Biochem Pharmacol 2007;73:1051–62. [4] Huang Y, Wright CD, Merkwan CL, Baye NL, Liang Q, Simpson PC, O’Connell TD. An alpha1A-adrenergic-extracellualr signalregulated kinase signaling pathway in cardiac myocytes. Circulation 2007;115:763–72. [5] Woodcock EA, Du X-J, Reichelt ME, Graham RM. Cardiac α1-adrenergic drive in pathological remodelling. Cardiovasc Res 2008;77:452–62. [6] Schwinn DA. Novel role of α1-adrenerigc receptors in lower urinary tract symptoms. BJU Int 2000;86(Suppl. 2):11–22. [7] Docherty JR. Subtypes of functional alpha1-adrenoceptor. Cell Mol Life Sci 2010;67:405–17. [8] Cotecchia S. The α1-adrenergic receptors: diversity of signaling networks and regulation. J Recept Signal Transduct 2010;30:410–9. [9] Lorand L, Graham RM. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 2003;4:140–56. [10] Finch A, Sarramegna V, Graham RM. Ligand binding, activation and agonist trafficking. In: Perez DM, editor. The Adrenergic Receptors in the 21st Century. : Humana Press Inc; 2006. p. 25–85. [11] Plascik MT, Kusiak JW, Barron KW. Alpha 1-adrenoceptor subtypes and the regulation of peripheral hemodynamics in the conscious rat. Eur J Pharmacol 1990;186:273–8.


C H A P T E R

10 a2-Adrenergic Receptors Qin Wang

PHARMACOLOGY AND FUNCTIONS OF a2-AR SUBTYPES

to be critical for agonist-mediated lowering of blood pressure, sedation, anesthetic sparing, and working memory. The a2A-AR also appears to respond to endogenous catecholamines to suppress epileptogenesis (kindling) and depressive symptoms, the latter measured in mouse behavioral studies. The a2C-AR elicits depressive behaviors, behaving functionally the opposite of the a2A-AR subtype. The a2B-AR, in contrast to the a2A-AR, is involved in the vascular hypertensive effect of a2-AR agonists. Taken together, these findings suggest that subtype-selective agonists may be useful in manipulating one versus another adrenergic response in vivo. An even more refined therapeutic selectivity might be achieved using partial agonists of a2-ARs. It has been observed that agonist-mediated lowering of blood pressure can occur in mice heterozygous for the a2A-AR, whereas agonist-evoked sedation cannot. These data suggest that partial agonists with less than 50% intrinsic activity (efficacy) might be useful in a number of therapeutic settings, such as treatment of attention deficit/hyperactivity disorder (ADHD) and improvement of cognition in the elderly, where sedation as a side effect would undermine the therapeutic value of these agents. Interestingly, imidazoline compounds, for which there is evidence of a role in the central nervous system in regulating blood pressure, nonetheless appear to lower blood pressure via a2-ARs when administered peripherally. Thus, moxonidine and rilmenidine, developed as imidazoline I1-selective agents, are unable to lower blood pressure in D79N a2A-AR or a2A-AR knockout mice. Also of interest is the finding that these agents are partial agonists at the a2A-AR, which may explain their ability to lower blood pressure without evoking sedative side effects.

Alpha2-adrenergic receptors (a2-ARs) bind to their endogenous ligands, epinephrine and norepinephrine, and are blocked by the antagonist yohimbine. There are three subtypes of a2-AR, encoded by three independent, intronless genes. These subtypes are denoted as a2A- (human chromosome 10), a2B- (human chromosome 2), and a2C(human chromosome 4). Ligand selectivity for pharmacological agents does exist for the various a2-AR subtypes, although this selectivity has been observed principally in vitro, as not all of these ligands have been evaluated for their pharmacokinetic properties in vivo (see Table 10.1). The lack of truly specific ligands for each of the a2-AR subtypes, particularly antagonists, has prevented the unequivocal assignment of the differing a2-AR subtypes to various physiological responses. However, tools to genetically manipulate the mouse genome to create mutant (e.g. D79N a2A-AR) or null alleles of each of these subtypes shed light into subtype-specific functions of the different a2-AR subtypes, as outlined in Table 10.2. For example, both the a2A- and a2C-ARs are involved in suppression of catecholamine release from central neurons. However, the a2A-AR serves as the primary presynaptic autoreceptor. The a2C-AR is also critical for suppression of epinephrine release from the adrenal chromaffin cells. Moreover, the a2A-AR appears TABLE 10.1* Relative Selectivity for Ligands at the Three a2Adrenergic Receptor Subtypes Agonists

Shared: norepinephrine, epinephrine, aproclonidine

a2-AR SIGNALING AND TRAFFICKING

Selective: oxynetazoline (A  C  B), clonidine, guanabenz (A,C), UK 14304 (A  C), dexmedetomidine (A,B) Antagonists

All three subtypes of the a2-AR share the same signaling pathways in native cells: decrease in adenylyl cyclase activity, suppression of voltage-gated Ca currents, and activation of receptor-operated K currents and MAP kinase activity. In heterologous cells, the a2A-AR has been shown to activate phospholipase A2 and phospholipase D,

Shared: yohimbine, rauwolscine, phentolamine, idazoxan, RX 821002, atipamezole Selective: ARC 239 (B  C  A), prazosin (B,C  A), BRL 44408 (A  C), mianserin (A,B)

*Modified from Saunders and Limbrid, Pharmacology & Therapeutics 84:193–205, 1999.


55


56

10. a2-ADRENERGIC RECEPTORS

TABLE 10.2*

Subtype-Specific Functions of a2-AR Derived From Gene-Targeted Mouse Models α2-AR Subtypes α2A-AR

Physiological Functions

α2B-AR

α2C-AR

CENTRAL EFFECTS Presynaptic inhibition of norepinephrine, dopamine, serotonin release

Xa

Hypotensive effect

X

Sedative effect

X

Antinociceptive effects of a2-adrenergic receptor agonist

X

Antinociceptive effects of moxonidine

X

Analgesic effect of nitrous oxide

X

X X

Anesthetic-sparing effects of dexmedetomidine

X

Adrenergic-opioid synergy in spinal antinociception

X

Anesthetic-sparing effects of dexmedetomidine

X

Hypothermic effects of dexmedetomidine

X

X

PERIPHERAL EFFECTS Inhibition of noradrenaline release from nerve terminal invaded into tissues

X

X

Inhibition of adrenaline release from adrenal cortex

X

Vasoconstriction, hypertensive effect

X

Salt-induced hypertension

X

Placenta angiogenesis

X

Platelet aggregation

X

X

Prevention of respiratory failure

X

BEHAVIOR EFFECTS Antiepileptogenic effect

X

Special working memory

X

Inhibition of startle responses

X

Antianxiety

X

Inhibition of locomotor stimulation of D-amphetamine

X

Latency to attack after isolation

X

*Above data summarized in Kable et al. J. Pharmacol. Exp. Ther. 2000;293(1):1–7; Brede et al. Biol Cell. 2004;96(5):343–8; and Knaus et al. Neurochem Int 2007;51(5):277–81. Also from Haubold et al. J Biol. Chem. 2010;285(44)34213–19. a Primary autoreceptor.

although these responses have yet to be observed in native target cells. Despite the similarity of the signaling pathways of the a2-AR subtypes, there are interesting differences in the trafficking itineraries of these receptors. Whereas the a2A- and a2B-AR subtypes are enriched on the surface at steady state, the a2C-AR is distributed between the surface and intracellular compartments. Upon agonist stimulation, both the surface a2A-AR and a2B-AR undergo internalization in an arrestin-dependent manner. a2A-AR internalization can be selectively promoted by different agonists such as clonidine and guanfacine, which, at least in part, account for distinct duration of signaling action of these drugs.

REGULATION BY INTERACTING PROTEINS a2-AR trafficking and signaling are tightly regulated by non-G protein interacting partners including GPCRs, kinases and scaffolding proteins such as arrestin and spinophilin (see Table 10.3). Some of these interactions serve to scaffold a2-ARs to particular cellular micro-compartments or to tether them to defined signaling molecules, while other receptor-protein interactions control a2-AR internalization and post-endocytotic sorting as well as the kinetics of a2AR-mediated signaling transduction. For example, whereas arrestin promotes a2A-AR and a2B-AR phosphorylation and


a2-AR POLYMORPHISMS

TABLE 10.3*

57

a2-AR-Interacting Proteins α2-AR Subtype(s)

Functional Role(s) Implicated

a2A

Precoupled to G proteins

a2C-AR

a2A

Attenuating agonist-induced a2A-AR to the a2A-AR phosphorylation, reducing arrestin binding

β1-AR

a2A

Altering ligand binding properties of β1-AR, leading to internalization of β1-AR in response to a2-agonist

β2-AR

a2C

Enhancing surface expression and internalization of a2C-AR, enhancing ERK activation by a2C-AR

μOR

a2A

Enhancing morphine-induced GTPγ S binding and ERK activation; however, no transactivation of G proteins, no interdependent internalization

δOR

a2A

Enhancing δOR-mediated neurite outgrowth

GRK2

a2A, a2B

Mediating agonist-induced phosphorylation and homologous desensitization

PKC

a2A

Mediating heterologous desensitization; Modulating constitutive activity of a2A-AR

14-3-3ζ

a2A, a2B, a2C

Function unknown, competed by phosphorylated Raf peptide

APLP1

a2A, a2B, a2C

Increasing a2A-AR-mediated inhibition of adenylyl cyclase activity

arrestin 2

a2B

Receptor affinity for arrestin 2 is lower than arrestin 3

arrestin 3

a2A, a2B

Stabilizing receptor phosphorylation, mediating endocytosis and desensitization, accelerating ERK signaling rate, enhancing sensitivity of in vivo response

arrestin 3

a2C

Mediating endocytosis

eIF-2B

a2A, a2B, a2C

Function unknown

Rab8

a2B

Promoting transport to cell surface

spinophilin

a2A, a2B

Stabilizing receptor at surface, attenuating phosphorylation, decelerating ERK signaling rate, decreasing in vivo response sensitivity

spinophilin

a2C

Function unknown

Uch-L1

a2A

Decreasing a2A-AR-mediated activation of ERK

Interacting Protein(s) GPCR DIMERIZATION HOMO-DIMERIZATION

HETERO-DIMERIZATION

KINASES

OTHER PROTEINS 14-3-3ζ

*Modified from Wang and Limbird, Biochem Pharm 2007;73(8):1135–1145; also from Weber et al. Cell Signal 2009;21(10):1513–21; Dong C et al. J Biol Chem 2010;285(26):20369–80.

internalization and accelerates the initial rate of signaling and desensitization, spinophilin blocks these activities by competing for GRK and arrestin interaction with these two a2-AR subtypes. Importantly, spinophilin antagonism of arrestin functions in regulating a2-ARs has in vivo relevance as manifested by reciprocal modulation of a2A-AR-evoked sedation by these two proteins in vivo.

a2-AR POLYMORPHISMS A number of individual human polymorphisms have been identified for each of the a2-AR subtypes. Some of

these have resulted in alterations in receptor density, G protein coupling, desensitization, or G protein receptor kinasemediated phosphorylation. Genetic association studies have linked a2-AR polymorphisms with a number of disease states and variations in drug responses in human populations. For example, a2A-AR polymorphisms have been linked to increased risk of ADHD, hypertension and type 2 diabetes. Additionally, genetic variants in all three subtypes have been associated with various forms of cardiovascular dysfunction. The study of a2-AR polymorphisms and their association with human diseases provides insights whose functional relevance can, in the future, be assessed by evaluating a variety of a2-AR in genetically engineered mouse


58

10. a2-ADRENERGIC RECEPTORS

models that introduce each polymorphism into the genome via homologous recombination. Given the new understanding of the a2-AR subtypes in manipulating different physiological effects, refinement of this understanding with regard to partial agonists at the receptor, and the impact of receptor interactions with other proteins in target cells beyond G protein coupling, it would seem possible that molecular entities might be developed to interfere with particular a2-AR–protein interactions or with a2-AR activation of various pathways and evaluated for efficacy as therapeutic interventions in disease settings still lacking appropriate a2-AR-system regulation.

Acknowledgement I am grateful to Dr. Lee Limbird (Vanderbilt University) for her critical reading of this chapter and invaluable suggestions.

Further Reading Flordellis C, Manolis A, Scheinin M, Paris H. Clinical and pharmacological significance of alpha2-adrenoceptor polymorphisms in cardiovascular diseases. Int J Cardiol 2004;97:367–72.

Kable JW, Murrin LC, Bylund DB. In vivo gene modification elucidates subtype-specific functions of alpha(2)-adrenergic receptors. J Pharmacol Exp Ther 2000;293:1–7. Knaus AE, Muthig V, Schickinger S, Moura E, Beetz N, Gilsbach R, et al. a2-Adrenergic subtypes-unexpected functions for receptors and ligands derived from gene-targeted mouse models. Neurochem Int 2007;51:277–81. Lu R, Li Y, Zhang Y, Chen Y, Shields AD, Winder DG, et al. Epitopetagged receptor knock-in mice reveal that differential desensitization of alpha2-adrenergic responses is due to ligand-selective internalization. J Biol Chem 2009;284:13233–243. Moore CA, Milano SK, Benovic JL. Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 2007;69:451–82. Rosengren AH, Jokubka R, Tojjar D, Granhall C, et al. Overexpression of alpha2A-adrenergic receptors contributes to type 2 diabetes. Science 2010;327:217–20. Saunders C, Limbird LE. Localization and trafficking of alpha2adrenergic receptor subtypes in cells and tissues. Pharmacol Ther 1999;84:193–205. Small KM, Liggett SB. Identification and functional characterization of alpha(2)-adrenoceptor polymorphisms. Trends Pharmacol Sci 2001;22:471–7. Tan CM, Wilson MH, MacMillan LB, Kobilka BK, Limbird LE. Heterozygous Alpha 2A-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists. Proc Natl Acad Sci USA 2002;99:12471–12476. Wang Q, Zhao J, Brady AE, Feng J, Allen PB, Lefkowitz RJ, et al. Spinophilin Blocks Arrestin Actions in vitro and in vivo at G proteinCoupled Receptors. Science 2004;304:1940–4.


C H A P T E R

11 β-Adrenergic Receptors C.Michael Stein INTRODUCTION

and norepinephrine, results from having different sub-types of β-ARs that are differentially expressed in particular tissues and that have differences in their affinity for agonists and signal transduction mechanisms, as well as the frequent concomitant presence of α-ARs – additional targets for epinephrine and norepinephrine. The three subtypes of β-ARs share approximately 60% sequence homology and are widely distributed in many cells and organs where they mediate several physiological responses (Figure 11.1; Table 11.1). Stimulation of β1-ARs mediates increased heart rate and cardiac contractility, β2-ARs mediate bronchodilation, vasodilation and presynaptic norepinephrine release, and β3-ARs mediate lipolysis and thermogenesis, relaxation of bladder, uterus and gut smooth muscle, and attenuated cardiac contractility (Table 11.1) [1,2].

There are three subtypes of β-adrenergic receptors (β1ARs, β2-ARs and β3-ARs) that mediate a wide range of physiological responses to the adrenergic agonists epinephrine and norepinephrine, and thus play an important role in regulating cardiovascular responses in health and disease (Table 11.1, Fig. 11.1). These receptors are also the targets of commonly used classes of drugs that block or stimulate signaling. β-Blockers are often used to treat hypertension and heart failure, and to control heart rate in atrial fibrillation, whereas β-agonists are used to treat asthma. There is a wide range of variation in responses to physiological and pharmacological stimulation or blockade of β-ARs; some of this variability is due to genetic variation among individuals in genes encoding β-ARs and their signal transduction proteins.

β-AR SUBTYPES AND THEIR DISTRIBUTION

β-ARs SIGNAL TRANSDUCTION β-ARs are 7-transmembrane spanning G proteincoupled receptors with an extracellular aminoterminus, an intracellular carboxyterminus, and three intracellular and three extracellular loops (Fig. 11.1). The binding of agonist

β-ARs mediate a wide range of responses (Table 11.1). The wide range of physiological responses mediated by just two primary endogenous adrenergic agonists, epinephrine TABLE 11.1 β Adrenergic Receptor Subtypes Subtype

Tissue Distribution

Functions

Gene Localization

Common Coding Variants

β1-AR

Heart Kidney Adipocytes

Positively inotropic and chronotropic Renin release Lipolysis

10q24-q26

Arg389Gly Ser49Gly

β2-AR

Lung and bronchial Vascular smooth muscle Heart Uterus Bladder Adipocytes Eye Liver Skeletal muscle Sympathetic terminal

Bronchodilation Vasodilation Positively inotropic and chronotropic Relaxation Relaxation Lipolysis Increase aqueous humor formation Glycogenolysis Glycogenolysis Norepinephrine release

5q31-q32

Gly16Arg Gln27Glu Thr164Ile

β3-AR

Adipocytes Uterus Bladder Heart

Lipolysis Relaxation Relaxation Negatively inotropic

8p12

Trp64Arg


59


60

11. β-ADRENERGIC RECEPTORS

β1AR Ser or Gly β2AR Arg or Gly

NH2

49

β2AR Gln or Glu

16

27

agonist binding β1AR Gly or Arg 389

β3AR Trp or Arg

64

164

β2AR Thr or IIe

subsequent responses. This occurs by three mechanisms – uncoupling of the receptor from its G-protein, a process mediated by G protein-coupled receptor kinases (GRKs) that phosphorylate the receptor; movement of receptors away from the cell membrane, known as internalization or sequestration; and loss of receptors (downregulation) mediated by several mechanisms including decreased transcription and increased ubiquitination and degradation [4]. β3-ARs are activated at higher concentrations of catecholamines than β1- and β2-ARs; thus they are more likely to activate under conditions of high sympathetic activity and they are also less likely to desensitize [5].

HOOC

THERAPEUTIC IMPORTANCE OF β-ARs

G-protein coupling

desensitization

FIGURE 11.1 Structure of β adrenergic receptors.

to receptor is translated into physiological response by complex signal transduction mechanisms that involves heterotrimeric GTP-binding regulatory proteins (termed G-proteins) and their associated second messenger and effector systems. G-proteins are made up of three subunits, an α subunit that is important for recognition and effector functions, and β and γ subunits that are important for membrane localization but also have effector functions. The α subunit may be stimulatory, for example Gsα stimulates adenylate cyclase and increases cyclic AMP production, or inhibitory, for example Giα decreases cyclic AMP production. All three β-ARs couple with Gsα, but β2- and β3-ARs can also couple to Giα. Receptors exist in a conformational equilibrium between the inactive and active states and this equilibrium shifts with the binding of a ligand. There are three types of ligands: agonists that shift the equilibrium towards the active conformation; inverse agonists that favor the inactive conformation; and neutral antagonists that do not affect the equilibrium. When agonist binds to the receptor there is a conformational change that allows the intracellular part of the receptor to couple with a G-protein. For example, the activated β-AR forms a complex with Gsα and guanosine triphosphate (GTP) and stimulates the production of cyclic AMP, that in turn affects secondary effectors such as protein kinase A (PKA). The signal transduction mechanism is modulated by many other mechanisms, for example desensitization (see later), phosphodiesterases that break down cyclic AMP, and regulators of G-protein signaling (RGS) proteins that promote GTP hydrolysis [3].

RECEPTOR DESENSITIZATION Exposure of β-ARs to agonist and consequent activation triggers a process known as desensitization that acts to limit

From a therapeutic perspective β1-ARs in the heart (the target for β-blockers), and β2-ARs in the lungs (the target for β2 agonists), are most important. β-blockers comprise more than 20 different drugs that vary in their selectivity for β1and β2-ARs, as well as their lipophilicity, distribution, elimination, and intrinsic sympathomimetic effects. The primary target of β-blockers in heart disease is the β1-AR, and drugs that preferentially block this receptor are termed cardioselective β-blockers (e.g. atenolol) whereas non-selective agent such as propranolol block both β1- and β2-ARs. Initially β-blockers were considered to be absolutely contraindicated in patients with heart failure. This was because the marked increase in sympathetic activity that accompanies heart failure was considered to be a beneficial compensatory response; therefore, agents such as β-blockers that antagonized the effects of catecholamines on the heart were considered harmful. However, interventions to increase sympathetic activity increased mortality in heart failure, and in vitro studies showed that β-AR numbers were decreased and the receptors were uncoupled (i.e. desensitization had occurred) [6]. These observations led to the current view that excessive sympathetic activation is harmful in heart failure and clinical studies confirmed that careful treatment with β-blockers could improve cardiac function and decrease mortality [2]. Available β-blockers such as metoprolol, atenolol and propranolol have much lower affinity for the β3-AR than they do for β1-ARs; thus, specific β3-AR antagonists were developed. These agents resulted in weight loss in animal models of obesity but were generally not effective in human clinical trials. Their lack of efficacy has been ascribed to poor selectivity for the β3-AR, and also to the fact that β3ARs appear to play a smaller role in mediating lipolysis in human adipose tissue than they do in rodents [1]. Potential therapeutic clinical applications for future β3-AR antagonists are premature labor, irritable bowel syndrome and overactive bladder [1]. β-agonists, particularly β2-AR selective agonists, are used primarily to treat bronchoconstriction associated with asthma and other diseases of the airways. Hydrophilic drugs such as albuterol have a rapid onset and relatively short


GENETIC VARIABILITY IN β-ARS

duration of action (4–6 hours), whereas lipophilic drugs such as formoterol and salmeterol partition into the membrane and have a prolonged action [4]. β2-AR selective agonists are also used to prevent preterm labor. Agonists that are more selective for the β1-AR, for example dobutamine, are used for their ability to increase cardiac contractility.

GENETIC VARIABILITY IN β-ARS Many studies reported ethnic differences in response to adrenergic agonists and antagonists but until recently the potential mechanisms for these observations were unclear. The identification of common genetic variants in all the adrenergic receptors and studies showing that some of these variations affect function in vitro and in vivo led to work to establish the genetic contribution to variability in responses among individuals, and the clinical significance of such variation.

61

changes: Gly16Arg, Gln27Glu, and Thr164Ile. In vitro the Gly16 increased desensitization, Glu 27 decreased desensitization, and the Ile164 receptor had decreased responses to agonist. The clinical pharmacogenetic studies in this area are difficult to interpret because the variants at positions 16 and 27 are in linkage disequilibrium and there are many other non-coding variants that may affect function. Some studies suggest that patients with asthma who carry the Arg16 allele, particularly homozygotes, are less responsive to β-agonist bronchodilators, but there is little consensus regarding the clinical importance of these observations [7,8].

ADRB3 There is a one common coding region variant in the β3AR gene (ADRB3) that results in an amino acid change: Trp64Arg. However, studies performed in vitro and in vivo have been inconsistent in defining functional and clinical consequences of this variant [7,8].

ADRB1

Acknowledgement

There are two common polymorphisms in the β1AR gene (ADRB1) that result in amino acid changes: Arg389Gly and Ser49Gly. The Gly389 variant has impaired coupling to Gs and therefore less response to agonist, and the Gly49 variant has increased desensitization after exposure to agonist in vitro. Thus, the Ser49 and Arg389 alleles code for a β1-AR likely to be associated with greater response. Concordantly, studies in healthy subjects, and patients with hypertension, studied under strictly controlled conditions found that carriers of the Arg389 and the Ser49 alleles were more responsive to β-blockers. Similarly, patients with heart failure who carried the Gly389 allele were less responsive to a β-blocker with a smaller improvement in ejection fraction. In the Beta Blocker Evaluation of Survival Trial (BEST) there was an interaction between Arg389Gly genotype and outcomes of bucindolol treatment. Patients with the Gly389 allele did not benefit, but Arg389 homozygotes receiving bucindolol had improved outcomes. However, other heart failure and hypertension studies found no effect of ADRB1 genotypes on responses – thus, the clinical significance of genotype in β-blocker therapy is uncertain. Genetic variation in other adrenergic receptors that may also affect sympathetic responses (e.g. α2C-AR), and in GRKs and other proteins important in regulating adrenergic responses, may also play a role [2,7].

The previous version of this chapter was written by Dr Stephen B. Liggett and forms the framework for the present chapter.

References [1] Ursino MG, Vasina V, Raschi E, Crema F, De PF. The beta3adrenoceptor as a therapeutic target: current perspectives. Pharmacol Res 2009;59:221–34. [2] Dorn GW. Adrenergic signaling polymorphisms and their impact on cardiovascular disease. Physiol Rev 2010;90:1013–62. [3] McGraw DW, Liggett SB. Molecular mechanisms of beta2-adrenergic receptor function and regulation. Proc Am Thorac Soc 2005;2:292–6. [4] Johnson M. Molecular mechanisms of beta(2)-adrenergic receptor function, response, and regulation. J Allergy Clin Immunol 2006;117:18–24. [5] Dessy C, Balligand JL. Beta3-adrenergic receptors in cardiac and vascular tissues emerging concepts and therapeutic perspectives. Adv Pharmacol 2010;59:135–63. [6] Feldman DS, Carnes CA, Abraham WT, Bristow MR. Mechanisms of disease: beta-adrenergic receptors – alterations in signal transduction and pharmacogenomics in heart failure. Nat Clin Pract Cardiovasc Med 2005;2:475–83. [7] Kirstein SL, Insel PA. Autonomic nervous system pharmacogenomics: a progress report. Pharmacol Rev 2004;56:31–52. [8] Small KM, McGraw DW, Liggett SB. Pharmacology and physiology of human adrenergic receptor polymorphisms. Annu Rev Pharmacol Toxicol 2003;43:381–411.

ADRB2 There are three relatively common coding region variants in the β2-AR gene (ADRB2) that result in amino acid


C H A P T E R

12 Dopaminergic Neurotransmission Graeme Eisenhofer, Heinz Reichmann

Dopamine is produced as a neurotransmitter in the central nervous system (CNS) and as an intermediate in the synthesis of norepinephrine in peripheral sympathetic and CNS noradrenergic nerves and of epinephrine in adrenal chromaffin cells. Dopamine is also produced as a catecholamine end product in chromaffin-like glomus cells of the hypoxia-sensing carotid bodies as well as in numerous other peripheral tissues. Synthesis of dopamine within dopaminergic neurons depends on the rate-limiting conversion of tyrosine to L-dihydroxyphenylanaline (L-dopa) by tyrosine hydroxylase, an enzyme confined to catecholamine-producing cells (Fig. 12.1). L-dopa is then converted to dopamine by L-aromatic amino acid decarboxylase, an enzyme with a wide tissue distribution. After this step, dopamine is translocated by vesicular monoamine transporters into storage vesicles from where the amine is available for release by exocytosis. Reuptake back into neurons by the cell membrane dopamine transporter represents the main mechanism for terminating the actions of dopamine at receptor sites, including D2 presynaptic autoreceptors, which act as a break on secretion. Complete inactivation requires metabolism by intraneuronal deamination to dihydroxyphenylacetic acid or extraneuronal O-methylation and deamination to homovanillic acid, the major metabolic end product of dopamine. Dopaminergic neurons within the brain are sparse, representing less than one thousandth of a percent of all CNS neurons. Nevertheless, dopaminergic neurotransmission is crucial for regulating numerous aspects of brain function. Deranged CNS dopaminergic neurotransmission also contributes to several devastating neurological and psychiatric disorders. Consequently the various components of dopamine neuronal systems, such as dopamine transporters, receptors and second messenger systems, represent important therapeutic targets that continue to receive intense scientific attention. CNS dopaminergic neurons can be broadly divided into two major groups: neurons in the mesencephalon project to the striatum and forebrain and subserve diverse functions from the control of movement to modulation of cognition, mood, attention, reward seeking behavior and learning; the other major group located in the arcuate nucleus of the


hypothalamus has projections that release dopamine in the portal vessels with important neuroendocrine regulatory influences that include inhibition of the release of prolactin and thyroid stimulating hormone. In addition to these two major groups, there are also smaller groups in other CNS

FIGURE 12.1 Schematic representation of a slow synaptic transmission neuromodulatory dopaminergic nerve ending impinging on a dendritic spine of a fast synaptic transmission inhibitory GABAergic neuron. Abbreviations: DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; MTY, 3-methoxytyramine; TH, tyrosine hydroxylase; AADC, aromatic acid decarboxylase; MAO, monoamine oxidase; COMT, catechol-O-methyltransferase; DAT, dopamine transporter; EMT, extracellular monoamine transporter; D1, dopamine D1 receptor; D2 dopamine D2 receptor.

63


64

12. DoPAmINERgIC NEuRoTRANsmIssIoN

regions, such as the dopaminergic neurons in the retina and olfactory bulb that have ultra-short projections that transmit signals within these neuronal centers for vision and smell. Mesencephalon dopaminergic neurons localized in the substantia nigra project mainly to the striatum to form the neostriatal pathway, whereas those of the ventral tegmental area project to cortical and limbic regions to form respective mesocortical and mesolimbic pathways. Neostriatal dopaminergic pathways regulate the initiation and maintenance of motor function, while mesocortical and mesolimbic pathways respectively regulate cognitive activity and reward seeking behavior. The importance of neostriatal dopaminergic pathways for control of extrapyrimidal (involuntary) movement is reflected by the loss of motor control when nigro-striatal neurons degenerate in Parkinson’s disease. Rather than acting as a classic excitatory or inhibitory neurotransmitter, dopamine released from midbrain projections acts as a neuromodulator that alters responses of target neurons to other neurons (Fig. 12.1). Such neuromodulatory influences occur by slow modes of synaptic transmission involving complex second messenger systems rather than the ligand-operated ion-channels that characterize fast synaptic transmission. Accordingly, accumulating evidence also indicates that signals from dopamine are generated in a volume mode of transmission in which the amine transmitter does not act rapidly within classical synapses, but instead diffuses from sites of release to act at receptors on more distant neurons. These include glutamatergic and γ-aminobutyric acidergic (GABA) neurons, which respectively release the fast acting excitatory and inhibitory neurotransmitters, glutamate and GABA. Awareness of the above concepts has important implications for understanding the biology and pharmacology of neurological diseases involving dopamine systems, including an appreciation of therapeutic principles underlying the efficacy of L-dopa and dopamine receptor agonists for treatment of the movement disorder characteristic of Parkinson’s disease. In this disorder it is the overall absence of dopamine, not just of the dopaminergic neurons, that disrupts the patterning of firing of intact CNS networks and cell assemblies crucial for control of contextdependent movement. Amelioration of the motor symptoms of Parkinson’s disease, therefore, does not require restoration of the degenerated neural connections, but can be largely achieved at least initially by correcting for the absence of the transmitter itself. Apart from Parkinson’s disease and related syndromes (e.g., multiple system atrophy, progressive supranuclear palsy, diffuse Lewy body disease), derangements in CNS dopamine neuronal systems also appear to be involved in numerous other neuropsychiatric conditions including Huntington's disease, drug addictions, depression, obsessive compulsive behavior, attention deficit/hyperactivity disorder and schizophrenia. The wide-ranging nature of these disorders illustrates the diverse functions subserved by CNS dopamine, with different pathologies and clinical manifestations explained by differences in the nature or

locations of the abnormalities of dopaminergic systems and associated dysfunctions in other neuronal systems. Elucidation of some of the underlying CNS dopaminergic abnormalities associated with the above neuropsychiatric conditions has been achieved through advances in brain imaging technology using positron emission tomographic (PET) or single photon emission computed tomographic (SPECT) imaging with ligands that target components of dopamine neuronal systems (e.g., dopamine receptors and transporters). Whereas the dysfunction of Parkinson’s disease is mainly presynaptic, the lesions associated with Parkinson plus syndromes involve both pre- and postsynaptic deficiencies, explaining why patients with the latter disorders are often unresponsive to therapy with L-dopa and dopamine receptor agonists. PET or SPECT imaging utilizing ligands specific for dopamine D1 and D2 receptors has indicated that in patients with Huntington’s disease there are reductions in striatal D1 and D2 receptor binding indicating postsynaptic dysfunction. In patients with attention-deficit/hyperactivity the disorder appears to involve presynaptic deficits associated with increases in dopamine transporter binding. Patients with chronic drug abuse problems appear to have lowered dopamine release and D2 receptor numbers in dorsal or ventral striatal regions. Furthermore, in these regions and particularly the nucleus accumbens reward centers, many of drugs of abuse – including cocaine, amphetamine, heroin, alcohol and nicotine – stimulate dopamine release, thereby reinforcing drug consumption. A central role of CNS dopaminergic systems in the pathogenesis of schizophrenia followed recognition in the 1950s that antipsychotic drugs interfere with brain dopamine function. Indeed even today, all neuroleptic drugs approved to treat schizophrenia appear to exert their antipsychotic effects through blocking the dopamine D2 receptor. In line with this, PET and SPECT imaging studies have indicated that mid-brain regions in schizophrenia are characterized by increased subcortical dopamine release and sensitivity to D2 receptor activation. Beyond the generally accepted view that psychotic episodes in schizophrenia are associated with a hyperdopaminergic state, the precise neuroanatomical sites and underlying alterations of CNS dopamine systems remain imprecisely elucidated. It is often presumed that production of dopamine within the brain accounts for most of the production of this catecholamine by the body. From measurements of the overflow of dopamine metabolites from the brain into jugular venous blood it is now clear, however, that the contribution of the brain to circulating levels and urinary excretion of dopamine metabolites represents less than 20% of the overall production of dopamine within the body (Fig. 12.2). Furthermore, almost all of the dopamine formed in sympathetic nerves and the adrenal medulla is converted to norepinephrine and epinephrine. Thus, most of the dopamine and dopamine metabolites in the circulation and excreted into urine are derived from other peripheral sources. However, in contrast to norepinephrine, which functions


DoPAmINERgIC NEuRoTRANsmIssIoN

FIGURE 12.2 Schematic representation of the main sources of dopamine and the principal metabolites of dopamine in plasma and urine. The brain makes a relatively minor contribution, whereas dopamine synthesized in the gastrointestinal tract or derived from the diet contributes substantially to dopamine metabolites in the bloodstream and urine. This contrasts with the free dopamine excreted in urine, which is derived almost entirely from renal extraction of circulating L-dihydroxyphenylalanine and local decarboxylation to dopamine by L-aromatic amino acid decarboxylase. Abbreviations: HVA, homovanillic acid; DOPAC, dihydroxyphenylacetic acid; DA, Dopamine; DA-SO4, dopamine-sulfate; L-Dopa, L-dihydroxyphenylalanine; MTY; 3-methoxytyramine; MTY-SO4, 3-methoxytyramine-sulfate; AADC, aromatic acid decarboxylase.

as an important neurotransmitter in both the CNS and the peripheral sympathetic nervous system, evidence that dopamine acts as a neurotransmitter outside of the CNS is weak. Instead, available evidence indicates that dopamine in the periphery functions not as a neurotransmitter or circulating hormone, but rather as an autocrine or paracrine substance. Findings of increased plasma concentrations of L-dopa, dopamine and dopamine metabolites after consumption of foods indicate that dietary constituents also represent an important source of peripheral dopamine. Such food sources do not, however, account for the substantial amounts of dopamine produced in peripheral tissues outside of the digestive tract. Diet also does not account for findings in fasting individuals in which large arterial to portal venous increases in plasma concentrations of dopamine and its metabolites indicate substantial production of dopamine within mesenteric organs. These findings are consistent with morphological studies demonstrating the presence of cells in the gastrointestinal tract that contain dopamine and express components of dopamine signaling pathways, including catecholamine biosynthetic enzymes and specific dopamine receptors and transporters. In the

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stomach, tyrosine hydroxylase is expressed in epithelial cells, including acid secreting parietal cells. In the small intestine, cells of the lamina propria, including immune cells, also express tyrosine hydroxylase. The enzyme is additionally found in pancreatic exocrine cells. Dopamine and dopamine receptor agonists stimulate bicarbonate secretion and protect against ulcer formation, whereas dopamine antagonists augment secretion of gastric acid and promote ulcer development. Dopamine also appears to influence GI motility, sodium transport, and gastric and intestinal submucosal blood flow. In the pancreas, dopamine may modulate secretion of digestive enzymes and bicarbonate. Thus, dopamine appears to act in mesenteric organs as an enteric neuromodulator or paracrineautocrine substance. In the kidneys, dopamine is an established autocrine and/or paracrine effector substance contributing to the regulation of sodium excretion. Unlike other catecholamine systems, production of dopamine in the kidneys is largely independent of local synthesis of L-dopa by tyrosine hydroxylase. Instead, production of dopamine in the kidneys depends mainly on proximal tubular cell uptake of L-dopa from the circulation. The L-dopa is then converted to dopamine by L-aromatic amino acid decarboxylase, the activity of which is up-regulated by a high-salt diet and down-regulated by a low-salt diet. The presence of a renal dopamine paracrine-autocrine system explains the considerable amounts of free dopamine excreted in the urine. Most derives from renal uptake and decarboxylation of circulating L-dopa and reflects the plasma levels of this amino acid and the function of the renal dopamine paracrine/ autocrine system.

Further Reading Carlsson A, Waters N, Waters S, Carlsson ML. Network interactions in schizophrenia – therapeutic implications. Brain Res Brain Res Rev 2000;31:342–9. Carlsson A. Thirty years of dopamine research. Adv Neurol 1993;60:1–10. Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism: a contemporary view with implications for physiology and medicine. Pharmacol Rev 2004;56:331–49. Flagel SB, Clark JJ, Robinson TE, Mayo L, Czuj A, Willuhn I, et al. A selective role for dopamine in stimulus-reward learning. Nature 2011; 469:53–7. Girault JA, Greengard P. The neurobiology of dopamine signaling. Arch Neurol 2004;61:641–4. Nikolaus S, Antke C, Kley K, Poeppel TD, Hautzel H, Schmidt D, et al. Investigating the dopaminergic synapse in vivo. I. Molecular imaging studies in humans. Rev Neurosci 2007;18:439–72. Reichmann H. Long-term treatment with dopamine agonists in idiopathic Parkinson’s disease. J Neurol 2000;247(Suppl 4):IV/17–19. Rice ME, Cragg SJ. Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res Rev 2008;58:303–13. Sotnikova TD, Beaulieu JM, Gainetdinov RR, Caron MG. Molecular biology, pharmacology and functional role of the plasma membrane dopamine transporter. CNS Neurol Disord Drug Targets 2006;5:45–56. Volkow ND, Fowler JS, Wang GJ, Swanson JM, Telang F. Dopamine in drug abuse and addiction: results of imaging studies and treatment implications. Arch Neurol 2007;64:1575–9.


C H A P T E R

13 Dopamine Receptors Sean M. Peterson, Nikhil Urs, Marc G. Caron

INTRODUCTION

receptors, both temporally and spatially, and is cataloged online at GENSAT [1].

Dopamine (DA) is one of the major monoamine neurotransmitters in the mammalian brain. It is generated by hydroxylation and decarboxylation of the amino acid tyrosine, and further metabolized into epinephrine and norepinephrine (collectively referred to as catecholamines). The role of DA in brain function was first proposed by Nobel Laureate, Arvid Carlsson, whereas Oleh Hornykeiwicz and colleagues first demonstrated a DA deficit in the brain of Parkinson’s patients. DA has been shown to regulate a variety of functions such as movement, reward, cognition and emotion. Dysfunction of DA neurotransmission has been implicated in numerous other disease states such as addiction, schizophrenia, ADHD and Tourette’s syndrome. These multiple effects of DA are mediated through its ability to bind to five distinct G protein-coupled receptors (GPCRs) that belong to two subfamilies, the D1-like (comprised of D1R and D5R) and the D2-like (comprised of D2R, D3R, and D4R) receptor families. These families are distinguishable not only by their unique pharmacology but also by their gene structure, expression patterns, protein structure, and signal transduction pathways. A large number of DA receptor (DAR) studies have focused on the receptors in the central nervous system (CNS); however the basic principles apply to both the CNS and autonomic nervous system (ANS).

Autonomic Nervous System DA neurons from the hypothalamus project to the anterior pituitary gland where D1 and D2 family receptors are expressed leading to an inhibition of prolactin release. All DAR subtypes are expressed in the kidney, blood vessels and heart. In the kidney, DARs regulate renin release as well as fluid and sodium reabsorption.

DOPAMINE RECEPTOR STRUCTURE Gene Structure Two distinct receptor families have evolved to bind DA. The D1 family is comprised of D1R, D5R, and the D5R pseudogenes, D5ψ1 and D5ψ2, which are not expressed. The D1 family is intronless in the protein coding region, as are approximately 50% of all GPCRs. The D2 family, which includes D2R, D3R, and D4R, has a more complex gene structure than the D1 family. The D2R gene, DRD2, contains six introns and is alternatively spliced to generate two functional proteins: D2LR (the long isoform) and D2SR (the short isoform lacking a 29 amino acid exon in the third intracellular loop of the receptor). D2SR is expressed predominantly on DA neurons, and functions as an autoreceptor to regulate extracellular levels of DA and modulate neuronal firing, while D2LR is expressed predominantly postsynaptically. The D3R gene, DRD3, contains five introns in the protein coding region and is alternatively spliced, however, DRD3 isoforms are not as well characterized, which may be due to nonsense mediated decay of mRNA products. The D4R gene, DRD4, contains three introns in the protein coding region, and contains a variable number tandem repeat (VNTR) of 48 nucleotides in exon 3 of the gene (which occurs in the intracellular loop 3 of the receptor). The VNTR can be 2–11 repeats, however, the four repeats (4R) is by far the most common, while 7R and 2R occur less frequently.

DISTRIBUTION AND EXPRESSION OF DOPAMINE RECEPTORS Central Nervous System Dopamine receptor distribution in the brain has been extensively studied. In the mammalian brain D1Rs and D2Rs are the most highly expressed receptors of all the five DARs. D1R and D2R expression is highest in areas of DA neuron innervation, whereas D3R, D4R and D5R expression is more specialized. The advent of BAC (bacterial artificial chromosome) recombineering technology has led to advances in clarifying the distribution patterns of these


67


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13. DoPAmInE RECEPToRs

The DRD4 7R allele has been proposed to be a contributing factor to the development of ADHD, and has undergone strong positive selection throughout all of modern human history [2].

Receptor Structure Dopamine receptors belong to the superfamily of GPCRs and are all considered to be in the Rhodopsin-like Class A family of 7-transmembrane receptors based on sequence homology and function. For all the five DARs the N-terminus and extracellular loops are glycosylated and cysteine residues between the loops form disulfide bonds. The intracellular loops interact with G proteins, whereas these loops and C-terminal tails are phosphorylated by G protein-coupled receptor kinases (GRKs) and interact with β-arrestins and other kinases and signaling molecules (Table 13.1; Fig. 13.1). The D2 family of DARs have an especially long intracellular loop 3 (IC3) and a short C-terminal tail, while the D1 family has a relatively short IC3 and longer C-terminal tail. Recently, the crystal structure of D3R was solved [3]

TABLE 13.1 Dopamine Receptor signal Transduction Pathways

Receptor

Signaling Molecule

Downstream Effector

Second Messenger System

D1-like

Gαs

AC activation

Increased cAMP

D2-like

Gαi

AC inhibition

Decreased cAMP

D2R

Gαz

AC inhibition

Decreased cAMP

D2R/D1R and D2R/D5R dimers

Gαq

PLC activation

Increased IP3, DAG, and Ca2

D2R

Gβγ

GIRK

K

D1R

β-arrestin

ERK

N/A

D2R

β-arrestin

Akt/GSK3

N/A

Receptors are designated as their family (D1- or D2-like) or their specific receptor subtype. Canonical G protein signaling is compared and contrasted to non-canonical β-arrestin signaling. The details and relationship between these signal transduction pathways are further illustrated in Figure 13.1.

FIGURE 13.1 Relationship of dopamine receptor signal transduction pathways. Dopamine binds to dopamine receptors (DARs) to cause the exchange of GTP for GDP at the Gα subunit and the dissociation of Gβγ, which goes on to interact with effectors like ion channels. Gα hydrolyzes GTP (with the help of RGS proteins in the case of Gαi and Gαq family members) and then re-associates with Gβγ. In addition, binding of dopamine to DARs causes their phosphorylation and the recruitment of β-arrestin, which mediates desensitization of G protein signaling as well as internalization and recycling of competent DARs to the plasma membrane. The DAR/arrestin complex can initiate G protein independent receptor mediated signaling in its own right. Functional selectivity refers to agonists that are able to activate either G protein or β-arrestin pathways to a much greater extent than the other. The intracellular loop 3 and C-terminus of the receptor is represented as a dotted line because of structural differences between D1- and D2-family DARs (see text).


DoPAmInE RECEPToR FunCTIon: sIgnAl TRAnsDuCTIon PATHwAys

in complex with a selective D3R antagonist. This insight will undoubtedly provide an avenue for the design of highly potent D3R ligands. The importance of DAR pharmacological agents will be discussed in more detail in later sections.

DOPAMINE RECEPTOR FUNCTION: SIGNAL TRANSDUCTION PATHWAYS G protein-coupled receptors have long been known for transducing signals by acting as guanine nucleotide exchange factors for heterotrimeric G proteins, however, recent work has shown that most GPCRs signal through noncanonical pathways both in vitro and in vivo. Dopamine receptors are no exception and the following section summarizes both canonical and noncanonical DAR signal transduction pathways. Figure 13.1 provides a summary of the general relationship between these signaling pathways, while Table 13.1 provides specific details of the important DAR signaling molecules.

G Protein Dependent Signaling The D1 family of DARs activates members of the Gαs family, which are so named because they stimulate adenylate cyclase (AC) leading to the production of adenosine 3,5-cyclic monophosphate (cAMP) as a second messenger. The D2 family of DARs couples to both the pertussis sensitive Gαi and the pertussis insensitive Gαz families resulting in an inhibition of AC and lower cAMP. The D2 family is also known to signal through Gβγ, which activates G protein-coupled inward rectifying potassium channels, phospholipase Cβ, and other proteins.

69

molecules and promote signal transduction [6]. Recent studies from our laboratory have shown a role for β-arrestin dependent signaling through both D1R and D2R. D1Rs have been shown to promote β-arrestin dependent ERK signaling upon morphine-stimulated DA release and this signaling pathway regulates morphine/DA-induced psychomotor activation but not its rewarding effects [7]. D2Rs have been shown to promote β-arrestin dependent Akt and GSK3 signaling and regulate several DA sensitive behaviors in mice that can be modeled as endophenotypes of schizophrenia [8].

Oligomerization Higher order GPCR signaling structures have long been hypothesized to exist, however there remains some controversy over the biological significance of such structures. Evidence for oligomers (mostly dimers) between the D1 family and the D2 family has been provided from both in vitro and in vivo approaches. D2R was shown to interact with both D1R or D5R the consequence of which causes the coupling of the receptors to switch from Gαs or Gαi to a different family of G proteins, Gαq, which actives phospholipase C to cause the release of IP3 and Ca2 [9]. Receptor oligomerization adds a layer of complexity to an already complex signaling model, and new investigational tools have and will continue to illuminate these intricacies.

Dopamine Receptor Pharmacology Dopamine’s role in the regulation of complex functions in both the CNS and ANS makes DARs key targets in developing therapeutics for many diseases associated with DA function. Extensive work has been done in characterizing DAR agonists and antagonists.

Signal Regulation

Ligand Specificity

Regulator of G protein signaling (RGS) proteins function to accelerate GTP hydrolysis by the Gα subunit of G proteins, thereby attenuating GPCR signaling. D2Rs are regulated by RGS9, which has been implicated in Parkinson’s disease [4]. Receptor regulation is also accomplished through phosphorylation of the intracellular residues of the receptors by GRKs, which stabilize the receptor in a conformation conducive to β-arrestin binding. All five DARs interact with GRK2, GRK3, GRK5 and GRK6 as well as β-arrestin 1 and 2. Upon binding, β-arrestins desensitize GPCR signaling and induce internalization of receptors by interacting with clathrin-coated pits [5].

Many ligands have been shown to differentiate between the D1 and D2 family of receptors. Classically, the specificity of agonists and antagonists can be used to delineate the contribution of each receptor family in in vitro and in vivo studies. Selective ligands are also used as therapeutic agents in conditions like Parkinsonism and schizophrenia.

β-Arrestin Dependent Signaling In addition to its desensitizing and internalizing functions, recent work has shown a role for β-arrestin dependent signaling through its ability to scaffold signaling

Functional Selectivity The significance of arrestin dependent signaling for GPCRs may be many fold [7]. First, arrestin dependent signaling is usually more persistent than G protein signaling. Second, it has been shown that for certain GPCRs a given compound can act as an agonist at one signaling pathway and an antagonist at the other or vice versa. Moreover, examples are beginning to accumulate demonstrating that these two signaling modes can sub-serve distinct biological functions mediated by the same GPCR.


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Interestingly, all clinically effective antipsychotics for the treatment of schizophrenia interact with D2R. Whereas antipsychotics have a mixed agonist/antagonist profile at D2R/G protein signaling, they appear to behave uniformly as antagonists at the D2R/β-arrestin interactions [10]. This principle is commonly referred to as functional selectivity or biased agonism/antagonism and should become an important element in the development of newer, more selective therapeutic agents.

References [1] The gene expression nervous system atlas (GENSAT) project. New York, NY: The Rockefeller University. [2] Wang E, Ding YC, Flodman P, Kidd JR, Kidd KK, Grady DL, et al. The genetic architecture of selection at the human dopamine receptor D4 (DRD4) gene locus. Am J Hum Genet 2004;74(5):931–44. [3] Chien EY, Liu W, Zhao Q, Katritch V, Han GW, Hanson MA, et al. Structure of the human dopamine d3 receptor in complex with a d2/d3 selective antagonist. Science 2010;330(6007):1091–5. [4] Gold SJ, Hoang CV, Potts BW, Porras G, Pioli E, Kim KW, et al. RGS9-2 negatively modulates L-3,4-dihydroxyphenylalanineinduced dyskinesia in experimental Parkinson’s disease. J Neurosci 2007;27(52):14338–48.

[5] Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG, et al. The beta2-adrenergic receptor/beta-arrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci USA 1999;96(7):3712–7. [6] Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, et al. beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem 2006;281(2):1261–73. [7] Urs NM, Daigle TL, Caron MGA. Dopamine D1 Receptordependent beta-arrestin signaling complex potentially regulates morphine-induced psychomotor activation but not reward in mice. Neuropsychopharmacology 2011; 36(3):551–8. [8] Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG. An Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 2005;122(2):261–73. [9] So CH, Verma V, Alijaniaram M, Cheng R, Rashid AJ, O’Dowd BF, et al. Calcium signaling by dopamine D5 receptor and D5-D2 receptor hetero-oligomers occurs by a mechanism distinct from that for dopamine D1-D2 receptor hetero-oligomers. Mol Pharmacol 2009;75(4):843–54. [10] Masri B, Salahpour A, Didriksen M, Ghisi V, Beaulieu JM, Gainetdinov RR, et al. Antagonism of dopamine D2 receptor/betaarrestin 2 interaction is a common property of clinically effective antipsychotics. Proc Natl Acad Sci USA 2008;105(36):13656–61.


C H A P T E R

14 Cholinergic Neurotransmission Brett A. English, Carrie K. Jones

ACETYLCHOLINE BIOSYNTHESIS AND METABOLISM

enzymatic inactivation by the enzyme acetylcholinesterase (AChE), which hydrolyzes ACh into acetate and choline. AChE operates at near diffusion-limited rates, thus this enzyme is not rate limiting in overall ACh homeostasis. The byproduct of ACh hydrolysis, choline, is then recycled by the presynaptic cholinergic terminal in a carrier-mediated mechanism of high-affinity choline uptake (HACU) process, mediated by the high-affinity choline transporter (CHT) and which appears to be the critical step in modulating the rate and extent of ACh production. Within cholinergic nerve terminals, choline is required for the biosynthesis of ACh since these neurons cannot synthesize de novo, hence the requirement for a HACU process. Recent studies in transgenic mice exhibiting a genetic reduction in CHT demonstrated age-related reductions in cardiac function and histologic changes consistent with heart failure.

Acetylcholine (ACh), one of the first identified neurotransmitters, was originally termed vagusstoff due to its actions in supporting vagal slowing of heart rate (HR), and has been the focus of intense research for decades. ACh has been shown to regulate many physiologic functions within the central nervous system (CNS) modulating diverse functions including cognition, attention and arousal. Within the autonomic nervous system (ANS), ACh serves as the principal neurotransmitter mediating fast synaptic neurotransmission at the preganglionic junction by both the sympathetic (SNS) and parasympathetic (PNS) nervous systems. The PNS (via the vagus nerve) uses ACh as its primary postganglionic neurotransmitter modulating a number of physiologic processes including heart rate, gastrointestinal motility, exocrine gland secretions and smooth muscle tone (Tables 14.1 and 14.2). In addition to mediating a number of peripheral autonomic effects, recent experimental evidence has demonstrated a role of the PNS (vagus nerve) in mediating cytokine production in response to inflammation. The availability of ACh for cholinergic transmission involves a highly co-ordinated process of ACh synthesis, vesicular packaging, vesicular release, hydrolysis and reuptake into the presynaptic nerve terminal. Within the cholinergic presynaptic terminal, the enzyme choline acetyltransferase (ChAT) synthesizes ACh from the precursors choline and acetyl coenzyme-A (acetyl-CoA). ChAT is not believed to serve as the rate-limited step in the biosynthesis of ACh as the presynaptic concentrations of choline are much lower than the Km (affinity) for ChAT. Upon synthesis, ACh is packaged into synaptic vesicles by the vesicular ACh transporter (VAChT), and released into the synaptic cleft upon depolarization of the neuron by an action potential mediated by voltage-sensitive Ca2channels. Once released into the synaptic cleft, ACh interacts with either nicotinic acetylcholine receptors (nAChRs) or muscarinic acetylcholine receptors (mAChRs). ACh then rapidly dissociates from its receptor and undergoes


ACETYLCHOLINE RECEPTORS The postsynaptic effects of ACh neurotransmission are mediated by two major types of acetylcholine receptors. At the preganglionic junction, both the SNS and PNS use ACh to activate nicotinic acetylcholine receptors (nAChRs) on the postganglionic neuron. The postganglionic effects of ACh by the PNS occur by activating a number of postsynaptic muscarinic acetylcholine receptors (mAChRs) expressed in several target organs (Table 14.1). The classification of the different acetylcholine receptor subtypes is primarily established based upon binding of specific ligands, their respective effector-coupling systems and their primary amino-acid sequence homology. Within the muscarinic acetylcholine receptor family, there are five receptor subtypes that have been identified, termed M1-M5), each with unique tissue distribution and pharmacologic characteristics (Table 14.2). All mAChRs subtypes are monomeric proteins with seven transmembrane spanning domains and mediate their effects by coupling to G-proteins. M1, M3 and M5 receptors couple to Gq/11 and increase phospholipase C (PLC) activity subsequently increasing cytosolic concentrations of

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72

14. CHolINERgIC NEuRoTRANsmIssIoN

TABLE 14.1 Cholinergic Pharmacology Target

Localization

Effect

Mechanism

Agonist

Antagonist

Nicotinic (N)N

Autonomic ganglia, CNS

Fast depolarization of postganglionic neuron

↑ Na/K channel conductance

Succinylcholine

Trimethaphan

Nicotinic (N)M

Neuromuscular junction (NMJ)

Motor end-plate depolarization, muscle contraction

↑ Na/K channel conductance

Succinylcholine

Tubocurarine

Muscarinic (M1)

CNS, autonomic ganglia

Attention, memory, arousal

Gq/11; ↑ PLC, PKC

Oxotremorine, cevimeline, xanomeline

Scopolamine, pirenzepine

Muscarinic (M3)

Smooth muscle, exocrine glands

Smooth muscle contraction, increased glandular secretions

Gq/11; ↑ PLC, PKC

Cevimeline, pilocarpine

Atropine, solifenacin

Muscarinic (M5)

CNS

?

Gq/11; ↑ PLC, PKC

Sabcomeline

Atropine

Muscarinic (M2)

CNS, heart (SA and AV node)

Reduced HR, AV nodal conduction

Gi; βγ, ↓ AC and ↑ K channel conductance

Carbachol, pilocarpine

Scopolamine, atropine

Muscarinic (M4)

CNS

Cognition

Gi; βγ, ↓ AC and ↑ Kchannel conductance

Xanomeline

Atropine, pirenzepine

Choline transporter (CHT)

Presynaptic; CNS, autonomic, lung

Rate-limiting step for synthesis of ACh

None Na/K-coupled high affinity choline transport

Choline acetyltransferase (ChAT)

Presynaptic terminal

Synthesis of ACh from choline & acetylCoA

AcetylCoA  choline → ACh

None

None

Vesicular acetylcholine transporter (VAChT)

Presynaptic vesicles

Storage of ACh in presynpatic vesicles

H-coupled

None

Vesamicol

Postsynaptic

Hydrolysis of ACh

ACh→choline  acetate

None

Physostigmine, donepezil

RECEPTOR



SYNTHESIS/STORAGE Hemicholinium-3

METABOLISM Acetylcholinesterase (AChE)

inositol-triphosphate (IP3) and diacylglycerol (DAG). Increased IP3 and DAG results in increased release of intracellular Ca2 from endoplasmic reticulum stores and activation of protein kinase C (PKC) respectively. M1 receptors are primarily expressed within the CNS and mediate a number of cognitive functions, while M3 receptors are involved in exocrine gland secretion and smooth muscle function in the PNS. M5 receptors are expressed only within the CNS where they serve to modulate midbrain dopamine release and cerebral vasculature tone. M2 and M4 receptors are coupled to Gi, which results in inhibition of adenylyl cyclase (AC) and activation of inwardly rectifying K-channels (IKACh) and suppression of voltage-gated Ca2-channels. These actions of G-protein coupling to Gi partly explain the cardiovascular effects of M2 activation in the heart via IKACh. Additionally, studies conducted in M2 knockout mice show increased sensitivity to the β-agonist, isoproterenol, developing abnormal ventricular functioning consistent with heart failure. Within the CNS, M2 and M4 receptors provide the major autoreceptor function for control of Ach release. Nicotinic acetylcholine receptors are also divided into nicotinic receptors that mediate neuromuscular junction

activation (NM) or neuronal activation (NN) at the autonomic ganglia and within the CNS. However, unlike muscarinic receptors, nAChRs are ligand-gated ion channels. Upon binding of Ach, these receptors undergo conformational changes that permit the rapid influx of Na with subsequent cellular depolarization. These distinctions are important pharmacologically as nicotinic receptors are an assembly of five heterologous subunits (α1–9 and β1–4) arranged symmetrically around a central ionic selectivity pore, each with different subunit arrangement and pharmacologic binding properties (nicotinic neurotransmission is discussed in detail in Chapter 15). While nAChRs do not directly affect peripheral organ systems, they mediate ganglionic transmission and can facilitate neurotransmission of postganglionic nerve fibers.

CHOLINERGIC PHARMACOLOGIC AGENTS Cholinomimetic drugs can be classified into three categories. Direct-acting cholinomimetics, indirect-acting agents and allosteric modulating compounds. Direct-acting agents bind to and activate either nAChRs or mAChRs; for


CHolINERgIC PHARmACologIC AgENTs

TABLE 14.2 Responses of Effectors organs to Cholinergic Transmission of the Autonomic Nervous system Effector Organ

Receptor

Effect

Sinoatrial node

M2

Bradycardia

Atrium

M2, M3

↓ lusitropy

Atrioventricular node

M2

↓ conduction velocity

Arteriole

M2

Vasodilation (via release of nitric oxide, NO)

HEART

GASTROINTESTINAL/UROLOGIC GI Motility

M2, M3

Increased

GI secretion

M3

↑ secretions

Gallbladder

M2

Contraction

Urinary detrusor

M2, M3

Contraction

Urinary sphincter

M2

Relaxation

Penis

M

Erection

M3, M1

Constriction; ↑ secretions

Sweat glands

M3

Diaphoresis

Salivary glands

M3

↑ secretions

Lacrimal glands

M3

↑ secretions

Nasopharyngeal glands

M3

↑ secretions

Iris (pupillae sphincter)

M3

Contraction (miosis)

Ciliary muscle

M3

Contraction (accommodation)

RESPIRATORY Bronchial muscle GLANDULAR

OCULAR

73

(iii) phosphoric acid organic derivatives (e.g. organophosphates). Organophosphates are considered “irreversible” inhibitors, while the other chemical classes of AChEIs are “reversible” acting. Recently, several novel cholinomimetic compounds have been identified that function as positive allosteric modulators (PAMs) or allosteric agonists instead of traditional orthosteric agonists at different mAChRs. Allosteric modulators and agonists of mAChRs do not bind to the orthosteric binding site of Ach but rather interact with topographically distinct binding sites on the receptor that are separate from the orthosteric sites. Because these allosteric modulators and agonists interact at less highly conserved regions of the mAChR, these compounds offer a tremendous advantage in conferring a high degree of subtype selectivity. In addition, since PAMs potentiate the effects of Ach and have no effect in the absence of the endogenous ligand, the physiological signal and transduction is preserved resulting in less receptor desensitization and development of tolerance. A number of mAChR selective PAMs, including the selective M1 PAM BQCA, are being investigated in several preclinical models of cognitive disorders associated with schizophrenia and Alzheimer’s disease. Similarly, cholinergic receptor antagonists are categorized based upon their acetylcholine receptor selectivity. There are few nAChR antagonists used clinically and these consist primarily of the non-depolarizing ganglionic blockers (e.g. hexamethonium) and the depolarizing blocker (e.g. succinylcholine). The prototypical muscarinic antagonist, scopolamine is a non-selective antagonist at all five mAChR subtypes resulting in a reversible blockade that is surmountable by increasing concentrations of ACh. Other muscarinic antagonists display relative selectivity for specific mAChR subtypes, but exhibit dose-dependent effects (e.g. pirenzepine, M1; oxybutynin, M3). Since muscarinic receptors are constitutively active, muscarinic antagonists shift the receptor to an inactive state preventing actions of IP3/DAG and AC.

Further Reading example chemical esters of choline such as carbachol or alkaloid compounds, including pilocarpine. Many of these compounds exhibit preferential selectivity for either nicotinic or muscarinic receptors. However, in the case of the mAChRs, subtype selective compounds within the mAChRs have been difficult to identify and develop due to the high conservation of the orthosteric binding site of ACh across M1–M5. Lack of subtype selectivity of orthosteric compound, especially for the mAChRs, has resulted in a number of adverse side effects due to activation of peripheral mAChRs. Indirect-acting agents increase synaptic concentrations of ACh by inhibiting the hydrolytic metabolism by AChE. While these agents all work by inhibition of AChE, they exhibit significant differences in their chemical properties and pharmacokinetics. There are three chemical groups of AChE inhibitors: (i) quaternary ammonium alcohols (e.g. edrophonium); (ii) carbamic acid esters (e.g. neostigmine);

Dhein S, Van Koppen CJ, Brodde OE. Muscarinic receptors in the mammalian heart. Pharmacol Res 2001;44(3):161–82. Digby GJ, Shirey JK, Conn PJ. Allosteric activators of muscarinic receptors for treatment of CNS disorders. Mol Biosyst 2010;6(8):1345–54. English BA, Appalsamy M, Diedrich A, Ruggiero AM, Lund D, Wright J, Keller NR, Louderback KM, Robertson D, Blakely RD. Tachycardia, reduced vagal capacity and age-dependent ventricular dysfunction arising from diminished expression of the presynaptic choline transporter. Am J Physiol Heart Circ Physiol 2010;299(3):H799–810. Ferguson SM, Blakely RD. The choline transporter resurfaces: new roles for synaptic vesicles. Mol Interv 2004;4:22–7. LaCroix C, Freeling J, Giles A, Wess J, Li YF. Deficiency of M2 muscarinic acetylcholine receptors increases susceptibility of ventricular function to chronic adrenergic stress. Am J Physiol Heart Circ Physiol 2008;294:H810–820. Langmead CJ, Watson J, Reavill C. Muscarinic acetylcholine receptors as CNS drug targets. Pharmacol Therap 2008;117:232–43. Olshanksy B, Sabbah HN, Hauptman PJ, Colucci WS. Parasympathetic nervous system and heart failure: pathophysiology and potential implications for therapy. Circulation 2008;118(8):863–71.


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Rosas-Ballina M, Tracey KJ. Cholinergic control of inflammation. J Intern Med 2009;265:663–79. Shirey JK, Brady AE, Jones PJ, Davis AA, Bridges TM, et al. A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning. J Neurosci 2009;29(45):14271–14286.

Westfall TC, Westfall DP. Neurotransmission: the autonomic and somatic motor nervous system. In: Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman’s the pharmacological basis of therapeutics. New York: McGraw-Hill; 2006. p. 137–83.


C H A P T E R

15 Acetylcholine and Muscarinic Receptors Joan Heller Brown, Nora Laiken The synthesis of acetylcholine (ACh) is catalyzed by choline acetyltransferase, a soluble cytoplasmic enzyme that transfers an acetyl group from acetyl coenzyme A to choline. The activity of choline acetyltransferase is much greater than the maximal rate at which ACh synthesis occurs, and inhibitors of this enzyme have little effect on ACh levels in cholinergic nerve terminals. The rate-limiting step in ACh synthesis is the transport of choline into the nerve terminal by the high-affinity choline transporter (CHT1), the activity of which is regulated in response to neuronal activity. Following its synthesis, ACh is transported into synaptic vesicles by the vesicular ACh transporter (VAChT). The depolarization of the cholinergic nerve terminal by an action potential activates voltage-gated calcium channels; the resulting calcium influx initiates the release of ACh into the junctional space by exocytosis. ACh release can be blocked by botulinum toxin, the etiologic agent in botulism. Botulinum toxin has been used to treat a variety of movement disorders and other conditions, both cosmetic (e.g., glabellar frown lines [facial wrinkles]) and pathological (e.g., strabismus [crossed eyes]); injected locally, the toxin can inhibit ACh release for as long as 3 to 4 months. The ACh released into the synapse associates with postsynaptic cholinergic receptors, triggering various physiological responses. The actions of ACh are terminated by its rapid hydrolysis into choline and acetic acid by acetylcholinesterase (AChE), which is found in the junctional space at all cholinergic junctions; thus, responses mediated by ACh tend to be transient and localized. The choline liberated by AChE can be taken back up into the nerve terminal by CHT1 and resynthesized into ACh. While not important for the hydrolysis of endogenous ACh, a nonspecific cholinesterase (pseudocholinesterase or butyrylcholinesterase) is present in plasma and some organs and important for metabolism of some drugs. Drugs that normally are hydrolyzed by pseudocholinesterase, such as succinylcholine (a neuromuscular blocking agent used in anesthesiology to produce skeletal muscle paralysis), are poorly metabolized by this variant enzyme; succinylcholine can produce a prolonged paralysis in affected patients. Cholinesterases are discussed in detail in Chapter 132 of this Primer. Cholinergic neurotransmission and drugs that affect it are further discussed in Chapter 14 and summarized in Table 15.1.


TABLE 15.1 Cholinergic Neurotransmission: Drug Mechanisms Cholinergic neurotransmission can be modified at several sites: (a)

Precursor transport blockade

Hemicholinium (blocks CHT1)

(b)

Choline acetyltransferase inhibition

No clinical example

(c)

Promote transmitter release

Black widow spider venom (latrotoxin)

(d)

Prevent transmitter release

Botulinum toxin

(e)

Storage

Vesamicol (blocks VAChT)

(f)

Cholinesterase inhibition

Physostigmine, neostigmine

(g)

Receptors

mAChR and nAChR agonists/antagonists

CHOLINERGIC NEUROTRANSMISSION: SITES AND RECEPTORS Cholinergic transmission occurs at five important locations: (i) all effector sites innervated by parasympathetic postganglionic neurons; (ii) a small number of effector sites innervated by sympathetic postganglionic neurons (most importantly, most sweat glands); (iii) all autonomic ganglia (including the adrenal medulla), innervated by parasympathetic and sympathetic preganglionic neurons; (iv) all motor end plates on skeletal muscle, innervated by somatic motor neurons; and (v) certain synapses in the CNS. As might be expected for an ancient and ubiquitous neurotransmitter, a variety of ACh (cholinergic) receptor types have emerged in evolution. Based on their responsiveness to the agonists muscarine and nicotine, cholinergic receptors can be divided into two groups, nicotinic cholinergic receptors (nAChRs) and muscarinic cholinergic receptors (mAChRs). nAChRs are found at the following locations: l l l

Autonomic ganglia (including the adrenal medulla). Neuromuscular junction of skeletal muscle. Central nervous system.

There are two nAChR subtypes, NN (neural nicotinic) and NM (muscle nicotinic). NN receptors mediate neurotransmission in autonomic ganglia, while NM receptors

75


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15. ACETylCHolINE AND MusCARINIC RECEPToRs

TABLE 15.2 Muscarinic Receptor-Mediated Responses at Autonomic Effector sites Iris sphincter muscle

Contraction (miosis)

Ciliary muscle

Contraction (near vision)

Sinoatrial node

Bradycardia

Atrium

Decreased contractility

Atrioventricular node

Decreased conduction velocity

Arteriole

Dilation (through nitric oxide)

Bronchial smooth muscle

Contraction

Gastrointestinal motility

Increased

Gastrointestinal secretion

Increased

Gallbladder

Contraction

Bladder (detrusor)

Contraction

Bladder (trigone, sphincter)

Relaxation

Penis

Erection (but not ejaculation)

Sweat glands

Secretion

Salivary glands

Secretion

Lacrimal glands

Secretion

Nasopharyngeal glands

Secretion

mediate skeletal muscle contraction; thus, NN and NM antagonists are called ganglionic blockers and neuromuscular blockers, respectively. nAChRs in the CNS resemble NN receptors and are primarily presynaptic. nAChRs and the responses that they mediate are discussed in Chapter 16 of this Primer. mAChRs are found at the following locations: l

l

l

l

l

Effector sites innervated by parasympathetic postganglionic neurons. Effector sites innervated by sympathetic cholinergic postganglionic neurons (most sweat glands). Presynaptic sites on noradrenergic and cholinergic nerve terminals. Non-innervated sites in blood vessels (vascular endothelium, smooth muscle). Central nervous system.

Because of their physiological and therapeutic significance, it is important to review the specific responses mediated by mAChRs at autonomic effector sites. All of these sites are innervated by parasympathetic postganglionic neurons except for the sweat glands, which are innervated by sympathetic cholinergic postganglionic neurons (this innervation is sympathetic because of the thoracolumbar origin of the preganglionic neurons and is cholinergic because the postganglionic neurons release ACh). See Table 15.2. There are five mAChR subtypes, designated M1–M5. All mAChRs are G protein-coupled receptors. Stimulation of M1, M3, and M5 receptors generally results in the hydrolysis of phosphoinositides by activating the Gq-phospholipase C

pathway, resulting in mobilization of intracellular calcium; stimulation M2 and M4 receptors generally leads to the inhibition of adenylyl cyclase and the activation of potassium channels by activating Gi and Go. The five mAChR subtypes are widely distributed in both peripheral tissues and the CNS, with most cells expressing at least two subtypes. Due to the paucity of agonists and antagonists that are truly subtype-specific, identifying which subtype(s) mediate a particular mAChR response has been difficult. Studies in knockout mice have helped to elucidate the functions of specific mAChR subtypes. For example, M1 receptors modulate neurotransmitter signaling in the cortex and hippocampus and also have an important role in modulating cholinergic transmission in autonomic ganglia; M2 receptors are the predominant subtype mediating the parasympathetic control of the heart and also mediate mAChR agonist-induced tremor, hypothermia, and presynaptic inhibition of neurotransmitter release; M3 receptors are the predominant subtype mediating the parasympathetic control of smooth muscle contraction and glandular secretion, the sympathetic cholinergic control of sweating, and mAChR agonist-induced increases in food intake and body weight; M4 receptors modulate dopaminergic activity in motor tracts and also mediate the presynaptic inhibition of neurotransmitter release; and M5 receptors modulate central dopaminergic function and the tone of cerebral blood vessels.

MUSCARINIC AGONISTS ACh itself is rarely used as a mAChR agonist because of its rapid hydrolysis after oral or intravenous administration, but several choline esters that are resistant to hydrolysis (methacholine, carbachol, and bethanechol) are available for clinical use. Bethanechol has the additional favorable property of an overwhelmingly high mAChR (vs. nAChR) specificity. In addition, several natural alkaloids are mAChR agonists, including muscarine, pilocarpine, and arecoline; pilocarpine and arecoline (and their synthetic congeners) are used clinically. mAChR agonists are used in the treatment of urinary tract motility disorders and xerostomia (dry mouth due to decreased salivary secretion) and in the diagnosis of bronchial hyperreactivity; they also are used in ophthalmology to produce miosis and to treat glaucoma. Bethanechol, which primarily affects the urinary and GI tracts, is used to facilitate urination in patients with postoperative urinary retention, diabetic autonomic neuropathy, or neurogenic bladder (it formerly was used to increase GI motility in patients with postoperative abdominal distension, gastroparesis, and other motility impairments; more efficacious therapies are now available for these disorders). Pilocarpine is used to stimulate salivary gland secretion in patients with xerostomia resulting from damage to the salivary glands (e.g., due to head and neck radiation treatments) or Sjögren’s syndrome, an autoimmune disorder characterized by decreased secretions


MusCARINIC ANTAgoNIsTs

(particularly salivary and lacrimal); antibodies to mAChRs of the M3 subtype have been reported in some patients. Methacholine, administered by inhalation, is used in the diagnosis of bronchial airway hyperreactivity; elicitation of significant bronchoconstriction with inhaled methacholine challenge sometimes leads to the diagnosis of asthma in patients with little baseline abnormality in pulmonary function. Pilocarpine and carbachol are used topically in ophthalmology as miotic agents and to treat glaucoma. Most side effects of mAChR agonists are predictable and include increased salivation, increased sweating, worsening of asthma, diarrhea, nausea, hypotension, and bradycardia; occasionally mAChR agonists cause hiccups. In rare cases, high doses of bethanechol may cause myocardial ischemia in patients with a predisposition to coronary artery spasm; therefore, chest pain in a patient taking bethanechol should be taken seriously.

MUSCARINIC ANTAGONISTS The classical mAChR antagonists are alkaloids derived from plants in the Solanaceae family, including the deadly nightshade (Atropa belladonna), jimson weed (Datura stramonium), and henbane (Hyoscyamus niger). Atropine, scopolamine, and hyoscine are notable examples of the plant-derived mAChR antagonists; these compounds often are termed belladonna alkaloids. The name belladonna (Italian for “beautiful lady”) originates from the historic use of drops prepared from such plants by women who wished to dilate their pupils for beauty. mAChR antagonists cause pupillary dilation (mydriasis) by blocking mAChRs on the iris sphincter muscle; such compounds currently are used in ophthalmology as mydriatics. The hallucinogenic effects of the belladonna alkaloids have been known for centuries; for example, the tiny dark seeds from jimson weed pods were used in sacred ceremonies by aboriginal Americans. In addition to the belladonna alkaloids atropine and scopolamine, the mAChR antagonists used clinically include (i) semisynthetic derivatives of these alkaloids, which differ from their parent compounds primarily in their distribution in the body and/or duration of action; and (ii) synthetic derivatives of the belladonna alkaloids, some of which exhibit some selectivity for specific mAChR subtypes. Notable drugs in these two categories are homatropine and tropicamide, which have a shorter duration of action than atropine; ipratropium, tiotropium, and methscopolamine, which are quaternary amines and, therefore, do not cross the blood–brain barrier or readily cross membranes and epithelial cell barriers; and agents with some mAChR subtype selectivity, such as pirenzepine (selective for M1 receptors), darifenacin (selective for M3 receptors), and solifenacin (selective for M3 receptors). mAChR antagonists are used to inhibit parasympathetic stimulation of the urinary tract, respiratory tract, GI tract, heart, and eye; because of their CNS effects, they also are used to treat Parkinson’s disease, manage extrapyramidal

77

(motor) symptoms caused by antipsychotic drugs, and prevent motion sickness. Oxybutynin and tolterodine are examples of drugs used to treat overactive bladder; these agents also can be used to reduce urinary frequency in patients with spastic paraplegia and to treat enuresis in children. Ipratropium and tiotropium, administered by inhalation, are important drugs in the treatment of chronic obstructive pulmonary disease. Ipratropium, administered via a nasal inhaler, is used for the treatment of rhinorrhea associated with the common cold or perennial rhinitis. Pirenzepine (not available in the US) is used for the treatment of acid-peptic disease; however, H2 antihistamines and proton pump inhibitors are more efficacious and more widely used for this purpose. Atropine, hyoscyamine, and other mAChR antagonists are used to treat irritable bowel syndrome and other conditions in which GI motility is increased, although their efficacy is limited in most patients. Atropine also is used to increase heart rate and/ or atrioventricular conduction during situations in which parasympathetic stimulation of the heart is enhanced (e.g., acute myocardial infarction involving the inferior or posterior wall of the left ventricle). Homatropine, tropicamide, and other mAChR antagonists are used in ophthalmology to dilate the pupils and paralyze the accommodation reflex. Benztropine and trihexyphenidyl are used in Parkinson’s disease, especially for the treatment of tremor; these agents also can be used to treat extrapyramidal side effects (such as dystonia and parkinsonian symptoms) caused by antipsychotic drugs. Scopolamine is widely used in a transdermal preparation to prevent motion sickness. Most side effects of mAChR antagonists are predictable and include constipation, xerostomia, anhidrosis (decreased sweating), urinary retention, precipitation of attacks of angle-closure glaucoma in susceptible patients, tachycardia, decreased lacrimation, and decreased respiratory secretions.

Further Reading Abrams P, Andersson K-E, Buccafusco J, et al. Muscarinic receptors: their distribution and function in body systems, and the implications for treating overactive bladder. Br J Pharmacol 2006;148:565–78. Barnes PJ, Hansel TT. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 2004;364:985–96. Brown JH, Laiken N. Muscarinic receptor agonists and antagonists. In: Brunton LL, editor. Goodman and Gilman’s the pharmacological basis of therapeutics. New York: McGraw-Hill; 2011. p. 219–37. Bymaster FP, McKinzie DL, Felder CC, Wess J. Use of Ml-M5 muscarinic receptor knockout mice as novel tools to delineate the physiological roles of the muscarinic cholinergic system. Neurochem Res 2003;28:437–42. Caulfield MP, Birdsall NJ. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 1998;50:279–90. Conn PJ, Christopoulos A, Lindsley CW. Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nature Rev Drug Discov 2009;8:41–54. Conn PJ, Jones CK, Lindsley CW. Subtype-selective allosteric modulators of muscarinic receptors for the treatment of CNS disorders. Trends in Pharmacol Sci 2009;30:148–55. Ferguson SM, Blakely RD. The choline transporter resurfaces; new roles for synaptic vesicles? Mol Interv 2004;4:22–37.


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Higgins CB, Vatner SF, Braunwald E. Parasympathetic control of the heart. Pharmacol Rev 1973;25:119–55. Taylor P, Brown JH. Acetylcholine. In: Siegel GJ, Albers RW, Brady ST, Price DL, editors. Basic neurochemistry: molecular, cellular, and medical aspects. Burlington, Massachusetts: Elsevier Academic Press; 2006. p. 185–209. Waterman SA, Gordon TP, Rischmueller M. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjögren’s syndrome. Arthritis Rheum 2000;43:1647–54.

Wellstein A, Pitschner JF. Complex dose-response curves of atropine in man explained by different functions of M1- and M2-cholinoceptors. Naunyn Schmiedebergs Arch Pharmacol 1988;338:19–27. Wess J, Eglen RM, Gautam D. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nature Rev Drug Discov 2007;6:721–33.


C H A P T E R

16 Nicotinic Receptors Palmer Taylor Nicotinic acetylcholine receptors are members of a large pentameric super family of ligand-gated ion channel receptors that include the 5-hydoxytryptamine3 (5-HT3), glycine, γ-aminobutyric acid (GABA), and Zn-activated families of receptors along with several invertebrate and prokaryotic receptors [1–4]. Less closely related are the ligand-gated ion channels that respond to the excitatory amino acids and adenosine. Owing to the abundance of nicotinic receptors in the electric fish and findings showing that several peptide toxins that block motor activity bind to subtypes of nicotinic receptor with high affinity and selectivity, the nicotinic receptor was the first pharmacologic receptor to be purified and the cDNAs encoding its subunits cloned. Appropriately, the nicotinic receptor became the prototype for the ligand-gated ion channel family.

conformational states that lead to activation and desensitization [1–5]. More recently, a soluble protein, termed the acetylcholine binding protein, exported from glial cells of snails, has been shown to bind acetylcholine and many of the classical nicotinic agonists and antagonists [6]. Homologous proteins have been found in other invertebrate species as well. This protein, with identical subunits of slightly over 200 amino acids, is composed of residues with homology to the amino-terminal, extracellular domain of family of nicotinic receptors. Its structural characterization by X-ray crystallography (Fig. 16.3) shows it to be pentameric and to contain an arrangement of amino acid residues consistent with findings of protein modification and site-specific mutagenesis studies conducted with acetylcholine receptors isolated from neuronal and muscle systems [5,6]. The binding protein protein is homomeric, and its five binding sites reside at the five subunit interfaces with binding site determinants coming from both of the proximal subunit surfaces. The ligands bind from an outer radial direction [6,7] and enlodge behind a loop that contains selective binding determinants and proximal cysteines at or near its tip (Fig. 16.3). Hence, consistent with other proteins that exhibit homotropic cooperativity, the binding protein and the nicotinic receptors have their binding sites located at the subunit interfaces.

STRUCTURAL CONSIDERATIONS Nicotinic receptors assemble as pentamers of individual subunits. Assembly occurs in a precise manner and order such that the assembled subunits encircle an internal membrane pore and the extracellular vestibule leading to the pore. The replicated pattern of front to back assembly of subunits insures that identical subunit interfaces are formed from the pentameric assembly of identical subunits. Moreover, the amino acid residues found at homologous positions in the receptors with heteromeric subunit assemblies should occupy the same position in three dimensional space (Fig. 16.1A). Each subunit encodes a protein with four transmembrane spans. The amino-terminal ~210 amino acids lie in the extracellular domain followed by three tightly threaded transmembrane spans. A relatively large cytoplasmic loop is found between transmembrane spans 3 and 4 with a short carboxyl terminus winding up on the extracellular side. The overall structure has been detailed in a series of electron microscopy imaging studies (Fig. 16.2) and reveals a large extracellular domain, a wide diameter vestibule on the extracellular side and the gorge constriction region controlling the gating function to exist within the transmembrane spanning region [3]. Rapid and slow binding of ligands appear to induce shape changes within the receptor structure reflective of the individual


SUBTYPE DIVERSITY OF NICOTINIC RECEPTORS The receptor from skeletal muscle and its homologous forms in the fish electric organ consist of four distinct subunits, where the pentamer has two copies of the α subunit and one copy of β, γ and δ subunits. When muscle becomes innervated, the γ subunit is replaced by an  subunit. Within this pentameric structure, the two acetylcholine binding sites exist at the αδ and αγ() subunit interfaces. Opposing faces of the homologous α and γ, δ or  subunits make up the two binding sites. The subunit compositions of the receptors expressed in the nervous system are far more complex where we find nine different α subtypes and four different β subtypes [8] (Table 16.1). The α subtypes have been defined as those containing vicinal cysteines on a loop on which

79


80

16. NICoTINIC RECEPToRs

TABLE 16.1 subtypes of Nicotinic Acetylcholine Receptors found in Muscle and Neuronal systems Composition α1, β1, γ, δ,  α6 α7 α9

β2 β3 β4

α2βγδ or α2βδ Various pentameric assemblies of α and β or α alone

α1 (A)

β2

β2

δ

α4

αβγδAChR

β2

α

α αAChR

αβNeuronal AChR

α10

α

α

H2N

ELECTROPHYSIOLOGIC EVENTS ASSOCIATED WITH RECEPTOR ACTIVATION All of the nicotinic receptors that are well characterized, to date, are cationic channels resulting in depolarization after activation. They differ substantially with respect to the permeabilities of the open channel state to Na and Ca. Na permeability is most effective in initiating a rapid depolarization, whereas Ca permeability could

2

COOH

1

reside several binding site determinants. All α subunits, except α5, use the face with the vicinal cysteines to form the binding site. The β subunits which lack this sequence and presumably the α5 subunit use the opposite or complementary face to form the binding site (Fig. 16.1). The α subunits, α2, α3, α4 and α6, combine with certain β subunits, mainly β2 and β4, to form pentameric receptors. α5 has the capacity to substitute for a β subunit in the pentameric assembly. Typically, it is thought that two of the neuronal α subunits assemble with three β subunits. Hence, typical stoichiometries (subscripted) would be α2β3 or α2α51β3 where the generic α subtypes are usually α3 or α4 and the β subtypes β2 or β4. Each of these heteromeric receptors would have two binding sites. Receptors composed of α7 subunits appear as functional homomeric entities where five copies of a single subunit confer function, when assembled. α9 and α10 subunits assemble uniquely in a pentamer and may control nociceptive responses. While the assembly patterns of the neuronal nicotinic receptor subunits are diverse, only certain combinations yield functional receptors, and particular combinations seem to be prevalent within regional areas of the nervous system. For example, pentamers of α3β4 are prevalent in ganglia of the autonomic nervous system, α4β2 is most prevalent in the central nervous system, and α7 appears widely distributed [8]. Central and peripheral nicotinic receptors play a discrete role in nicotine addiction and cardiovascular actions [9]. Mutations in nicotinic receptors give rise to congenital myasthenic syndromes [10] and other manifestations that affect autonomic and central nervous system function.

4

α2 α3 α4 α5

β1

γ

3

Muscle Neuronal

Assembly

α

α4

α1

(B)

FIGURE 16.1 Structure of the nicotinic acetylcholine receptor. (A) Arrangement of receptor subunits as pentamers in muscle and neuronal receptors. The muscle receptor exists as a pentamer with two copies of α1 and one of β, γ and δ, as seen for the receptor found in embryonic muscle. The binding sites as designated are found at the αγ and αδ interfaces. In the case of the innervated receptor in skeletal muscle, an  subunit of different composition replaces γ at the same position. Two types of neuronal receptors are shown: the heteromeric type is thought to have usually two copies of α, where α is α2,α3,α4, or α6, and three copies of β, where β can be β2, β3 or β4. α5 is thought to substitute for a β subunit at one of the non-binding positions. The homomeric neuronal receptors are made up of α7 pentamers. α7 subunits may form heteromeric subtypes with certain β subunits remains open, but not proven. α9 and α10 subunits associate with each other in presumed variable ratios to form pentamers. (B) Threading pattern of the α-carbon chain of the subunits of the nicotinic receptor. The first ~210 amino acids form virtually all of the extracellular domain, contain the residues on the primary and complementary subunit interfaces forming the binding site determinants and govern the assembly process. Transmembrane span 2 from the five subunits forms the inner perimeter of the channel and is involved in the ligand gating. The other transmembrane segments contribute to the structural integrity of the receptor. The extended segment between transmembrane spans 3 and 4 forms the bulk of the cytoplasmic domain.


81

DIsTRIbuTIoN of NICoTINIC RECEPToRs

(A)

(B)

synapse

synaptic face

M

cytoplasm

N

S

I

S

S

S

L

L

T

G P

F

V

T

L

V

L

L

L

E

V

I

C

S

P

I

cytoplasmic face

(C)

M2

E T

V L K D

FIGURE 16.2 Overall dimensional characteristics of the nicotinic receptor. Image reconstruction of electron micrographs shows the receptor to be some ~140 Angstroms in length and perpendicular to the membrane. Its diameter is 80–90 Angstroms and contains a large central channel on the membrane surface. Rapid and prolonged exposure to acetylcholine followed by rapid freezing has yielded distinctive structures for the agonist bound, unligated and desensitized states of the receptor [3,6].

serve as an intrinsic activating function in cell signaling or in triggering an excitation step. However, depolarization per se through an increase in Na permeability can also mobilize Ca through voltage sensitive Ca channels. Typically postsynaptic nicotinic receptors, such as those found in ganglia and the neuromuscular junction, function through the simultaneous occupation of the receptor by more than a single agonist molecule. Agonist association and the ensuing channel opening reveals positive cooperativity in ligand binding. These rapid binding events cause channel openings to occur in a millisecond time frame. Several openings and closings may occur during the short interval of agonist occupation, and the intrinsic efficacy of an agonist may relate to whether the ligand-receptor complex favors an open or closed state. Upon continuous exposure to agonists, most receptors desensitize. Desensitization confers a receptor state wherein the agonist has a high affinity, but the receptor is locked in a closed channel state. Desensitization provides an additional means by which temporal responses to an agonist can be regulated. Antagonists may be competitive or non-competitive. Competitive antagonists show binding that is mutually exclusive with agonists and exhibit a surmountable block of the receptor in which the dose–response curve to the agonist is shift rightward in a parallel fashion. Occupation by a single antagonist molecule is sufficient to block function. Non-competitive antagonists typically block the channel through which ions pass in the open state of the receptor. In the case of ganglionic nicotinic receptors,

trimethaphan is a competitive agonist, while hexamethonium and mecamylamine are non-competitive, therein blocking channel function. Great interest has developed in allosteric sites where binding of a ligand at a non-agonist site, enhances or diminishes agonist function and are termed allosteric activators or inhibitors. Hence, they are distinguished from orthosteric ligands that occupy the primary agonist or antagonist sites.

DISTRIBUTION OF NICOTINIC RECEPTORS Nicotinic receptors are widely distributed in the central and peripheral nervous systems. In innervated skeletal muscle they are found in very high density localized to the motor end plate. Also, certain motor neurons may contain receptors at the presynaptic nerve ending to control release. In ganglia, the primary nicotinic receptor is found on the postsynaptic dendrite and nerve cell body. Others may exist presynaptically to control release from the presynaptic nerve ending. In the CNS, one finds that the majority of receptors are presynaptic or prejunctional. As such, they control the release of other transmitters or in the case of released acetylcholine, they play an autoreceptor role. Presynaptic nicotinic receptors found in the spinal cord and in higher centers of the brain have a functional role in modulating central control of autonomic function and sensory to autonomic control of reflexes and other functions.


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16. NICoTINIC RECEPToRs

(A)

(B)

FIGURE 16.3 X-ray crystallographic structure of the acetylcholine binding protein. The structure was produced from crystallographic coordinates of Sixma and colleagues [5]. Surfaces are represented as Connolly surfaces. Left, colors are used to delineate the subunit interfaces in the homomeric pentamer. The vestibule on the extracellular surface that represents the entry to the internal channel in the receptor is shown by the arrow. Right, a single subunit interface Note the residues that have been found to be determinants in the binding of agonists and alkaloid and peptidic antagonists to the receptor. Hence, the numbers delineate the probable binding surface(s) for large and small antagonists. These residues come from seven distinct segments of amino acid sequence in the subunit, as determined from extensive mutagenesis, labeling and crystallographic studies [3,5,6]. The right hand panel shows a superimposition of the acetylcholine binding protein with the presumed structure of the muscle receptor.

References [1] Changeux J-P, Edelstein SJ. Nicotinic acetylcholine receptors. New York: Odie Jacob; 2005. [2] Karlin A. Emerging structures of nicotinic acetylcholine receptors. Nat Rev Neurosci 2002;3:102–14. [3] Changeux J-P. Allosteric receptors: From electric organ to cognition. Annu Rev Pharmacol Toxicol 2010;50:1–38. [4] Thompson AJ, Lester HA, Lummis SCR. The structural basis of function in Cys-loop receptors. Q Rev Biophys 2010;43:449–99. [5] Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 2005;346:967–89. [6] Brejc K, van Dijk WJ, Klaasen RV, Shuurmans M, van der Oost J, Smit AB, et al. Crystal structure of an acetylcholine binding protein

[7]

[8]

[9] [10]

reveals the ligand binding domain of nicotinic receptors. Nature (London) 2001;411:269–76. Hibbs RE, Sulzenbacher G, Shi J, Talley T, Conrod S, Kem WR, et al. Structural determinants for interaction of partial agonist with the acetylcholine binding protein and the neuronal alpha7 acetylcholine receptor. EMBO J 2009;28:3040–51. Various authors. Neuronal nicotinic receptors. In: Clementi F, Fornasi D, Gotti C, editors. Handbook of experimental pharmacology, vol. 144. Berlin: Springer-Verlag, 2000. 821 pp. Benowitz NL. Nicotine Addiction. N Eng J Med 2010;362:2295–303. Engel AG, Shen X-M, Selcen D, Sine SM. What we have learned from congenital myasthenic syndromes. J Mol Neurosci 2010;40:143–53.


C H A P T E R

17 Serotonin Receptors and Neurotransmission Elaine Sanders-Bush, Charles D. Nichols Tryptophan hydoxylase, the rate limiting enzyme, is not saturated under normal conditions, rendering 5-HT levels sensitive to changes in blood tryptophan. This translates into the remarkable finding that brain levels of this neurotransmitter can be regulated by the dietary intake of tryptophan. Clinical studies have often utilized a tryptophan-free diet to lower brain 5-HT and, by inference, evaluate its role in a particular behavior or drug effect. The principal metabolism of 5-HT is mediated by the ubiquitous enzyme, monoamine oxidase (MAO), to form an inactive product, 5-hydroxyindole acetic acid (5-HIAA). 5-HIAA is subsequently secreted in the CSF and urine. MAO is a family of mitochondrial enzymes that metabolize all biogenic amines.

Serotonin (5–hydroxytryptamine) is a neurotransmitter as well as a circulating hormone. Serotoninergic neurons in the brain synthesize and store serotonin at axon terminals where it is released and interacts with cell-surface receptors on adjacent neurons. The action of serotonin is terminated by re-uptake into presynaptic terminal mediated by the serotonin transporter or by metabolism by monoamine oxidase. Of the 14 different receptor subtypes, most generate intracellular messengers by coupling to G-proteins and modulate, rather than mediate, fast neurotransmission. This multitude of receptors explains the variety of actions of serotonin in normal and abnormal states and provides ample opportunity for drug development for treatment of nervous system diseases.

NEUROTRANSMISSION

LOCALIZATION

In the central nervous system, the entire pathway of synthesis and metabolism exists at axon terminals. The life cycle of 5-HT at the synapse is illustrated in Figure 17.3. Both synthetic enzymes are present in the presynaptic terminal; MAO is also highly expressed in adjacent cells. Newly synthesized 5-HT is accumulated in synaptic vesicles to protect it from degradation by MAO. Uptake into vesicles is mediated by the vesicular monoamine transporter (VMAT). Stored 5-HT is released from synaptic vesicles by a complex series of phosphorylation-dependent protein-protein interactions initiated by the influx of calcium. Once released, 5-HT is inactivated by MAO in the synaptic cleft or its action is terminated by re-uptake into the presynaptic terminal by the 5-HT transporter (SERT). Once in the presynaptic terminal, 5-HT is either accumulated in synaptic vesicles via VMAT or metabolized by MAO. The two transporters in 5-HT nerve terminals, VMAT and SERT, belong to different gene families and have markedly different properties. SERT is a sodium-dependent carrier that translocates 5-HT into the presynaptic nerve terminal. VMAT is driven by a proton gradient; it is promiscuous, present in 5-HT and catecholamine vesicles. SERT is expressed exclusively in serotoninergic neurons

Serotonin, also referred to as 5-hydroxytrytamine (5-HT), is a simple indoleamine (Fig. 17.1), discovered over five decades ago. Since then, 5-HT has been shown to function as a neurotransmitter in central nervous system and also as circulating hormone [1]. The principal source of circulating 5-HT is the intestinal enterochromaffin cells, where 5-HT is synthesized, stored and released into the bloodstream. 5-HT in blood is concentrated in platelets by an active transport mechanism. In the pineal gland, 5-HT is converted by a two-step process to melatonin (5-methoxy-N-acetyltryptamine), a hormone that regulates ovarian function and has been implicated in the control of biological rhythms. The brain 5-HT containing neurons are localized in raphe nuclei of the brainstem, which project diffusely throughout the brain and spinal cord.

SYNTHESIS AND METABOLISM The pathway of synthesis is common throughout the body. 5-HT is synthesized from tryptophan, an essential amino acid obtained in the diet. 5-HT synthesis requires the action of two synthetic enzymes, as in Figure 17.2.


83


84

17. SERoToNIN RECEPToRS ANd NEuRoTRANSmISSIoN

FIGURE 17.1 The chemical structure of serotonin (5–hydroxytryptamine).

FIGURE 17.2 Biosynthetic and metabolic pathway of serotonin. TABLE 17.1 Serotonin Receptor Subtypes and Pharmacology Receptor Family 5-HT1

Primary Transduction Subtype Pathway

Activation of DOI phospholipase BW723C86 Lorcaserin C (Gaq)

MDL100907 RS127445 RS102221

5-HT3

Ligand gated ion channel

m-CPBG

Ondansetron

5-HT4

Activation of adenylate cyclase (Gas)

Cisapride

GR113808

5-HT6

RECEPTORS 5-HT in the synaptic cleft interacts with cell-surface receptors localized at the postsynaptic membrane or on the

WAY100635 SB224289 BRL15572

5-HT2A 5-HT2B 5-HT2C

5-HT5A 5-HT5B

in the CNS and is also found in the enteric nervous system and in blood platelets. Platelets are devoid of 5-HT synthetic enzymes; transport into platelets by SERT is responsible for the high level of 5-HT found in platelets.

U92016A Anpirtoline PNU-142633

Inhibition of adenylate cyclase (Gai)

5-HT5

5-HT is stored in synaptic vesicles to avoid metabolism to 5-HIAA by mitochondrial MAO. Released 5-HT interacts with receptors in the postsynaptic membrane or on autoreceptors in the presynaptic membrane. The principal mechanism of inactivation is reuptake into the presynaptic terminal by the 5-HT transporter.

Pharmacology: Antagonist

5-HT1A 5-HT1B 5-HT1D 5-HT1E 5-HT1F

5-HT2

FIGURE 17.3 Schematic of 5-HT nerve terminal. Newly synthesized

Pharmacology: Agonist

5-HT7

LY344864

Inhibition of adenylate cyclase (Gai) Activation of adenylate cyclase (Gas) Activation of adenylate cyclase (Gas)

SB699551

EMD386088

SB399885

AS-19

SB258719

presynaptic terminal. The 14 serotonin receptors, reclassified in 1994 [2], segregate into seven families (Table 17.1). All but one are members of the superfamily of G proteincoupled receptors (GPCRs), which are predicted to span the plasma membrane seven times with the N-terminus on the outside of the cell and the C-terminus, intracellular [3]. The intracellular loops and C-terminal tail interact directly with G proteins (Fig. 17.4). As illustrated in Figure 17.3, most of the G protein-coupled 5-HT receptors are localized on postsynaptic membranes and modulate


85

RolE IN AuToNomIC PRoCESSES

TABLE 17.2 Clinically Available 5-HT drugs

FIGURE 17.4 5-HT receptors couple to multiple G-proteins. G proteins are classified based on their alpha subunits. 5-HT receptors have been definitively shown to couple to four different families of G-proteins.

neurotransmission via second messenger pathways. In contrast, the 5-HT3 receptor is a multimeric 5-HT gaited cation channel; it is primarily localized on presynaptic terminals of non-serotoninergic neurons where it regulates the release of other neurotransmitters such as acetylcholine. GPCRs generate intracellular second messengers such as cyclic AMP and calcium, which stimulate or inhibit various kinases and phosphatases that, in turn, regulate proteins by changes in phosphorylation state [4]. As such these receptors act as neuromodulators, modulating other receptors and ion channels that mediate fast neurotransmission. The 5-HT3 receptor is the only 5-HT receptor that gates ions, hence directly altering membrane potential. The other 5-HT receptors indirectly modify the membrane potential by regulating voltage-gated ion channels (such as Ca or K channels) or ligand-gated ion channels (such as glutamate receptors). For example, presynaptic 5-HT1B receptors inhibit N-type calcium channels via Go G-protein, thereby decreasing 5-HT release. This receptor, as well as other G protein-coupled 5-HT receptors, are expressed on the presynaptic terminals of other neurotransmitter releasing neurons, where these so-called heteroreceptors inhibit or potentiate neurotransmitter release. Thus, the possibilities of neurotransmitter cross-talk are numerous and widespread.

PHARMACOLOGY AND ROLE IN DISEASE The large number of receptors translates into a complex pharmacology and a myriad of targets for drug development. Specific drugs do exist, but they are rare. The drugs listed in Table 17.1 are at least 50-fold more potent at their primary target, which translates into reasonable specificity in vivo. Readers interested in more information about pharmacological properties should access the extensive database at http://pdsp.med.unc.edu/indexR.html. Serotonin plays a role in a myriad of behaviors [5]. Advances in genetically modified mice have advanced our understanding of the role of specific receptors in behaviors and drug actions [6]. The clinically available drugs that target 5-HT neurotransmission (Table 17.2) have a range of disease targets

Target

Action

Clinical Use

Examples

MAO

Antagonist

Major depressive Tranylcypromine illness

5-HT Transporter Channel blocker

Major depressive Fluoxetine, illness, panic, escitalopram anxiety, OCD

5-HT1A receptor

Agonist

Anxiety

Buspirone

5-HT1D receptor

Agonist

Migraine

Sumatriptan

5-HT2 receptor

Antagonist

Migraine

Methysergide

5-HT2A receptor

Antagonist

Schizophrenia

Clozapine, risperidone

5-HT4 receptor

Agonist

Irritable bowel syndrome

Prucalopride (Europe)

5-HT3 receptor

Channel blocker

Nausea/emesis

Ondansetron

and varying degrees of specificity. Esitalopram, for example, is a highly specific inhibitor of 5-HT transporter with three orders of magnitude lesser affinity for secondary targets (catecholamine transporters). On the other hand, tranylcypromine blocks the degradation of serotonin, dopamine and norepinephrine equally well. One of the most exciting areas of current research deals with genetic variations in 5HT receptor and transporter genes and their association with human diseases, such as schizophrenia and major depressive illness. Research has focused on more common genetic alterations referred to as single nucleotide polymorphisms (SNPs) and, although inconsistent, the early results suggest that SNPs in serotoninrelated genes may be associated with disease symptoms as well as drug response [7].

ROLE IN AUTONOMIC PROCESSES 5-HT serves a key role in the regulation of gut function and motility [8]. Enterochromaffin cells synthesize and store 5-HT in granules, and release 5-HT upon stimulation by norepinephrine released from the myenteric plexus. This 5-HT can then bind to 5-HT4 and 5-HT2 receptors and activate secretion from enterocytes into the intestinal lumen. Furthermore, 5-HT also binds to 5-HT3/4 receptors on the submucosal plexus of the enteric nervous system to modulate smooth muscle contractility and peristalsis. Too much 5-HT in the gut, resulting from a carcinoid tumor or cisplatin chemotherapy for example, induces diarrhea and emesis. Too little 5-HT in the gut results in slow transit times and constipation. Antagonists of 5-HT3 receptors, including ondansetron, are effective anti-emetics to block the nausea associated with chemotherapy. Although agonists of 5-HT4 receptors like cisapride have had some success in the clinic to increase gut motility associated with irritable bowel syndrome and constipation, they have been


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withdrawn from the market due to their ability to produce fatal cardiac arrhythmias in some patients. Other 5-HT4 agonists are available in Europe. Many cardiovascular functions, including heart rate and blood pressure, are modulated by 5-HT acting at a variety of receptors located in the sympathetic ganglia, parasympathetic ganglia, the vagus nerve, and the heart itself [9]. The interactions of 5-HT with its receptors are complex in mediating cardiovascular effects. Increasing circulating 5-HT levels primarily produces tachycardia. Receptor selective drugs, however, can produce either bradycardia or tachycardia, influence heart rate, cardiac outflow, or blood pressure, as well as have adverse developmental effects like cardiac valvulopathies depending on the receptor targeted and site of action.

References [1] Sanders-Bush E, Hazelwood L. 5-Hydroxytryptamine (serotonin) and dopamine. In: Brunton LL, editor. The pharmacological basis of therapeutics. New York: McGraw-Hill; 2011. p. 335–62.

[2] Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, et al. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol Rev 1994;46:157–203. [3] Nichols DE, Nichols CD. Serotonin Receptors. Chem Rev 2008;46:1614–41. [4] Millan MJ, Marin P, Bockaert J, Mannoury la Cour C. Signaling at G-Protein-coupled serotonin receptors: recent advances and future research directions. Trends Pharmacol Sci 2008;29:454–64. [5] Lucki I. The spectrum of behaviors influenced by serotonin. Biol Psychiatry 1998;44:151–62. [6] Murphy DL, Wichems C, Li Q, Heils A. Molecular manipulations as tools for enhancing our understanding of 5-HT neurotransmission. Trends Pharmacol Sci 1999;20:246–52. [7] Hariri AR, Mattay VS, Tessitore A, Kolachana B, Fera F, Goldman D, et al. Serotonin transporter genetic variation and the response of the human amygdala. Science 2002;297:400–3. [8] Spiller R. Serotonin and GI clinical disorders. Neuropharmacology 2008;55:1072–80. [9] Villalon CM, Centurion D. Cardiovascular responses produced by 5-hydroxytriptamine: a pharmacological update on the receptors/mechanisms involved and therapeutic implications. NaunynSchmiedeberg’s Arch Pharmacol 2007;376:45–63.


C H A P T E R

18 Purinergic Neurotransmission and Nucleotide Receptors Geoffrey Burnstock

PURINERGIC NEUROTRANSMISSION

Another concept that has had a significant influence on our understanding of purinergic transmission was that of cotransmission. Burnstock wrote a Commentary in Neuroscience in 1976 [4] entitled: “Do some nerves release more than one transmitter?” This position challenged the single-neurotransmitter concept, which became known as “Dale’s Principle”, even though Dale himself never defined it as such. The commentary was based on hints about cotransmission in the early literature describing both vertebrate and invertebrate neurotransmission, and, more specifically, with respect to purinergic cotransmission, on the surprising discovery in 1971 that ATP was released from sympathetic nerves supplying the taenia coli as well as from NANC inhibitory nerves. The excitatory junction potentials (EJPs) recorded in the vas deferens were blocked by α,β-meATP, a selective desensitizer of P2X receptors (Fig. 18.3a and b). This clearly supported the earlier demonstration of sympathetic cotransmission in the vas deferens in the laboratory of Dave Westfall, following an earlier report of sympathetic cotransmission in the cat nictitating membrane. Sympathetic cotransmission was later described in a variety of blood vessels. The proportions of ATP and noradrenaline (NA) vary in different tissues and species, during development and ageing and in different pathophysiological conditions. Acetylcholine (ACh) and ATP are cotransmitters in parasympathetic nerves supplying the urinary bladder. Subpopulations of sensory nerves have been shown to utilize ATP in addition to substance P and calcitonin gene-related peptide; it seems likely that ATP cooperates with these peptides in “axon reflex” activity. ATP, vasoactive intestinal polypeptide and nitric oxide (NO) are cotransmitters in NANC inhibitory nerves. ATP and NA act synergistically to release vasopressin and oxytocin from the hypothalamus, which is consistent with ATP cotransmission in the hypothalamus involved in central nervous system (CNS) control of autonomic functions. Release of ATP from autonomic nerves is by vesicular exocytosis and after release it is broken down by ectonucleotidases. Much is now known about the

The existence of non-adrenergic, non-cholinergic (NANC) neurotransmission in the gut was established in the mid 1960s (Fig. 18.1a). Several years later, after many experiments, a study was published that suggested that the NANC transmitter in the guinea-pig taenia coli and stomach, rabbit ileum, frog stomach and turkey gizzard was adenosine 5-triphosphate (ATP) [1]. The experimental evidence included mimicry of the NANC nervemediated response by ATP (Fig. 18.1b); measurement of release of ATP during stimulation of NANC nerves with luciferin-luciferase luminometry; histochemical labeling of subpopulations of neurons in the gut with quinacrine, a fluorescent dye known to selectively label high levels of ATP bound to peptides; the later demonstration that the slowly-degradable analog of ATP, α,β-methylene ATP (α,β-meATP), which produces selective desensitization of the ATP receptor, blocked the responses to NANC nerve stimulation. Soon after, evidence was presented for ATP as the neurotransmitter for NANC excitatory nerves in the urinary bladder (Fig. 18.1c and d). The term “purinergic” was proposed in a short letter to Nature in 1971 and the evidence for purinergic transmission in a wide variety of systems was presented in Pharmacological Reviews in 1972 [2] (Fig. 18.2). This concept met with considerable resistance for many years. Perhaps understandably, this was probably partly because it was felt that ATP was established as an intracellular energy source involved in various metabolic cycles and that such a ubiquitous molecule was unlikely to be involved in extracellular signaling. However, ATP was one of the biological molecules to first appear and, therefore, it is not really surprising that it should have been utilized for extracellular, as well as intracellular, purposes early in evolution. The fact that potent ectoATPases were described in most tissues in the early literature was also a strong indication for the extracellular actions of ATP. Purinergic neurotransmission is now generally accepted (see [3]).


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18. PuRINERgIC NEuRoTRANsmIssIoN ANd NuClEoTIdE RECEPToRs

FIGURE 18.1 (a) Sucrose gap recording of membrane potential changes in smooth muscle of guinea-pig taenia coli in the presence of atropine (0.3 μM) and guanethidine (4 μM). Transmural field stimulation (0.5 ms, 0.033 Hz, 8 V) evoked transient hyperpolarizations, which were followed by rebound depolarizations. Tetrodotoxin (TTX, 3 μM) added to the superfusing Kreb’s solution (applied at arrow) rapidly abolished the responses to transmural field stimulation, indicating that they were inhibitory junction potentials in response to stimulation of NANC inhibitory nerves. (Reproduced from [19] with permission of Blackwell Publishing.) (b) Mechanical responses of the guinea-pig taenia coli to intramural nerve stimulation (NS, 1 Hz, 0.5 ms pulse duration, for 10 s at supramaximal voltage) and ATP (2  10–6 M). The responses consist of a relaxation followed by a “rebound contraction”. Atropine (1.5  10–7 M) and guanethidine (5  10–6 M) were present. (Reproduced from [20] with permission of the Nature Publishing Group). (c) A comparison of the contractile responses of the guinea-pig bladder strip to intramural nerve stimulation (NS: 5 Hz, 0.2 ms pulse duration and supramaximal voltage) and exogenous ATP (8.5 μM). Atropine (1.4 μM) and guanethidine (3.4 μM) were present throughout. (Reproduced from [21] with permission of the Nature Publishing Group). (d) Effect of changing the calcium ion (Ca2) concentration on the release of ATP from the guinea pig isolated bladder strip during stimulation of intramural nerves. Upper trace: mechanical recording of changes in tension (g) during intramural nerve stimulation (NS: 20 Hz, 0.2 ms pulse duration, supramaximal voltage for 20 seconds). Lower trace: concentration of ATP in consecutive 20 s fractions of the superfusate. The Ca2 concentration in the superfusate varied as follows: (i) 2.5 mM (normal Krebs); (ii) 0.5 mM; (iii) 0.25 mM; (iv) 2.5 mM. The successive contractions were separated by 60 min intervals as indicated by the breaks in the mechanical trace. Atropine (1.4 μM) and guanethidine (3.4 μM) were present throughout. The temperature of the perfusate was between 22°C and 23°C. (Reproduced from [21] with permission).

ectonucleotidases that break down ATP released from neurons and non-neuronal cells. Several enzyme families are involved: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) of which NTPDase 1, 2, 3 and 8 are extracellular; ectonucleotide pyrophosphatase (E-NPP) of three subtypes; alkaline phosphatases; ecto-5-nucleotidase and ecto-nucleoside diphosphokinase (E-NDPK). NTPDase1 hydrolyses ATP directly to AMP and uridine 5-triphosphate (UTP) to uridine diphosphate (UDP), while NTPDase 2 hydrolyses ATP to adenosine 5-diphosphate (ADP) and 5-nucleotidase AMP to adenosine (see [5]). Purinergic receptors have been cloned and characterized in amoeba, Schistosoma and green algae, which resemble P2X receptors found in mammals, suggesting that purinergic signaling was present early in evolution (see [6]). This perhaps explains the wide distribution of purinergic receptors in most non-neuronal as well as neuronal cell types (see [7]).

An important conceptual step was when, in addition to purinergic neuromuscular transmission, purinergic nerve– nerve synaptic transmission was described in coeliac ganglion in 1992 by Silinsky, Surprenant and colleagues. Synaptic transmission has also been demonstrated in the enteric plexuses and in various sensory sympathetic, parasympathetic and pelvic ganglia (see [3]). A hypothesis was proposed that purinergic mechanosensory transduction occurred in visceral tubes and sacs, including ureter, bladder and gut, where ATP, released from epithelial cells during distension, acted on P2X3 homomultimeric and P2X2/3 heteromultimeric receptors on subepithelial sensory nerves initiating impulses in sensory pathways to pain centers in the CNS. Subsequent studies of bladder, ureter, gut, tongue and tooth pulp have produced evidence in support of this hypothesis (see [8]).


RECEPToRs foR PuRINEs ANd PyRImIdINEs

89

FIGURE 18.2 Purinergic neuromuscular transmission depicting the synthesis, storage, release and inactivation of adenosine 5-triphosphate (ATP). ATP, stored in vesicles in nerve varicosities, is released by exocytosis to act on postjunctional ATP receptors on smooth muscle. ATP is broken down extracellularly by ATPases and 5-nucleotidase to adenosine, which is taken up by varicosities to be re-synthesized and restored in vesicles. If adenosine is broken down further by adenosine deaminase to inosine and hypoxanthine, they are removed by the circulation. (Reproduced from [2] with permission from the American Society for Pharmacology and Experimental Therapeutics.)

While the early emphasis was on short-term purinergic signaling in neurotransmission, neuromodulation and secretion, it was later recognized that ATP, released from autonomic nerves and by paracrine or autocrine release from non-neuronal cells, is involved in long-term (trophic) signaling, involved in cell proliferation, differentiation and death in development and regeneration [9]. Purinergic signaling in the brain stem is involved in control of autonomic functions, including cardiovascular and respiratory control and in the regulation of hormone secretion and body temperature at the hypothalamic level. The nucleus tractus solitarius (NTS) is a major integrative center of the brain stem involved in reflex control of the cardiovascular system; stimulation of P2X receptors in the NTS evokes hypotension. P2X receptors expressed in neurons in the trigeminal mesencephalic nucleus might be involved in the processing of proprioceptive information.

RECEPTORS FOR PURINES AND PYRIMIDINES Implicit in the purinergic neurotransmission hypothesis was the presence of purinoceptors. A basis for distinguishing two types of purinoceptor, identified as P1 and P2 for adenosine and ATP/ADP, respectively, was recognized in

FIGURE 18.3 (a) Excitatory junction potentials in response to repetitive stimulation of adrenergic nerves (white dots) in the guinea pig vas deferens. The upper trace records the tension, the lower trace the electrical activity of the muscle recorded extracellularly by the sucrose gap method. Note both summation and facilitation of successive junction potentials. At a critical depolarization threshold an action potential is initiated which results in contraction. (Reproduced from [22] with permission from Springer). (b) The effect of various concentrations of α,β-methylene ATP (α,β-meATP) on EJPs recorded from guinea pig vas deferens (intracellular recordings). The control responses to stimulation of the motor nerves at 0.5 Hz are shown on the left. After at least 10 min in the continuous presence of the indicated concentration of α,β-meATP, EJPs were recorded using the same stimulation parameters. (Reproduced from [23] with permission from Elsevier.)

1978 [10]. This helped resolve some of the ambiguities in earlier reports, which were complicated by the breakdown of ATP to adenosine by ectoenzymes, so that some of the actions of ATP were directly on P2 receptors, whereas others were due to indirect action via P1 receptors. However, it was not until 1985 that a pharmacological basis for distinguishing two types of P2 receptors (P2X and P2Y) was proposed [11]. A year later, two further P2 receptor subtypes were named, a P2T receptor selective for ADP on platelets and a P2Z receptor on macrophages. Further subtypes followed, perhaps the most important of which being the P2U receptor, which could recognize pyrimidines such as UTP in addition to ATP. However, to provide a more manageable framework for newly identified nucleotide receptors, Abbracchio and Burnstock proposed in 1994 [12] that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G protein-coupled receptors. This was based on studies of transduction mechanisms and the cloning of nucleotide receptors: P2Y receptors were cloned first in 1993 and a year later P2X receptors were cloned (Fig. 18.4). This nomenclature has been widely adopted and currently seven P2X subtypes and eight P2Y


90


FIGURE 18.4 Membrane receptors for extracellular ATP. (a) The P2X family of receptors are ligand-gated ion channels (S-S; disulphide bond; M1 and M2, transmembrane domains), and (b) the P2Y family are G protein-coupled receptors (S-S; disulphide bond; pale blue circles represent amino acid residues that are conserved between P2Y1, P2Y2 and P2Y3 receptors; fawn circles represent residues that are not conserved; and purple circles represent residues that are known to be functionally important in other G protein-coupled receptors). (Part (a) reproduced from [24] with permission from Nature; Part (b) modified from [25] with permission from Elsevier.)

receptor subtypes are recognized, while four subtypes of P1 receptor have been cloned and characterized (see [13]; Table 18.1).

P2X Receptors Members of the family of ionotropic P2X1–7 receptors show a subunit topology of intracellular N- and C- termini possessing consensus binding motifs for protein kinases; two transmembrane-spanning regions (TM1 and TM2), the first involved with channel gating and the second lining the ion pore; a large extracellular loop, with 10 conserved cysteine residues forming a series of disulfide bridges and an ATP-binding site, which may involve regions of the extracellular loop adjacent to TM1 and TM2 (see [14]). The crystal structure of the P2X4 receptor was recently described [15]. Heteromultimers as well as homomultimers are involved in forming the trimer ion pore. P2X2/3, P2X1/2, P2X1/5, P2X2/6, P2X4/6 and P2X1/4 receptor heteromultimers have been identified. P2X7 does not form heteromultimers, and P2X6 will not form a functional homomultimer. Advances have been made by the preparation of knockout mice for P2X1, P2X2, P2X3, P2X4 and P2X7 receptors, and transgenic mice that over-express the P2X1 receptor. Adenoviral expression of a P2X1 receptor-green fluorescent protein construct in vas deferens shows the receptor to be localized in clusters, with larger ones apposing nerve varicosities. The P2X2 receptor is generally described as non-desensitizing, compared with the P2X1 and P2X3 receptors. P2X3 receptors are prominently expressed on nociceptive sensory neurons. P2X2/3 heteromer receptors have been identified in subpopulations of sensory neurons and sympathetic ganglion cells. Homomeric P2X4 receptors

are activated by ATP, but not by α,β-meATP. The most useful distinguishing feature of ATP-evoked currents at P2X4 receptors is their potentiation by ivermectin. The P2X5 receptor cDNA was first isolated from cDNA libraries constructed from rat coeliac ganglion and heart. Cells expressing the heteromeric P2X1/5 receptor are very sensitive to ATP, concentrations as low as 3 or 10 nM evoking measurable currents. The P2X6 subunit is only functionally expressed as a heteromultimer. P2X2/6 heteromeric receptors are prominently expressed by respiratory neurons in the brainstem. The main feature of the P2X7 receptor is that, in addition to the usual rapid opening of the cationselective ion channel, with prolonged exposure to high concentrations of ATP large pores form and this usually leads to cell death. Vesicles are shed after activation of P2X7 receptors, which release inflammatory cytokines.

P2Y Receptors At present, there are eight accepted human P2Y receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and P2Y14 (see [16] and Table 18.1). The missing numbers represent either non-mammalian orthologs, or receptors having some sequence homology to P2Y receptors, but for which there is no functional evidence of responsiveness to nucleotides. In contrast to P2X receptors, P2Y receptor genes do not contain introns in the coding sequence, except for the P2Y11 receptor. Site-directed mutagenesis of the P2Y1 and P2Y2 receptors has shown that some positively charged residues in TM3, TM6 and TM7 are crucial for receptor activation by nucleotides. From a phylogenetic and structural (i.e., protein sequence) point of view, two distinct P2Y receptor subgroups characterized by a relatively high level of sequence divergence have been


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RECEPToRs foR PuRINEs ANd PyRImIdINEs

TABLE 18.1 Characteristics of Purine-mediated Receptors Transduction Mechanisms

Receptor

Main Distribution

Agonists

Antagonists

P1 (adenosine) A1

Brain, spinal cord, testis, heart, autonomic nerve terminals

CCPA, CPA, S-ENBA

DPCPX, N-0840, MRS1754

Gi/o ↓cAMP

A2A

Brain, heart, lungs, spleen

CGS 21680, HENECA

KF17837, SCH58261, ZM241385

GS ↑cAMP

A2B

Large intestine, bladder

NECA (non-selective)

Enprofylline, MRE2029-F20, MRS17541, MRS 1706

GS ↑cAMP

A3

Lung, liver, brain, testis, heart

IB-MECA, 2-Cl-IB-MECA, DBXRM, VT160

MRS1220, L-268605, MRS1191, MRS1523, VUF8504

Gi/o Gq/11 ↓cAMP ↑IP3

P2X1

Smooth muscle, platelets, cerebellum, dorsal horn spinal neurons

α,β-meATP  ATP  2-MeSATP (rapid desensitization), L-β,γ-meATP

TNP-ATP, IP5I, NF023, NF449

Intrinsic cation channel (Ca2 and Na)

P2X2

Smooth muscle, CNS, retina, chromaffin cells, autonomic and sensory ganglia

ATP  ATPγS  2-MeSATP  α,β-meATP (pH  zinc sensitive)

Suramin, isoPPADS, RB2, NF770

Intrinsic ion channel (particularly Ca2)

P2X3

Sensory neurones, NTS, some sympathetic neurons

2-MeSATP  ATP  α,βmeATP  Ap4A (rapid desensitization)

TNP-ATP, PPADS, A317491, NF110

Intrinsic cation channel

P2X4

CNS, testis, colon

ATP  α,β-meATP, CTP, Ivermectin

TNP-ATP (weak), BBG (weak)

Intrinsic ion channel (especially Ca2)

P2X5

Proliferating cells in skin, gut, bladder, thymus, spinal cord

ATP  α,β-meATP, ATPγS

Suramin, PPADS, BBG

Intrinsic ion channel

P2X6

CNS, motor neurons in spinal cord

– (does not function as homomultimer)

–

Intrinsic ion channel

P2X7

Apoptotic cells in, for example, immune cells, pancreas, skin

BzATP  ATP  2-MeSATP  α,β-meATP

KN62, KN04, MRS2427 Intrinsic cation channel Coomassie brilliant and a large pore with blue G prolonged activation

P2Y1

Epithelial and endothelial cells, platelets, immune cells, osteoclasts

2-MeSADP  ADPβS  2-MeSATP  ADP  ATP, MRS2365

MRS2179, MRS2500, MRS2279, PIT

Gq/G11; PLC-β activation

P2Y2

Immune cells, epithelial and endothelial cells, kidney tubules, osteoblasts

UTP  ATP, UTPγS, INS 37217

Suramin  RB2, AR-C126313

Gq/G11 and possibly Gi; PLC-β activation

P2Y4

Endothelial cells

UTP  ATP, UTPγS

RB2  Suramin

Gq/G11 and possibly Gi; PLC-β activation

P2Y6

Some epithelial cells, placenta, T cells, thymus

UDP  UTP  ATP, UDPβS

MRS2578

Gq/G11; PLC-β activation

P2Y11

Spleen, intestine, granulocytes

AR-C67085MX  BzATP  ATPγS  ATP

Suramin  RB2, NF157, 5-AMPS

Gq/G11 and GS; PLC-β activation

P2Y12

Platelets, glial cells

2-MeSADP  ADP  ATP

CT50547, AR-C69931MX, INS49266, AZD6140, PSB0413, ARL66096, 2-MeSAMP

Gi/o; inhibition of adenylate cyclase

P2Y13

Spleen, brain, lymph nodes, bone marrow Placenta, adipose tissue, stomach, intestine, discrete brain regions

ADP  2-MeSADP  ATP & 2-MeSATP UDP glucose  UDP-galactose

MRS2211, 2-MeSAMP

Gi/o

–

Gq/G11

P2X

P2Y

P2Y14

Updated from [18] with permission from Elsevier. Abbreviations: BBG, Brilliant blue green; BzATP, 2-&3-O-(4-benzoyl-benzoyl)-ATP; cAMP, cyclic AMP; CCPA, chlorocyclopentyl adenosine; CPA, cyclopentyl adenosine; CTP, cytosine triphosphate; IP3, inosine triphosphate; Ip5I, di-inosine pentaphosphate; 2-MeSADP, 2-methylthio ADP; 2-MeSATP, 2-methylthio ATP; NECA, 5-N-ethylcarboxamido adenosine; PLC, phospholipase C; RB2, reactive blue 2.


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identified. The first subgroup includes P2Y1,2,4,6,11 and the second subgroup encompasses the P2Y12,13,14 subtypes. Selective antagonists have been identified for some P2Y receptor subtypes (see Table 18.1). P2Y1, P2Y2, P2Y4 and P2Y6 receptors couple to G proteins to increase inositol triphosphate (IP3) and cytosolic calcium. Activation of the P2Y11 receptor by ATP leads to a rise in both cAMP and in IP3, whereas activation by UTP produces calcium mobilization without IP3 or cAMP increase. The P2Y13 receptor can simultaneously couple to G16, Gi and, at high concentrations of ADP, to Gs. The activation of several P2Y receptors is commonly associated with the stimulation of several mitogen-activated protein kinases, in particular extracellular signal regulated protein kinase 1/2. In most species, ADP is a more potent agonist than ATP at P2Y1 receptors. Site-directed mutagenesis studies on the human P2Y1 receptor have shown that amino acid residues in TM3, TM6 and TM7 are critical determinants in the binding of ATP. P2Y2 receptors are fully activated by ATP and UTP, whereas ADP and UDP are much less effective agonists. Expression of P2Y2 receptor mRNA and protein has been detected in many peripheral tissues. UTP is the most potent activator of the recombinant human P2Y4 receptor. In human and mouse, P2Y4 mRNA and protein was most abundant in the intestine, but was also detected in other organs. The mouse, rat and human P2Y6 receptors are UDP receptors. A wide tissue distribution of P2Y6 mRNA and protein has been demonstrated, with the highest expression in spleen, intestine, liver, brain and pituitary. ATPγS is a more potent agonist at the P2Y11 receptor than ATP. ADP is the natural agonist of the P2Y12 receptor. It is heavily expressed in platelets where it is the molecular target of the active metabolite of the antiplatelet drug clopidogrel. The P2Y13 ADP-sensitive receptor is strongly expressed in the spleen, followed by placenta, liver, heart, bone marrow, monocytes, T-cells, lung and various brain regions. The P2Y14 receptor is activated by UDP, UDPglucose as well as UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine. The formation of oligomers by P2Y receptors is likely to be widespread and to greatly increase the diversity of purinergic signaling. P2X receptors are often expressed in the same cells as P2Y receptors. Thus, there is the possibility of bi-directional cross-talk between these two families of nucleotide-sensitive receptors. P2X receptors in general mediate fast neurotransmission, but are sometimes located prejunctionally to mediate increase in release of cotransmitters, for example glutamate in terminals of primary afferent neurons in the spinal cord. P2Y receptors are particularly involved in prejunctional inhibitory modulation of transmitter release, as well as cell proliferation. P2Y1,2,4,6 receptors have been described on subpopulations of sympathetic neurons, P2Y2 and P2Y4 receptors in intracardiac ganglia, P2Y1 and P2Y2 receptors on sensory neurons while P2Y1 receptors appear to be the dominant subtype on enteric neurons. P2Y2 (and/or P2Y4) receptors are expressed on enteric glial cells.

CONCLUSIONS Purinergic neurotransmission is now widely established for both autonomic neuromuscular transmission to smooth muscle and synaptic transmission in ganglia and it is a rapidly expanding field. There is particular interest in the physiology and pathophysiology of purinergic signalling in autonomic systems and therapeutic interventions are being explored [17]. The autonomic nervous system shows marked plasticity: that is, the expression of cotransmitters and receptors show dramatic changes during development and ageing, in nerves that remain after trauma or surgery and in disease conditions. There are several examples where the purinergic component of cotransmission is increased in pathological conditions. The parasympathetic purinergic nerve-mediated component of contraction of the human bladder is increased to 40% in pathophysiological conditions such as interstitial cystitis, outflow obstruction, idiopathic instability and also some types of neurogenic bladder. ATP also has a significantly greater cotransmitter role in sympathetic nerves supplying hypertensive compared to normotensive blood vessels. Receptors for purines and pyrimidines have been cloned and characterized. There are 4 subtypes of P1(adenosine) receptors, 7 subtypes of P2X ion channel receptors and 8 subtypes of P2Y G protein-coupled receptors. These are widely distributed in non-neuronal cells as well as neurons.

References [1] Burnstock G, Campbell G, Satchell D, Smythe A. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. Br J Pharmacol 1970;40:668–88. [2] Burnstock G. Purinergic nerves. Pharmacol Rev 1972;24:509–81. [3] Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 2007;87:659–797. [4] Burnstock G. Do some nerve cells release more than one transmitter? Neuroscience 1976;1:239–48. [5] Zimmermann H. Ectonucleotidases: some recent developments and a note on nomenclature. Drug Dev Res 2001;52:44–56. [6] Burnstock G, Verkhratsky A. Evolutionary origins of the purinergic signalling system. Acta Physiologica 2009;195:415–47. [7] Burnstock G, Knight GE. Cellular distribution and functions of P2 receptor subtypes in different systems. Int Rev Cytol 2004;240:31–304. [8] Burnstock G. Purine-mediated signalling in pain and visceral perception. Trends Pharmacol Sci 2001;22:182–8. [9] Burnstock G, Verkhratsky A. Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death. Cell Death Dis 2010;1:e9. [10] Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Straub RW, Bolis L, editors. Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach. New York: Raven Press; 1978. p. 107–18. [11] Burnstock G, Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol 1985;16:433–40. [12] Abbracchio MP, Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Therap 1994;64:445–75. [13] Burnstock G. Purine and pyrimidine receptors. Cell Mol Life Sci 2007;64:1471–83. [14] North RA. Molecular physiology of P2X receptors. Physiol Rev 2002;82:1013–67.


CoNClusIoNs

[15] Kawate T, Michel JC, Birdsong WT, Gouaux E. Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature 2009;460:592–8. [16] Abbracchio MP, Burnstock G, Boeynaems J-M, Barnard EA, Boyer JL, Kennedy C, et al. International Union of Pharmacology. Update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006;58:281–341. [17] Burnstock G. Pathophysiology and therapeutic potential of purinergic signaling. Pharmacol Rev 2006;58:58–86. [18] Burnstock G. Introduction: ATP and its metabolites as potent extracellular agonists. In: Schwiebert EM, editor. Current Topics in Membranes, vol 54. Purinergic Receptors and Signalling. San Diego: Academic Press; 2003. p. 1–27. [19] Burnstock G. The changing face of autonomic neurotransmission. (The First von Euler Lecture in Physiology). Acta Physiol Scand 1986;126:67–91. [20] Burnstock G, Wong H. Comparison of the effects of ultraviolet light and purinergic nerve stimulation on the guinea-pig taenia coli. Br J Pharmacol 1978;62:293–302.

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[21] Burnstock G, Cocks T, Crowe R, Kasakov L. Purinergic innervation of the guinea-pig urinary bladder. Br J Pharmacol 1978;63:125–38. [22] Burnstock G, Costa M. Adrenergic Neurones: Their Organization, Function and Development in the Peripheral Nervous System. London: Chapman and Hall; 1975. pp. 1–225 [23] Sneddon P, Burnstock G. Inhibition of excitatory junction potentials in guinea-pig vas deferens by α,β-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur J Pharmac 1984;100:85–90. [24] Brake AJ, Wagenbach MJ, Julius D. New structural motif for ligandgated ion channels defined by an ionotropic ATP receptor. Nature 1994;371:519–23. [25] Barnard EA, Burnstock G, Webb TE. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci 1994;15:67–70.


C H A P T E R

19 Adenosine Receptors and Autonomic Regulation Italo Biaggioni

Adenosine is an endogenous nucleoside formed by the degradation of adenosine-triphosphate (ATP) during energy-consuming processes. Adenosine modulates many physiological processes through activation of four subtypes of G-protein coupled membrane P1 purinergic receptors, A1, A2A, A2B and A3. Its physiological importance depends on the affinity of these receptors and the extracellular concentrations reached. Extracellular concentrations of adenosine are usually low, but adenosine may have tonic actions even during physiological conditions, mostly through activation of high affinity A2A and A1 receptors. Theoretically, adenosine can be formed within the synapse, from the degradation of ATP that is released as a co-transmitter (see Chapter 18). Significant increases of extracellular adenosine, however, occur mostly in ischemic tissues, when energy demands exceed oxygen supply. Thus, adenosine is considered a “retaliatory” metabolite, whose actions are physiologically relevant during ischemic conditions. Adenosine has perhaps the shortest half-live of all autacoids, particularly in humans. It is rapidly and extensively metabolized to inactive inosine by adenosine deaminase. It is also quickly transported back into cells by an energydependent uptake mechanism, which is part of a purine salvage pathway designed to maintain intracellular levels of ATP. The effectiveness of this adenosine transport system is species-dependent. It is particularly active in humans, and is mainly responsible for the extremely short half-life of adenosine in human blood, which is probably less than one second. Adenosine mechanisms are the target of commonly used drugs. Dipyridamole (Persantin, Aggrenox) acts by blockade of adenosine reuptake, thus potentiating its actions. Conversely, caffeine and theophylline are antagonists of adenosine receptors. Adenosine receptors are ubiquitous and, depending on their localization, may mediate opposite effects. This phenomenon is particularly evident in the interaction of adenosine and the autonomic nervous system; adenosine can produce either inhibition or excitation of autonomic


neurons [1]. I will first outline the effects of adenosine in the efferent, central, and afferent autonomic pathways, emphasizing those with clinical relevance. I will then propose an integrated view that may explain how these seemingly contradictory effects may work together. ATP shares many of the modulatory effects of adenosine on autonomic function and we will discuss these similarities when appropriate. A comprehensive review of the effects of ATP and its P2 purinergic receptors is found in Chapter 18.

POSTSYNAPTIC ANTI-ADRENERGIC EFFECTS OF ADENOSINE Adenosine A1 receptors are found in target organs innervated by the sympathetic nervous system. A1 receptors are coupled to inhibition of adenylate cyclase and their effects are opposite to those of β-adrenoreceptor agonists. For example, adenosine will oppose β-mediated tachycardia and lipolysis. This phenomenon is translated functionally as an “anti-adrenergic” effect. The physiological relevance of this effect is not entirely clear, but some studies suggest that adenosine is more effective in reducing heart rate during isoproterenol-induced tachycardia than in the baseline state or during atropine-induced tachycardia.

PRESYNAPTIC EFFECTS OF ADENOSINE ON EFFERENT NERVES AND GANGLIONIC TRANSMISSION Adenosine inhibits the release of neurotransmitters through putative presynaptic A1 receptors in both the brain and the periphery. Blockade of forearm adenosine receptors with intrabrachial theophylline potentiates sympathetically mediated forearm vasoconstriction, suggesting that endogenous adenosine inhibits noradrenergic neurotransmission in vivo in humans.

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19. AdEnosInE RECEPToRs And AuTonomIC REgulATIon

A few studies have investigated the effects of adenosine on ganglionic neurotransmission, and most of them show an inhibitory effect. Adenosine inhibits the release of acetylcholine presynaptically and blocks calcium current postynaptically in ganglia.

ADENOSINE AND CENTRAL AUTONOMIC REGULATION Adenosine acts as a neuromodulator within the central nervous systems, mostly through interaction with A1 and A2A receptors. Of particular relevance to this review are its actions on brainstem nuclei involved in autonomic cardiovascular regulation. In general, the central actions of adenosine result in inhibition of sympathetic tone through complex, and incompletely understood, mechanisms of action. Microinjection of adenosine into the nucleus tractus solitarii (NTS) evokes a dose-related decrease in blood pressure, heart rate, and renal sympathetic nerve activity. These effects appear to be mediated, at least partially, through A2A receptors. The NTS is the site of the first synapse of afferent fibers arising from baroreceptors. The NTS provides excitatory input to the caudal ventrolateral medulla, which in turn provides inhibitory input to the rostro ventrolateral medulla (RVLM), where sympathetic activity is thought to originate. Thus, stimulation of baroreceptor afferents (e.g., by an increase in blood pressure) activates the NTS and this results in inhibition of the RVLM and a reduction in sympathetic tone. The effect of adenosine in the NTS is similar to that of the excitatory neurotransmitter glutamate, implying that adenosine has excitatory neuromodulatory effects on the NTS. The precise mechanisms that explain this phenomenon are not known. It has been proposed that adenosine releases glutamate within the NTS [2,3], or blunts the release of the inhibitory neuromodulator GABA. The effects of adenosine in the NTS are also blunted by microinjection of the nitric oxide synthase inhibitor L-NAME, suggesting an interaction between adenosine and nitric oxide in the NTS. Regardless of the mechanism of action, microinjections of adenosine receptor antagonists into the NTS results in blunting of the baroreflex gain, suggesting a role of endogenous adenosine on central cardiovascular regulation. A1 and A2A receptors are also found in the RVLM and may modulate neuronal activity either directly, or through inhibition of GABA release [4]. ATP and P2 receptors are also important in central autonomic regulation of cardiovascular and respiratory functions [5].

carotid body chemoreceptors, renal afferents, and myocardial and skeletal muscle afferents. The neuroexcitatory actions of adenosine were first recognized in animals in the early 1980s when it was found that adenosine activates arterial chemoreceptors in rats and cats, and renal afferents in rats and dogs. The functional relevance of these findings, however, was not apparent until human studies were done. The most striking effect of intravenous adenosine in humans is a dramatic stimulation of respiration and sympathetic activation. This effect can be explained by carotid body chemoreceptor activation because it is observed when adenosine is injected into the aortic arch at a site proximal to the origin of the carotid arteries, but not if adenosine is injected into the descending aorta. Activation of vagal C-fibers may also contribute to adenosine-induced dyspnea. The effects of intravenous adenosine are dramatically different if autonomic nervous system is absent. For example, adenosine lowers blood pressure and heart rate in autonomic failure patients. Pain resembling angina has been reported with intravenous and intracoronary administration of adenosine presumably because of activation of sensory afferents. Intracoronary adenosine also elicits a pressor reflex in humans, which may be explained by activation of myocardial afferents. The few animal studies that have tested this hypothesis have yielded conflicting results, perhaps because of the confounding effects of anesthesia or to species differences. There is also controversy over whether adenosine activates skeletal muscle chemoreceptors that trigger sympathetic activation in response to ischemic exercise. Studies favoring [6] or opposing [7] the possibility that adenosine is a metabolic trigger of the exercise pressor reflex have been published. ATP has also been implicated as a trigger of skeletal muscle afferent activation [8]. In summary, whereas adenosine inhibits sympathetic efferents, it activates afferent nerves, including arterial chemoreceptors (in animals and humans), renal afferents (animals), and possibly cardiac and muscle afferents (humans). The autonomic excitatory actions of adenosine clearly predominate when adenosine is given intravenously to conscious human subjects [9]. This sympathetic activation may explain the usefulness of intravenous adenosine as a challenge test to diagnose neurogenic syncope during tilt table tests [10]. It remains speculative whether or not endogenous adenosine plays a role in the generation of spontaneous neurogenic syncope. The adenosine receptor antagonist theophylline is used in the treatment of neurogenic syncope, but controlled studies are lacking.

NEUROEXCITATORY ACTIONS OF ADENOSINE ON AFFERENT PATHWAYS

INTEGRATED VIEW OF ADENOSINE AND CARDIOVASCULAR AUTONOMIC REGULATION

In contrast to the “inhibitory” actions of adenosine in efferent pathways, adenosine excites a variety of afferent fibers that evoke systemic sympathetic activation including

These neuroexcitatory actions seem at odds with the postulated protective role of adenosine, which heretofore has been assigned to its “inhibitory” effects. We postulate,


InTEgRATEd VIEw of AdEnosInE And CARdIoVAsCulAR AuTonomIC REgulATIon

97

Sympathetic activation NE NE Systemic

Systemic vasoconstriction blood pressure perfusion pressure

NE

III Local inhibition of NE release and F– vasodilation I Adenosine

Local

F+ II. Activation of afferent fibers

ATP

Ischemic exercise

FIGURE 19.1 Postulated modulation by purines (adenosine and ATP) of autonomic cardiovascular regulation. See text for details.

however, that the neuroexcitatory actions of adenosine work in tandem with its inhibitory effects to provide protection against ischemia. It is also likely that ATP, in addition to or instead of adenosine, participate in these actions. This framework is presented in Figure 19.1. 1. Interstitial levels of adenosine increase under conditions of increased metabolic demand (exercise) and decreased energy supply (ischemia), reaching physiologically relevant concentrations. ATP can also be released as a co-transmitter from noradrenergic neurons. 2. Adenosine (or ATP) then activates sensory afferent fibers that produce pain, and muscle or myocardial afferents (metaboreceptors) that trigger an ischemic pressor reflex. Pain sensation is a primordial defense mechanism that signals the individual to stop exercising. Sympathetic activation leads to systemic vasoconstriction, increase in blood pressure and improved perfusion pressure. 3. This systemic vasoconstriction would be deleterious to the ischemic organ, if it were not for the simultaneous local inhibitory actions of adenosine that produce vasodilation and inhibit norepinephrine (NE) release. These actions are, for the most part, circumscribed to the local ischemic tissue so that it is protected from sympathetically-mediated vasoconstriction while it benefits from the improved perfusion pressure. ATP has been proposed as a mediator of this “functional sympatholysis” [11]. We propose, therefore, that the excitatory actions of adenosine and ATP work in tandem with their inhibitory effects to provide local protection against ischemia, even at the expense of the rest of the organism. Furthermore, we propose that adenosine (and ATP) provides a link between

local mechanisms of blood flow autoregulation and systemic mechanisms of autonomic cardiovascular regulation, heretofore thought to work independent from each other.

References [1] Biaggioni I. Contrasting excitatory and inhibitory effects of adenosine in blood pressure regulation. Hypertension 1992;20:457–65. [2] Phillis JW, Scislo TJ, O’Leary DS. Purines and the nucleus tractus solitarius: effects on cardiovascular and respiratory function. [Review] [29 refs]. Clin Exp Pharmacol Physiol 1997;24:738–42. [3] Biaggioni I, Mosqueda-Garcia R. Adenosine in cardiovascular homeostasis and the pharmacological control of its activity. In: Laragh J, Brenner BM, editors. Hypertension: pathophysiology, managements and diagnosis. New York: Raven Press; 1995. [4] Spyer KM, Thomas T. A role for adenosine in modulating cardiorespiratory responses: a mini-review. Brain Res Bull 2000;53:121–4. [5] Gourine AV, Wood JD, Burnstock G. Purinergic signalling in autonomic control. Trends Neurosci 2009;32:241–8. [6] Costa F, Diedrich A, Johnson B, Sulur P, Farley G, Biaggioni I. Adenosine, a metabolic trigger of the exercise pressor reflex in humans. Hypertension 2001;37:917–22. [7] Cui J, Leuenberger UA, Blaha C, Yoder J, Gao Z, Sinoway LI. Local adenosine receptor blockade accentuates the sympathetic responses to fatiguing exercise. Am J Physiol Heart Circ Physiol 2010;298:H2130–H2137. [8] McCord JL, Tsuchimochi H, Kaufman MP. P2X2/3 and P2X3 receptors contribute to the metaboreceptor component of the exercise pressor reflex. J Appl Physiol 2010;109:1416–23. [9] Biaggioni I, Killian TJ, Mosqueda-Garcia R, Robertson RM, Robertson D. Adenosine increases sympathetic nerve traffic in humans. Circulation 1991;83:1668–75. [10] Shen WK, Hammill SC, Munger TM, Stanton MS, Packer DL, Osborn MJ, et al. PA Adenosine: potential modulator for vasovagal syncope. J Am Coll Cardiol 1996;28:146–54. [11] Rosenmeier JB, Yegutkin GG, Gonzalez-Alonso J. Activation of ATP/UTP-selective receptors increases blood flow and blunts sympathetic vasoconstriction in human skeletal muscle. J Physiol 2008;586:4993–5002.


C H A P T E R

20 Nitric Oxide and Autonomic Regulation Alfredo Gamboa GMP causes activation of cyclic GMP-dependent protein kinases, with reduction of intracellular Ca2 and a decrease in the sensitivity of contractile elements to Ca2.

Since the discovery that nitric oxide (NO) was a naturally occurring signal molecule in blood vessels, it has become one of the most widely studied substances in biology. Now it is recognized as an endogenous mediator of numerous physiological processes ranging from regulation of cardiovascular function to memory formation. NO is probably one of the most important metabolic modulator of blood pressure; our previous studies suggest that NO tonically restrains blood pressure by at least 30 mmHg in healthy young adults [1]. The autonomic nervous system is a major regulatory mechanism for both short and long term adjustments of the cardiovascular system in general, and blood pressure in particular. In addition to its control of local vasomotor tone and arterial pressure, NO has been proposed to also modulate autonomic regulation of blood pressure through interactions with autonomic pathways in the central nervous system and at peripheral sites.

CENTRAL NO–AUTONOMIC NERVOUS SYSTEM INTERACTIONS In the central nervous system, nitric oxide functions mainly as a neuromodulator. Nitric oxide can have both central neural sympathoinhibitory and sympathoexcitatory actions. In humans, intravenous infusion of L-NMMA (a competitive stereospecific non-selective inhibitor of NOS) induces an increase of mean arterial pressure of about 10% without the expected compensatory inhibition of sympathetic firing rate mediated by baroreflex loading. In comparison, the same increase in blood pressure induced by phenylephrine produced a decrease of about 50% in sympathetic firing rate. When sodium nitroprusside was given in addition to L-NMMA in order to prevent the increase in mean arterial blood pressure, muscle sympathetic nerve activity increased more than double its baseline values [3]. Furthermore, it has been shown that acute and/or chronic (3 weeks) intracerebroventricular injection of L-NMMA, at a dose ineffective to produce systemic effects; increases mean arterial pressure in normotensive rats [4]. Taken together these findings suggest that NO tonically inhibits central sympathetic tone. Thus, NOS inhibition increases blood pressure in part by increasing sympathetic firing rate but this effect is partially masked by the baroreflex. The excitatory effects of NO on neural sympathetic outflow, and their physiological relevance, are less well understood. In rat brain slices, L-arginine, the substrate to produce NO, stimulates the neuronal activity of some nucleus tractus solitarius neurons and this effect can be blocked by co-infusion of L-NMMA [5]. It should be noted that the NTS provides inhibitory input (through the caudal ventrolateral medulla) to the rostral ventrolateral medulla (RVLM) where sympathetic tone is thought to originate. Therefore, neuroexcitation of NTS neurons by NO may contribute to its tonic inhibition of sympathetic tone. On the other hand, in vivo studies in rabbits have shown that NO has an excitatory effect on renal

NO SYNTHESIS AND ACTIONS NO is produced from L-arginine by three isoforms of the enzyme nitric oxide synthase (NOS). An inducible form is found mostly in macrophages (iNOS). Two isoforms are expressed constitutively, one in epithelial and neural cells (nNOS), and another in endothelial cells, platelets, and myocardial cells (eNOS). Both eNOS and nNOS consists of a C-terminal reductase domain and an N-terminal oxygenase domain, differing only in one aminoacid, namely, Asp597 in nNOS is Asn368 in eNOS [2]. The monomer of NOS is inactive, while the dimer is its active form and the dimerization requires tetrahydrobiopterin (BH4), heme and L-arginine binding. Overall, NOS catalyzes the oxidation of L-arginine to generate citrulline and NO. The synthesis of nitric oxide by these NOS isoforms is inhibited non-selectively by L-arginine analogs, including NG-monomethyl-L-arginine (L-NMMA), NG-nitro-Larginine (L-NA), L-NA methylester (L-NAME), and asymmetric dimethylarginine (ADMA). There are also selective nNOS inhibitors such as 7-nitroindazol (7-NI), S-methylL-thiocitrulline (SMTC) and N[omega]-propyl-L-arginine. Once formed, NO activates soluble guanylyl cyclase and produces cyclic guanosine monophosphate (GMP) from guanosine triphosphate (GTP). Accumulation of cyclic


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20. NITRIC OxIdE ANd AuTONOmIC REgulATION

FIGURE 20.1 Schematic illustration of the effects of NO and of NOS inhibition on blood pressure regulation both centrally and in the periphery.

sympathetic preganglionic neurons [6]. Additionally, in eNOS/KO mice, selective inhibition of neural NOS produces a drop in blood pressure and it has been hypothesized that NO released in the central nervous system and/or the baroreceptor pathways may increase sympathetic nerve activity and increase blood pressure, while the inhibition of NO may have the opposite effect [7]. In summary, nitric oxide in the central nervous system may have both excitatory and inhibitory effects on sympathetic activity, but the evidence from human studies suggest that tonic inhibition of sympathetic tone by NO predominates and may contribute to its role in lowering blood pressure.

NO–AUTONOMIC NERVOUS SYSTEM INTERACTIONS IN THE PERIPHERY In addition to its role as a central modulator of sympathetic activity, NO may also modulate sympathetic vasoconstrictor tone peripherally. Nitric oxide synthase activity has been localized by immunohistochemistry in preganglionic autonomic fibers and postganglionic parasympathetic nerves innervating vascular smooth muscle. There is

evidence that NO released from nitrergic nerves interferes with the release of norepinephrine from adrenergic nerve terminals and with its vasoconstrictive actions on smooth muscle. In anesthetized paralyzed, baroreflex denervated cats, systemic NOS inhibition by L-NAME produced a greater pressor response compared to intact cats [8]. In humans undergoing thoracic sympathectomy, the vasoconstrictor response to systemic infusion of L-NMMA has been evaluated and it has been found that sympathectomy potentiates the vasoconstrictor effect of NOS inhibition only in the denervated limb. This effect is thought to be NO-dependent since a similar increase in forearm vasoconstriction in both limbs was observed during phenylephrine infusion [9]. NO is tonically produced by endothelial cells and in disease states (e.g. during inflammation) also possibly by vascular smooth muscle cells (iNOS) to induce local vasodilatation and, simply through this mechanism, counteract sympathetically-mediated vasoconstriction. NO can be formed locally by either eNOS and nNOS and their relative contribution to endothelial dependent vasodilatation remains a work in progress driven by the availability of selective nNOS inhibitors. The importance of neuronally derived NO in regulating basal vascular tone independently


NO–AuTONOmIC NERvOus sysTEm INTERACTIONs IN HEART RATE CONTROl

of eNOS has been recently studied in humans [10] and in isolated mouse aorta [11]. These studies suggest a role for neurally derived NO in regulating basal vascular tone. In summary, NO acting in the CNS places a brake on sympathetic outflow decreasing sympathetic vasoconstriction. In the periphery, NO produced locally from endothelial and vascular smooth muscle cells, or nitrergic nerves innervating them, produces vasodilatation and counteracts sympathetically-mediated vasoconstriction. NOS inhibition has been shown to produce an increase in sympathetic outflow as well as an increase in NE release. These effects however are masked by the baroreflex. Thus the final effect of NOS inhibition on blood pressure will be the resultant of these effects (Figure 20.1).

NO–AUTONOMIC NERVOUS SYSTEM INTERACTIONS IN HEART RATE CONTROL In addition to modulation of vascular tone, NO has been implicated in the control of cardiac function but its exact role is not completely understood. The effects of NO on heart rate modulation appear to be not merely a baroreflex-mediated response to vasodilatation. NO donors have been shown to elicit a positive chronotropic response [12] that seems to be at least in part independent of the autonomic nervous system since this effect is also seen in heart rate transplant recipients with cardiac denervation [13]. Inhibition of NOS with non-isoform specific inhibitors has shown either no effect on baseline heart rate or a modest negative chronotropic effect. Chronic inhibition of NOS in animal models results in sustained bradycardia. Studies on eNOS/ or nNOS/ KO mice show conflicting results and it may be a reflection on the complex interactions between the different NOS isoforms, the regulation of heart rate by the autonomic nervous system and the specific localization of the different NOS isoforms.

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References [1] Gamboa A, Shibao C, Diedrich A, Choi L, Pohar B, Jordan J, et al. Contribution of endothelial nitric oxide to blood pressure in humans. HTN 2007;49:170–7. [2] Flinspach ML, Li H, Jamal J, Yang W, Huang H, Hah JM, et al. Structural basis for dipeptide amide isoform-selective inhibition of neuronal nitric oxide synthase. Nat Struct Mol Biol 2004;11:54–9. [3] Owlya R, Vollenweider L, Trueb L, Sartori C, Lepori M, Nicod P, et al. Cardiovascular and sympathetic effects of nitric oxide inhibition at rest and during static exercise in humans. Circulation 1997;96:3897–903. [4] Sakima A, Teruya H, Yamazato M, Matayoshi R, Muratani H, Fukiyama K. Prolonged NOS inhibition in the brain elevates blood pressure in normotensive rats. Am J Physiol 1998;275:R410–417. [5] Tagawa T, Imaizumi T, Harada S, Endo T, Shiramoto M, Hirooka Y, et al. Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices. Circ Res 1994;75:70–6. [6] Hakim MA, Hirooka Y, Coleman MJ, Bennett MR, Dampney RA. Evidence for a critical role of nitric oxide in the tonic excitation of rabbit renal sympathetic preganglionic neurones. J Physiol 1995;482:401–7. Pt 2 [7] Kurihara N, Alfie ME, Sigmon DH, Rhaleb NE, Shesely EG, Carretero OA. Role of nNOS in blood pressure regulation in eNOS null mutant mice. HTN 1998;32:856–61. [8] Zanzinger J, Czachurski J, Seller H. Inhibition of sympathetic vasoconstriction is a major principle of vasodilation by nitric oxide in vivo. Circ Res 1994;75:1073–7. [9] Lepori M, Sartori C, Duplain H, Nicod P, Scherrer U. Sympathectomy potentiates the vasoconstrictor response to nitric oxide synthase inhibition in humans. Cardiovasc Res 1999;43:739–43. [10] Melikian N, Seddon MD, Casadei B, Chowienczyk PJ, Shah AM. Neuronal nitric oxide synthase and human vascular regulation. Trends Cardiovasc Med 2009;19:256–62. [11] Capettini LS, Cortes SF, Lemos VS. Relative contribution of eNOS and nNOS to endothelium-dependent vasodilation in the mouse aorta. Eur J Pharmacol 2010;643:260–6. [12] Musialek P, Lei M, Brown HF, Paterson DJ, Casadei B. Nitric oxide can increase heart rate by stimulating the hyperpolarizationactivated inward current, I(f). Circ Res 1997;81:60–8. [13] Chowdhary S, Harrington D, Bonser RS, Coote JH, Townend JN. Chronotropic effects of nitric oxide in the denervated human heart. J Physiol 2002;541:645–51.


C H A P T E R

21 Glutamatergic Neurotransmission Deborah Bauer, Michael Robinson Unlike the neuromuscular junction, where acetylcholine mediates rapid excitatory signaling, glutamate mediates essentially all rapid excitatory signaling in the mammalian CNS. This excitatory signaling is counterbalanced by GABA- or glycine-mediated inhibition (see Chapter 22). The levels of glutamate are 1,000 to 10,000fold higher than most of the other “classical” neurotransmitters, including dopamine, serotonin, norepinephrine, and acetylcholine; they approach 10 mmol/Kg. Glutamatedependent signaling is required for essentially all sensory and motor processing. It contributes to neuronal migration and synapse formation during development. In addition, there is substantial evidence to suggest that plasticity of excitatory synapses contributes to memory formation [1]. In considering glutamate as a neurotransmitter, it may be helpful to remember that glutamate has multiple functions that are not necessarily unique to the nervous system. It is an amino acid and is incorporated into proteins. It is one metabolic step from α-ketoglutarate, an intermediate in the tricarboxylic acid cycle. This cycle is responsible for ~90% of the ATP generated from glycolysis. In the brain there is no intact urea cycle; waste nitrogen (ammonia) is incorporated into glutamate with the subsequent export of glutamine to the periphery. Glutamate is the only precursor for the inhibitory neurotransmitter, GABA. In addition, there are other substances in the brain, such as aspartate, that may serve as excitatory neurotransmitters in the CNS using many of the same receptors as glutamate. For the purposes of this review, we will refer to all of this as glutamatergic neurotransmission. From a disease perspective, stroke and head trauma cause an increase in extracellular glutamate, excessive activation of glutamate receptors, and cell death [2]. This form of cell death is referred to as “excitotoxicity”. Remarkably, evidence for an excitotoxic contribution to stroke-induced brain damage first emerged in the late 1980s, and no effective therapies have emerged in spite of significant efforts by pharmaceutical firms. Excitotoxicity may also contribute to the cell loss that accompanies many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [2]. An imbalance of excitatory and inhibitory signaling contributes to seizure disorders, and glutamate receptor antagonists reduce seizure activity [3]. Finally,


there is significant evidence that altered glutamatergic function contributes to psychiatric conditions and to developmental disorders. Several drugs targeting specific glutamate receptors have entered clinical trials for psychiatric conditions (anxiety, depression, schizophrenia) and Fragile X syndrome [4,5]. While therapeutics targeting glutamatergic neurotransmission are promising, it may be difficult to target glutamate in specific pathways without altering the myriad signals that are required for normal brain function. In this chapter, we provide an overview of our current understanding of glutamate signaling in the nervous system and describe a few specific roles for glutamate in the autonomic nervous system. A simplified schematic of an excitatory synapse is provided (Fig. 21.1).

SYNTHESIS AND VESICULAR RELEASE Although glutamate is available in the diet, it cannot efficiently cross the intact blood brain barrier and rapid signaling requires a robust mechanism to replenish vesicular pools [6]. Although there are several possible routes for glutamate synthesis, it is generally thought that neurotransmitter pools of glutamate are generated from glutamine by glutaminase [6]. Glutamate can also be synthesized from α-ketoglutarate by glutamate dehydrogenase [6]. There are also other routes for glutamate synthesis from proline or from the dipeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), but these are not thought to be robust sources of neurotransmitter pools of glutamate [7]. After synthesis, glutamate is actively transported into vesicles by a family of vesicular glutamate transporters (VGLUT1-3) [8]. These transporters utilize a proton electrochemical gradient to actively accumulate glutamate in synaptic vesicles to concentrations estimated to be as high as 100 mM. None of these vesicular transporters can package aspartate, but recently a distant member of the same gene family was shown to transport aspartate [9]. Interestingly, VGLUT1 and 2 have a complementary distribution in the nervous system with VGLUT1 found in the cerebrum and cerebellum and VGLUT2 enriched in brainstem areas. It is not known why there are two VGLUTs, but it has been suggested that the rhythmic activity of

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PRESYNAPTIC NEURON Gln Gln

Glutaminase

Glu

Glutamine Synthetase

VGLUTs

Glutamine Dehydrogenase

ASTROCYTE

Krebs Cycle

GLAST

mGluRII,III

mGluRII,III

Glu

Kainate Glu

Glu

GLT-1

Kainate

Glu

Glu Glu

GLT-1

EAAC1

mGluRI

Glu

AMPA NMDA

mGluRII

Gammaglutamylcysteine + Glutathione Synthetase

EAAT4

Glutathione

POSTSYNAPTIC NEURON

FIGURE 21.1 Schematic of the glutamate synapse. (Adapted from [27].) Glutamate is synthesized from glutamine with the enzyme glutaminase. Glutamate is packaged into presynaptic vesicles by vesicular glutamate transporters (VGLUTs) and released into the synaptic cleft where it can activate NMDA, AMPA, kainate, and metabotropic glutamate receptors (mGluRs). Glutamate is cleared from the synapse by the plasma membrane glutamate transporters EAAC1 in neurons, and GLAST and GLT-1 in astrocytes. Glutamate can then enter the Krebs cycle, be converted into glutathione, or be converted back into glutamine for the cycle to begin again.

brainstem neurons necessitates a specific vesicular transporter. VGLUT3 is mostly found in presynaptic termini of neurons that are not classically considered glutamatergic, including those that utilize serotonin, acetylcholine, and GABA [8]. This provides further evidence that Dale’s hypothesis, postulating that a neuron only uses one neurotransmitter, is no longer supported by experimental findings.

RECEPTORS Once released into the synapse, glutamate activates ionotropic (ligand-gated ion channel) or metabotropic (G-protein-coupled) glutamate receptors, called iGluRs or mGluRs. iGluRs are named for exogenous agonists that selectively activate subtypes of glutamate receptors, including N-methyl-aspartate (NMDA), α-amino-3hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and kainate [10]. Both groups of receptors are found on

various aspects of the synapse, including the postsynaptic bouton, presynaptic terminus, and nearby astrocytes. AMPA receptors are ligand-gated channels permeable to sodium and potassium, and under some circumstances calcium [11]. Thus, activation of AMPA receptors causes depolarization of the postsynaptic membrane. AMPA receptors are tetramers comprised of the subunits GluR1-4 [10]. These receptors are responsible for the majority of fast glutamate-mediated neurotransmission [2]. Like AMPA receptors, kainate receptors are also ligand-gated channels permeable to sodium and potassium [2]. Kainate receptors are comprised of GluR5-7 and KA1-KA2 [2]. They can exist as homotetramers or heterotetramers [10]. NMDA receptors are ligand-gated channels permeable to Na, K, and Ca2 [11]. Activation of these receptors requires two compounds, glutamate and glycine (or possibly D-serine) [2]. Therefore, in addition to its accepted role as an inhibitory neurotransmitter in brainstem and spinal cord, glycine also contributes to excitatory signaling. NMDA receptors are comprised of four subunits, two


GluTAmATE IN THE AuToNomIC NERvous sysTEm

105

of which must be NR1 subunits, and the other two can be comprised of either NR2 or NR3 subunits [2]. Thus, it is a dimer of dimers. The binding of both glutamate and glycine is not sufficient to activate NMDA receptors when the membrane is near 70 mV because physiologic concentrations of Mg2 bind to and block channel function [2]. This effect of magnesium is voltage-dependent, such that depolarization removes the Mg2 from the channel and allows current to pass through the channel. Thus, the NMDA receptor is considered a “coincidence detector”, it is activated when another receptor causes sufficient membrane depolarization, as would be expected to occur with high frequency stimulation. This unique property of the NMDA receptor is linked to synaptic plasticity that may underlie memory formation [12]. In addition to the ionotropic receptors, there also exist a series of seven transmembrane domain metabotropic glutamate receptors (mGluR1-8) [2]. These receptors are grouped into three families, based on the sequence similarity and their signaling properties [2]. Type I receptors include mGluR1 and 5. These receptors primarily activate phospholipase C and IP3 production. Type II receptors includes mGluR2 and 3. Type III receptors includes mGluR4, 6, 7, and 8. These two families of receptors primarily signal through inhibition of adenylate cyclase and a reduction in cAMP levels. The mGluRs are situated on all aspects of the synapse and might be considered sensors of glutamate; they regulate neurotransmitter release from the presynaptic nerve terminal, iGluR channel opening, and aspects of astrocytic function [13].

EAAC1/EAAT3 expression is enriched in neurons in the CNS [18]. It is found on postsynaptic excitatory neurons, GABAergic neurons, and on the presynaptic nerve terminal. On GABAergic neurons, it may directly provide glutamate for the synthesis of GABA as a complement to the normal source of carbon backbone, which is thought to be glutamine (see Chapter 22). EAAC1/EAAT3 participates in neuronal import of cysteine, the rate limiting ingredient for the synthesis of glutathione. Interestingly, these transporters also gate Cl– ions to a varying degree. In fact, EAAT4 and EAAT5 are quite slow as transporters, but gate substantial Cl– currents [14,19]. Rather than having significant roles in the clearance of glutamate, these two transporters may serve as receptors in the cerebellum and retina, respectively [19].

CLEARANCE OF GLUTAMATE

GLUTAMATE IN THE AUTONOMIC NERVOUS SYSTEM

Unlike some other neurotransmitters, there is no evidence that glutamate is metabolized by extracellular enzymes [14]; instead, it is cleared by a family of Nadependent transporters. In mammals, there are five related gene products that perform this function, including GLAST, GLT-1, EAAC1 (called EAAT1-3, respectively in human), EAAT4 and EAAT5 [14]. These transporters exist as homotrimers [15] and co-transport 3 Na and 1 H with each molecule of glutamate and counter-transport 1 K ion. With this stoichiometry, these transporters are capable of generating a 1 million-fold concentration gradient of glutamate across the membrane [16]. The vast majority of glutamate uptake is mediated by GLT-1 and GLAST with GLT-1 having a larger role in forebrain and GLAST a larger role in cerebellum [17]. These transporters are found on many cell types in the nervous system, but are highly enriched on glial cells. Therefore, unlike any of the other classical neurotransmitters that are recycled back into the presynaptic nerve terminal (see chapters on dopamine, serotonin, and norepinephrine; 6, 8, 12 and 17), glutamate is not substantially recycled back into the presynaptic nerve terminal.

GLUTAMATE METABOLISM After clearance into astrocytes, most, if not all of the glutamate carbon backbone is conserved, by conversion to glutamine by the astrocyte specific enzyme, glutamine synthetase [6]. Glutamine is then exported by a specific transporter, called System N [20]. Glutamine is thought to be imported back into the presynaptic nerve terminal and used for regeneration of glutamate. This is referred to as the glutamate-glutamine cycle. Glutamate can also be converted to α-ketoglutarate by either glutamate-dehydrogenase or a transaminase (either aspartate aminotransferase or glutamate oxalacetate transaminase), and enter the citric acid cycle [6].

In contrast to the CNS, glutamate is not the predominant excitatory neurotransmitter in the periphery. The sodium dependent glutamate transporters are found throughout the body and thus are not a good marker for glutamatergic signaling. However, glutamate receptors and vesicular glutamate transporters have been detected in a variety of peripheral tissues including the kidney, bone, heart, intestine, pancreas, and platelets [14,21]. Thus, while their role in signaling has not been fully elucidated, glutamate is involved in signaling outside of the CNS. For example, glutamate is critical in regulation of the enteric nervous system. Glutamatergic neurons innervate the gut and influence both motility and secretion [22]. Both ionotropic and metabotropic glutamate receptors have been identified in the gut. Although the sympathetic and parasympathetic nervous systems innervate the organs using norepinephrine and acetylcholine, respectively, glutamate is important for central control of these systems. The nucleus tractus solitarii (NTS) of the medulla, often considered the command center of the autonomic nervous system, controls


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cardiovascular and digestive tract reflexes and contains both glutamatergic afferents and efferents [23] (see Fig. 21.2). The importance of glutamatergic signaling in the NTS is clearly illustrated in the baroreceptor reflex (see Fig. 21.2). This reflex begins with activation of baroreceptors, pressure sensitive neurons, found in the aortic arch and carotid sinus that project to the NTS [24]. At this point, the reflex splits into two pathways, the cardioinhibitory pathway and the sympathoinhibitory pathway [24]. In the cardioinhibitory pathway, second order glutamatergic neurons in the NTS excite cholinergic neurons in the nucleus ambiguus (NA) that project to the cardiac ganglion. The cardiac ganglion projects cholinergically to the heart and controls heart rate. This activation of muscarinic acetylcholine receptors causes a decrease in heart rate [24]. In the sympathoinhibitory pathway, second order glutamatergic neurons in the NTS excite GABAergic interneurons in caudal ventrolateral medulla (CVL) [24]. These CVL interneurons project to and inhibit glutamatergic neurons in the rostral ventrolateral medulla (RVLM), which in turn innervate preganglionic sympathetic neurons in the spinal cord. These spinal cord neurons release acetylcholine at the sympathetic ganglion. Finally, noradrenergic neurons in the sympathetic ganglion activate vasoconstriction. However, because excitation of GABAergic interneurons in the CVL causes downstream inhibition of the pathway, the net result is decreased

-

GABA

CVL

Glutamate

+

Some estimates suggest that glutamate mediates upwards of 80% of all neuron–neuron communication in the mammalian CNS. It is required for virtually every physiologic function in mammals, including most brainstem pathways that contribute to autonomic control. In addition to normal physiologic function, excessive activation of glutamate receptors contributes to cell death observed in both acute and chronic neurodegenerative diseases. As our understanding of this system evolves, it seems likely that therapeutic targeting of this system will become more sophisticated.

NTS

+

Sympathoinhibitory

Cardioinhibitory

Glutamate

SUMMARY

Glutamate

+

Glutamate

Glutamate

RVLM

vasoconstriction. Thus, activation of baroreceptors activates two homeostatic pathways collectively known as the baroreceptor reflex, causing heart rate and blood pressure to decrease. Another major output of the NTS where glutamate plays a role is the hypothalamus. Glutamatergic activity in the hypothalamus also influences the baroreceptor reflex. Microinjection of glutamate in the paraventricular nucleus (PVN) of the hypothalamus results in baroreflex bradycardia through activation of NMDA receptors [25]. In fact, glutamate is the major excitatory neurotransmitter providing input to the PVN and supraoptic nucleus of the hypothalamus [26].

+

Heart Acetylcholine

Acetylcholine

NA

+ Cardiac Ganglion

-

+

Acetylcholine

+ SC

Sympathetic

Norepinephrine

Ganglion

FIGURE 21.2 Simplified baroreflex circuit. (Adapted from [24].) Baroreceptors in the aortic arch and carotid sinus project to the nucleus tractus solitarii (NTS) and release glutamate. In the cardioinhibitory pathway, second order glutamatergic neurons in the NTS excite cholinergic neurons in the nucleus ambiguus (NA) that project to the cardiac ganglion. Cholinergic neurons of the cardiac ganglion project to the heart and decrease heart rate. In the sympathoinhibitory pathway, second order glutamatergic neurons in the NTS excite GABAergic interneurons in caudal ventrolateral medulla (CVL). CVL interneurons project to and inhibit glutamatergic neurons in the rostral ventrolateral medulla (RVLM), which in turn innervate preganglionic sympathetic neurons in the spinal cord. These spinal cord neurons release acetylcholine at the sympathetic ganglion which activate noradrenergic neurons in the sympathetic ganglion causing vasoconstriction. The final result of the baroreceptor reflex is a decrease in heart rate and blood pressure.


summARy

References [1] Izquierdo I. Pharmacological evidence for a role of long-term potentiation in memory. FASEB J 1994;8:1139–45. [2] Lau A, Tymianski M. Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch 2010;460:525–42. [3] LaRoche SM, Helmers SL. The new antiepileptic drugs: scientific review. JAMA 2004;291:605–14. [4] Berry-Kravis E, Hessl D, Coffey S, Hervey C, Schneider A, Yuhas J, et al. A pilot open label, single dose trial of fenobam in adults with fragile X syndrome. J Med Genet 2009;46:266–71. [5] Olive MF. Metabotropic glutamate receptor ligands as potential therapeutics for addiction. Curr Drug Abuse Rev 2009;2:83–98. [6] Palmada M, Centelles JJ. Excitatory amino acid neurotransmission. Pathways for metabolism, storage and reuptake of glutamate in brain. Front Biosci 1998;3:d701–18. [7] Robinson MB, Blakely RD, Couto R, Coyle JT. Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain. J Biol Chem 1987;262:14498–14506. [8] Takamori S. VGLUTs: “exciting” times for glutamatergic research? Neurosci Res 2006;55:343–51. [9] Miyaji T, Echigo N, Hiasa M, Senoh S, Omote H, Moriyama Y. Identification of a vesicular aspartate transporter. Proc Natl Acad Sci USA 2008;105:11720–11724. [10] Kew JN, Kemp JA. Ionotropic and metabotropic glutamate receptor structure and pharmacology. Psychopharmacology (Berl) 2005;179:4–29. [11] Mayer ML, Armstrong N. Structure and function of glutamate receptor ion channels. Annu Rev Physiol 2004;66:161–81. [12] Tsien JZ. Linking Hebb’s coincidence-detection to memory formation. Curr Opin Neurobiol 2000;10:266–73. [13] Pin JP, Duvoisin R. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 1995;34:1–26. [14] Danbolt NC. Glutamate uptake. Prog Neurobiol 2001;65:1–105. [15] Gendreau S, Voswinkel S, Torres-Salazar D, Lang N, Heidtmann H, Detro-Dassen S, et al. A trimeric quaternary structure is conserved in bacterial and human glutamate transporters. J Biol Chem 2004;279:39505–39512. [16] Zerangue N, Kavanaugh MP. Flux coupling in a neuronal glutamate transporter. Nature 1996;383:634–7.

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[17] Robinson MB. The family of sodium-dependent glutamate transporters: a focus on the GLT-1/EAAT2 subtype. Neurochem Int 1998;33:479–91. [18] Shashidharan P, Huntley GW, Murray JM, Buku A, Moran T, Walsh MJ, et al. Immunohistochemical localization of the neuronspecific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody. Brain Res 1997;773:139–48. [19] Dehnes Y, Chaudhry FA, Ullensvang K, Lehre KP, StormMathisen J, Danbolt NC. The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: a glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia. J Neurosci 1998;18:3606–19. [20] Fei YJ, Sugawara M, Nakanishi T, Huang W, Wang H, Prasad PD, et al. Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na- and Hcoupled glutamine transporter. J Biol Chem 2000;275:23707–23717. [21] Morrell CN, Sun H, Ikeda M, Beique JC, Swaim AM, Mason E, et al. Glutamate mediates platelet activation through the AMPA receptor. J Exp Med 2008;205:575–84. [22] Kirchgessner AL. Glutamate in the enteric nervous system. Curr Opin Pharmacol 2001;1:591–6. [23] Baude A, Strube C, Tell F, Kessler JP. Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics. J Chem Neuroanat 2009;38:145–53. [24] Benarroch EE. The arterial baroreflex: functional organization and involvement in neurologic disease. Neurology 2008;71:1733–8. [25] Crestani CC, Alves FH, Busnardo C, Resstel LB, Correa FM. N-Methyl-D-aspartate glutamate receptors in the hypothalamic paraventricular nucleus modulate cardiac component of the baroreflex in unanesthetized rats. Neurosci Res 2010;67:317–26. [26] Iremonger KJ, Benediktsson AM, Bains JS. Glutamatergic synaptic transmission in neuroendocrine cells: Basic principles and mechanisms of plasticity. Front Neuroendocrinol 2010;31:296–306. [27] Bauer D, McCullumsmith RE, Meador-Woodruff JH. A role for glutamate receptors, transporters, and interacting proteins in cortical dysfunction in schizophrenia. In: O’Donnell P, editor. In Cortical Deficits in Schizophrenia. New York, NY: Springer; 2008. p. 113–48.


C H A P T E R

22 GABAergic Neurotransmission Andre H. Lagrange, Mark D. Grier OVERVIEW OF GABA MEDIATED SIGNALING GABA is the primary mediator of neuronal inhibition in the brain, as well as being a very important neurotransmitter in the spine and peripheral nervous system. Major components of the GABAergic neurotransmission system include two enzymes that synthesize GABA, two classes of receptors, two types of transporters, and an enzyme that degrades GABA. Nearly all GABA found in the mammalian brain is synthesized by decarboxlyation of glutamate by two forms of glutamic acid decarboxylase, GAD65 and GAD67. While GAD67 is present as a nearly constitutively active enzyme throughout the cell, GAD65 is localized primarily to the axon terminals and only becomes highly active in periods of increased demand. After GABA is synthesized, it is packaged into synaptic vesicles by the vesicular GABA transporter vGAT (also known as vesicular inhibitory amino acid transporter, VIAAT). Upon stimulation of the neuron, these vesicles fuse to the presynaptic terminal and release GABA into the synapse via calcium dependent exocytosis. GABA is removed from the synapse by another class of GABA transporter, GATs. It is important to note that while the function of vGAT and GAT are similar, their structure and mechanisms of transport are very different. To date, there have been four GAT isoforms identified, each with unique pharmacology and cellular localization. In the CNS, the main transporters are GAT1 and GAT3, expressed primarily on presynaptic terminals and astrocytes, respectively. Once GABA is cleared from the synapse it is degraded by GABA-transaminase. Inhibitors of GABA reuptake and breakdown effectively produce increased levels of extracellular GABA and are used clinically in conditions of CNS hyperexcitability, such as spasticity and epilepsy [1].

GABA RESPONSES ARE MEDIATED BY BOTH IONOTROPIC AND METABOTROPIC RECEPTORS The neuronal response to GABA is mediated through two populations of receptors, GABAA and GABAB receptors. While both populations generally produce neuronal


inhibition, they do so through wholly different mechanisms. GABAA receptors are primarily postsynaptic ligand-gated Cl ion channels, while GABAB receptors are G-protein coupled receptors found on both pre- and postsynaptic terminals. Activation of GABAA receptors produces Cl ion fluxes that directly alter membrane potential. In contrast, the effects GABAB receptor stimulation are mediated by G-proteins that produce a variety of responses, including activation of potassium channels, inhibition of presynaptic voltage-gated calcium channels and modulation of intracellular second messenger systems, including adenylate cyclase and phospholipase C. This review will focus primarily on GABAA receptors; however it is important to note that GABAB receptors also play an important role in inhibition in the central nervous system.

GABAA RECEPTORS MEDIATE MULTIPLE MODES OF NEURONAL SIGNALING GABAA receptors have the important ability to convey two different types of inhibition within the brain [2]. Phasic inhibition involves temporally and spatially precise responses to very brief, high levels of synaptic GABA (≈1 mM, 1 ms). In comparison, synaptic spillover produces prolonged low levels of GABA in the extrasynaptic space that activate highly sensitive GABAA receptor subtypes, thereby producing a tonic inhibitory current (Fig. 22.1). Despite its small amplitude, the prolonged duration of tonic inhibition produces an overall charge transfer that exceeds that of synaptic neurotransmission in certain brain regions. Furthermore, by effectively reducing membrane resistance, tonic inhibition reduces the ability of excitatory currents to depolarize the cell toward the action potential threshold. These dual effects allow tonic inhibition to serve as a critical determinant of overall network excitability in a number of important brain regions. While classically thought of as an inhibitory neurotransmitter, GABAA receptors also have the unusual capacity to convey excitatory responses, depending on the Cl reversal potential across the neuronal membrane [3]. In normal adult neurons, robust activity of the Cl transporter KCC2 results in low intracellular Cl levels

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Strong Synaptic Input

Weak Synaptic Input

Presynaptic Neurons

Presynaptic Neurons Glia

Glia

Metabolism

Metabolism

GABA-T

GABA-T

VGAT

GABA

GABA

Postsynaptic Neuron GABA A Receptor GAT

GABA B Receptor Synaptic Vesicle

Postsynaptic Neuron GABA A Receptor GAT

GABA B Receptor Synaptic Vesicle

FIGURE 22.1 GABA receptors mediate multiple forms of neurotransmission. During weak synaptic activity, brief surges of GABA activate the ionotropic GABAA receptors clustered within the synapse, producing transient inhibitory postsynaptic currents. However, during more pronounced synaptic activity, GABA can overflow beyond the synapse, activating highly sensitive GABAA receptor subtypes that exist outside the synapse to produce a long-lived tonic current. G-protein coupled GABAB receptors on both the pre- and postsynaptic neurons are also activated by synaptic overflow to help suppress neuronal activity.

compared to the extracellular fluid, thereby producing a hyperpolarizing influx of negative Cl ions when GABAA receptors are activated. In contrast, maturing neurons express very little KCC2, and anion homeostasis is largely determined by a different Cl transporter (NKCC1) that preferentially transports Cl into the cell. This produces high levels of intracellular Cl that allow GABAA receptor activation to produce a depolarizing efflux of anions [3] that can generate action potentials, intracellular calcium waves, and relieve Mg block of NMDA receptors. These excitatory responses regulate a number of important steps in brain development, including the formation of both excitatory and inhibitory synapses. Interestingly, similar depolarizing responses can be found in specific subcellular regions in the normal adult neurons, as well as more widely following brain injury. Furthermore, these excitatory GABA responses may be involved in the pathogenesis of some neurological disorders (e.g. epilepsy) and selective inhibition of NKCC1 with drugs like bumetanide, has been proposed as a potential future treatment for some of these conditions [4].

NEUROTRANSMISSION IS MEDIATED BY A DIVERSE SET OF GABAA RECEPTOR SUBTYPES GABAA receptors are heteropentameric protein complexes, generally composed of a combination of 2α, 2β and either a γ or δ subunit. To date, there are 6α, 3β, 3γ and a single δ isoform subtypes. Despite a vast array of possible subunit combinations, only a limited number of GABAA receptor subunit combinations are actually found in native tissue. The expression of individual subunit combinations is highly region and cell-type specific. While most synaptic responses throughout the brain are thought to be mediated by postsynaptic α1β2γ2 containing GABAA receptors, other

subunit combinations are highly expressed in select brain regions (thalamus, hypothalamus, hippocampus), as well as in specific subcellular domains, including the axon initial segment (α2βxγ) or extrasynaptic space (α4βxδ and α5β3γ2). Since the identity of the constituent subunit isoforms is the primary determinant of GABAA receptors’ sensitivity to GABA and current kinetic properties, specific subunit isoform combinations likely serve very different physiological functions. For example, α4βxδ containing receptors are more than 10–100 times more sensitive to low levels of GABA than some other subunit combinations, allowing them to respond to the very low levels of ambient GABA in the extrasynaptic space. In contrast, the less sensitive, but rapidly activating α1β2γ2 containing GABAA receptors are found clustered within inhibitory synapses, where they are able to convert extremely brief, high levels of GABA to more prolonged postsynaptic currents. However, these receptors also desensitize quickly, and respond poorly during repetitive input. Replacement of the α1 subunit with the α3 subunit, produces receptors that are even less sensitive to low GABA, but are more slowly desensitizing and deactivate extremely slowly, resulting in very prolonged IPSCs that can produce a summating response during repetitive stimulation [5,6]. More recently, it has become clear that these patterns of subunit expression can be profoundly disrupted in a variety of pathological states like ischemia and seizures [7,8]. These changes may be compensatory, but are also thought to contribute to some of the pathological plasticity that occurs after brain injury.

SUBTYPE PREFERRING DRUGS ALLOW FOR SELECTIVE MODULATION OF GABA SIGNALING Given that essentially every neuron in the mammalian brain expresses GABAA receptors, it is not surprising that


suBTyPE PREfERRING DRuGs Allow foR sElECTIvE moDulATIoN of GABA sIGNAlING

a number of the most useful medications used in neurology and psychiatry target these receptors. Nonselective GABAA receptor-active drugs, like barbiturates, and inhaled anesthetics, propofol and etomidate, are commonly used for surgery. At low concentrations, these agents enhance the effects of endogenous GABA, but at higher concentrations they directly and indiscriminately activate GABAA receptors throughout the brain. While this allows these agents to serve as general anesthetics, it also introduces the possibility of lethal effects on respiration and blood pressure. Fortunately, there are also a variety of subunit-preferring medications that allow selective modulation of specific GABAA receptor subunits combinations. For example, benzodiazepines increase the GABA affinity of α(1, 2, 3 or 5)βxγx containing GABAA receptors. Even more selective is the benzodiazepine-like hypnotic compound, zolpidem. This drug potently enhances α1βxγ2 mediated inhibition, has much weaker effects on α2,3βxγ2 mediated currents, and is virtually inactive at all other GABAR subunit combinations. Since synaptic GABAA receptors are usually exposed to saturating levels of GABA, the affinityenhancing effects of benzodiazepines and zolpidem produce prolonged IPSCs, without much effect on current amplitude. In contrast, extrasynaptic α5β3γ2 GABAA receptors on hippocampal neurons are exposed to prolonged, low levels of GABA. Consequently, benzodiazepines, but not zolpidem, produce increased tonic current amplitude in these cells. This form of inhibition is thought to be important in learning/memory, and suppression of tonic inhibition with α5βxγ-selective benzodiazepine site inverse agonists is currently being evaluated as a potential mechanism for cognitive enhancement. Similar agents that modulate α2 and α3 containing receptors are in more preliminary stages of development as anxiolytics, muscle relaxants and analgesics [9]. The other predominant mediators of tonic inhibition in the brain are α4βδ containing GABAA receptors. These relatively small GABA-evoked currents can be enhanced several fold by barbiturates and ethanol. While truly α4βδ selective agents are lacking, the drug gaboxadol is a superagonist with particularly high potency and efficacy at this particular subunit combination, and low concentrations of this drug are often used to selectively enhance α4βδ mediated tonic inhibition. Interestingly, these receptors are also especially prone to modulation by endogenous neuroactive steroid hormones produced in the gonads, adrenals and even in the CNS itself. While steroid hormones are generally associated with long-term changes at the genomic level, neuroactive steroids, like allopregnanolone (3α,5α-tetrahydroprogesterone) and THDOC (allotetrahydrocorticosterone) enhance GABAergic signaling within seconds. This enhancement is not mediated by the classic nuclear steroid hormone receptors, nor is it mediated by changes in protein synthesis. Instead, these neuroactive steroids are thought to interact directly with GABAA receptors, possibly involving specific binding site(s) in the transmembrane region. While allopregnanolone and THDOC enhance most GABAA receptor subunit

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combinations to some degree, αxβxδ containing receptors are especially sensitive to even extremely low neurosteroid concentrations. Moreover, αxβxδ containing receptors typically produce small currents due to a lower single channel conductance and mean open times. However, nanomolar concentrations of neurosteroids shift the activation properties of αxβxδ containing receptors, producing a nearly ten-fold increase in the maximal GABA evoked current [10]. In summary, GABA is a multifunctional neurotransmitter capable of producing brief, phasic responses or tonic currents that may be either excitatory or inhibitory, depending on the age, brain region, and cell-type being studied. In addition, the control of GABAergic signaling involves multiple proteins, many of which have multiple isoforms. The regulation of GABAergic signaling is a critical determinant of nervous system function, and disruption of this system is thought to play a crucial role in many neurological disorders. Fortunately, the availability of a number of isoform-selective drugs at these diverse preand postsynaptic targets provides an unparalleled potential for pharmacological manipulation of neuronal activity in the treatment of human illness.

References [1] Bialer M, Johannessen SI, Kupferberg HJ, Levy RH, Perucca E, Tomson T. Progress report on new antiepileptic drugs: a summary of the Seventh Eilat Conference (EILAT VII). Epilepsy Res 2004;61:1–48. [2] Farrant M, Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci 2005;6:215–29. [3] Ben Ari Y, Gaiarsa JL, Khazipov R. GABA: A pioneer transmitter that excites immature neurons and generates primitive oscillations. Phys Rev 2007;87:1215–84. [4] Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, et al. NKCC1 transporter facilitates seizures in the developing brain. Nat Med 2005;11:1205–13. [5] Cox CL, Huguenard JR, Prince DA. Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus. Proc Natl Acad Sci USA 1997;94:8854–9. [6] Rula EY, Lagrange AH, Jacobs MM, Hu N, Macdonald RL, Emeson RB. Developmental modulation of GABA(A) receptor function by RNA editing. J Neurosci 2008;28:6196–201. [7] Clarkson AN, Huang BS, Macisaac SE, Mody I, Carmichael ST. Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke. Nature 2010;468:305–9. [8] Zhang N, Wei W, Mody I, Houser CR. Altered localization of GABA(A) receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. J Neurosci 2007;27:7520–31. [9] Mohler H. GABA(A) receptor diversity and pharmacology. Cell Tissue Res 2006;326:505–16. [10] Wohlfarth KM, Bianchi MT, Macdonald RL. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. J Neurosci 2002;22:1541–9.


C H A P T E R

23 Renin-Angiotensin Amy C. Arnold, Debra I. Diz INTRODUCTION The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure under normal and pathophysiological conditions. The RAS interacts with the autonomic nervous system for blood pressure regulation, with angiotensin receptors localized to brain regions involved in modulation of both sympathetic and parasympathetic nervous system activity. The following provides a brief overview of the classical and brain RAS, focusing on interactions of this hormonal system with autonomic brain regions integral to cardiovascular control.

THE CLASSICAL RAS

Recent Advances in the Classical RAS

Components and Features of the Classical RAS As recently reviewed [1,2] the classical circulating RAS is a series of enzyme-substrate interactions that generates angiotensin peptides involved in cardiovascular, fluidelectrolyte and neuroendocrine homeostasis. The ratelimiting enzyme, renin, is synthesized in juxtaglomerular cells of the kidney and is secreted into the circulation in response to various stimuli. As illustrated in Figure 23.1, circulating renin acts upon hepatic-derived angiotensinogen to form angiotensin I. Angiotensin converting enzyme (ACE), a lung-derived dipeptidyl carboxypeptidase, subsequently cleaves angiotensin I into angiotensin II, the main effector peptide of the RAS. However, other enzymes including chymase are capable of forming angiotensin II in pathological conditions. Four subtypes of the angiotensin II g-protein coupled receptor (AT1–AT4) have been identified. The majority of angiotensin II effects are mediated by ubiquitously expressed AT1 receptors. Depending on the cell and tissue type, AT1 receptor activation is associated with inhibition of adenylyl cyclase, activation of phospholipase C or phosphoinositide hydrolysis. Angiotensin II stimulation of AT1 receptors induces vasoconstriction, sympathetic activation, suppression of baroreflex function, sodium and water reabsorption, cellular proliferation and hypertrophy, inflammation, oxidative stress and release of aldosterone, vasopressin and noradrenaline (Table 23.1). Angiotensin II also binds AT1 receptors on juxtaglomerular


cells to provide negative feedback suppression of renal renin release. These actions are collectively abrogated by ACE inhibitors or AT1 receptor antagonists, to prevent the formation or binding of angiotensin II, respectively. Angiotensin II also binds AT2 receptors, with expression of these receptors abundant during fetal life, but declining into adulthood. The AT2 receptor opposes AT1 activation with respect to blood pressure and cellular proliferation and is also involved in growth, repair, pain threshold and neuronal cell maturation and differentiation (Table 23.1). Angiotensin II is degraded by aminopeptidases A and N into the shorter fragments angiotensin III and IV, respectively (Fig. 23.2, Table 23.1).

New pathways for the production of angiotensin peptides have been recently revealed, including the formation of the heptapeptide angiotensin-(1-7) (Figs. 23.1 and 23.2). Angiotensin-(1-7) can be formed via cleavage of angiotensin I by various endopeptidases including neprilysin (NEP), thimet oligopeptidase (TOP) and prolyl oligopeptidase (POP) [3,4]. Alternatively, angiotensin-(1-7) is formed through cleavage of angiotensin II by ACE2, a novel homolog of ACE (Fig. 23.1) [3–5]. The actions of angiotensin-(1-7) are mediated by g-protein coupled mas receptors and are blocked by the selective receptor antagonist [D-Ala7]angiotensin-(1-7) [3]. Angiotensin-(1-7) is degraded into the inactive metabolite angiotensin-(1-5) by ACE [3]. The complexity of the RAS is further increased by the recent discovery of angiotensin-(1-12), a C-terminally extended peptide longer than angiotensin I (Figs. 23.1 and 23.2) [6]. Angiotensin-(1-12) is found in plasma and peripheral tissues and appears to serve as a renin-independent precursor for the production of angiotensin peptides [4].

The Classical RAS and Autonomic Regulation Systemic angiotensin II elicits vasoconstriction, sympathetic activation, baroreflex dysfunction and neuroendocrine and fluid-electrolyte changes (Table 23.1), all of which can contribute to blood pressure elevations [1,7]. The sympathetic and baroreflex actions of circulating angiotensin II involve interactions with AT1 receptors in specialized circumventricular organs lacking a functional

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23. REnIn-AngIoTEnsIn

FIGURE 23.1 Schematic diagram of the current view of the reninangiotensin system showing biochemical pathways involved in the formation, degradation and actions of the biologically active peptides angiotensin II and angiotensin-(1-7). Ang, angiotensin; ACE, angiotensin converting enzyme; NEP, neprilysin; TOP, thimet oligopeptidase; POP, prolyl oligopeptidase; AT1-R, angiotensin type 1 receptor; AT2-R, angiotensin type II receptor; mas-R, mas receptor.

TABLE 23.1 Receptors and Actions of Angiotensin Peptides Receptor

Actions

Potential Ligands

AT1

Increase blood pressure, vasoconstriction, sympathetic activation, decrease baroreceptor reflex sensitivity for heart rate control and increase baroreflex set-point, pro-inflammation, pro-oxidative, thirst behaviors, cell growth and hypertrophy, anxiety, stress, neuronal excitation, and stimulate aldosterone, norepinephrine and vasopressin release

Angiotensin II Angiotensin III Angiotensin-(1-12)– likely after conversion to Angiotensin II

AT2

Vasodilation, decrease in blood pressure, apoptosis, antiinflammation, neuronal development and differentiation

Angiotensin II Angiotensin III

AT1-7 [mas]

Vasodilation, decrease in blood pressure, anti-inflammation, anti-oxidant, increase baroreceptor sensitivity for heart rate control, neuronal excitation

Angiotensin-(1-7)

AT4

Memory, learning, Angiotensin IV depression, anxiety, prevents insulin-regulated aminopeptidase (IRAP)mediated metabolism of vasoactive peptides

FIGURE 23.2 Amino acid sequences for peptides of the renin-angiotensin system.

blood-brain barrier [7]. The importance of circulating angiotensin II to hypertension is illustrated by the finding that ACE inhibitors or AT1 receptor blockers lower blood pressure, reset the baroreflex set-point and improve baroreflex sensitivity in hypertensive populations [8]. These therapies also increase levels of angiotensin-(1-7), a peptide that stimulates mas receptor-mediated vasodilation and improvement of baroreflex function (Table 23.1) [1,3]. In rodents, low doses of angiotensin-(1-7) increase vasodilation through interactions with prostaglandinbradykinin-nitric oxide systems [3,4]. However, the systemic effects of angiotensin-(1-7) on blood pressure in humans are controversial. The counter-regulatory actions of angiotensin II and angiotensin-(1-7) suggest that the balance of these two peptides is important to cardiovascular physiology and pathophysiology. Indeed, emerging evidence suggests that cardiovascular diseases are associated with an imbalance in the angiotensin II/ACE and angiotensin-(1-7)/ACE2 axes [3–5]. Possibly shifting the balance of the RAS in the circulation, angiotensin-(1-12) acts as a precursor to angiotensin II for peripheral cardiovascular actions including vasoconstriction and elevations in blood pressure [4,6].

THE BRAIN RAS All components of the RAS are present in distinct, independently-regulated tissue systems including adipose, kidneys, blood vessels, heart and brain [2]. As early as 1961, studies demonstrated that angiotensin II acts centrally to increase blood pressure [7]. At this time, it was hypothesized that angiotensin II interacted with the brain through AT1 receptors in circumventricular organs. A few years later, the existence of an independent brain RAS was postulated by Ganten and colleagues. Indeed, all necessary precursors and enzymes required for the local formation and metabolism of angiotensins are found in brain [2,7]. However, controversy still exists regarding the cellular localization, independence from the circulating system and authenticity of brain RAS peptides [9].


THE BRAIn RAs

Components of the Brain RAS Angiotensinogen is widely expressed in the central nervous system with high levels in cerebrospinal fluid, hypothalamic and brainstem regions [2,7]. While angiotensinogen is primarily produced in astrocytes, it is also present in neuronal cells of key cardiovascular nuclei [7]. Renin immunoreactivity is described in discrete glial and neuronal cells of the pituitary, choroid plexus, medulla and hypothalamus. However, renin expression in brain is low suggesting alternate pathways exist for central production of angiotensin peptides [2,7]. All enzymes necessary for processing angiotensin I into bioactive peptides are found in brain (ACE, ACE2, NEP and aminopeptidases) [7]. While the exact mechanisms involved in their formation remains unclear, angiotensin peptides are all reported in brain, with the highest levels demonstrated for angiotensin II and angiotensin-(1-7) [7]. Angiotensin II AT1 receptors are highly localized to brain regions associated with autonomic outflow as well as areas involved in energy homeostasis, respiration, salt appetite, vasopressin release and thirst [2,7]. Low levels of AT2 receptors are also found in brain regions associated with cardiovascular, motor, sensory and limbic activity [7]. The active fragment angiotensin III binds central AT1 receptors to mediate actions attributed to angiotensin II including regulation of blood pressure and vasopressin release (Table 23.1) [1]. Central stimulation of AT4 receptors by angiotensin IV prevents insulin-regulated aminopeptidase-mediated metabolism of vasoactive peptides [7] and is also implicated in depression, learning, memory and anxiety (Table 23.1) [7].

Influence of the Brain RAS on Autonomic Regulation Angiotensin II AT1 and angiotensin-(1-7) mas receptors are abundant at each synaptic relay of the sympathetic and parasympathetic nervous systems, including spinal cord and ganglia [7,8]. AT1 receptors are distributed in sympathetic preganglionic neurons, sympathetic ganglia, sympathetic nerve terminals and sympathetic regulatory brain sites including hypothalamus, dorsal medullary and ventral medullary regions [10]. Central angiotensin II infusion increases arterial pressure and stimulates whole body and regional sympathetic activity in rodents, in part mediated by stimulatory interactions with other neurotransmitters including norepinephrine, dopamine and substance P [10]. Angiotensin II also acts at AT1 receptors within the solitary tract nucleus (NTS) to attenuate baroreflex restraint of sympathetic outflow to the heart, kidney and vasculature [8,10]. The NTS has descending projections to brainstem vasomotor nuclei, including the rostral and caudal ventrolateral medullas, which respond to angiotensin peptides to influence sympathetic activity, blood pressure and baroreflex function [8]. Importantly, a positive feedback loop exists between the RAS and sympathetic nervous system as increased renal sympathetic nerve activity is a major

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stimulus for β-adrenergic receptor-mediated renal renin release [10]. Angiotensin receptors are also distributed in vagal pathways influencing control of the parasympathetic nervous system [7,8,10]. High-affinity AT1 receptor binding sites are located on presynaptic vagal afferent terminals and on NTS cell bodies, with a small population of AT2 receptors in this brain region [8]. Central infusion or NTS microinjection of angiotensin II or angiotensin III impairs the baroreflex sensitivity for control of heart rate for bradycardia to increases in arterial pressure, an established marker of parasympathetic function, independent of changes in resting pressure [8,10]. Furthermore, NTS administration of AT1 receptor antagonists improves baroreflex sensitivity in rats suggesting that angiotensin II endogenous to the brain attenuates baroreflex function [8]. Illustrating the importance of brain angiotensin II to blood pressure regulation, transgenic rodents with increased brain angiotensin II generation are hypertensive and have impaired baroreflex sensitivity while those with reduced brain angiotensin II synthesis are hypotensive and have lower sympathetic and higher parasympathetic tone [2]. Studies in transgenic rodents also suggest that the source of angiotensin II for baroreflex modulation is of glial origin, whereas the source of the peptide for blood pressure regulation is neuronal [11]. Similar to the periphery, central actions of angiotensin-(1-7) oppose angiotensin II. However, both peptides can elicit similar pressor/depressor actions in specific brain nuclei and stimulate hypothalamic vasopressin release [3,4]. Numerous studies document that central administration of angiotensin-(1-7) lowers blood pressure, increases baroreflex sensitivity for control of heart rate and reduces norepinephrine release in hypertensive rodents [5]. Moreover, [D-Ala7]-angiotensin-(1-7) impairs baroreflex sensitivity in normotensive rats suggesting angiotensin-(1-7) endogenous to the brain facilitates baroreflex function [11]. Thus, the prevailing level of baroreflex sensitivity appears to reflect the balance of angiotensin II and angiotensin-(1-7) actions in the brain. This balance may in part depend on local levels of ACE2, the enzyme that converts angiotensin II to angiotensin-(1-7). Indeed, central genetic deletion or NTS pharmacologic inhibition of ACE2 reduces baroreflex sensitivity in rodents [5]. In contrast, central overexpression of ACE2 lowers blood pressure and restores baroreflex function in hypertensive animals demonstrating an emerging role for brain ACE2 in central cardiovascular regulation [5]. The angiotensinogen: renin mismatch is an ongoing topic of controversy for synthesis of angiotensin peptides within brain [9]. Levels of angiotensin-(1-12) are reported similar or higher to angiotensin II in brain [6]. In tissues, angiotensin-(1-12) can form either angiotensin II or angiotensin-(1-7) depending on the local enzyme milieu [4]. However, central immunoneutralization of angiotensin-(1-12) lowers blood pressure and improves baroreflex sensitivity in hypertensive rats [12] and NTS


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microinjection of angiotensin-(1-12) impairs baroreflex sensitivity in normotensive animals [13]. The baroreflex actions of angiotensin-(1-12) within the NTS are blocked by either an AT1 receptor antagonist or ACE inhibitor suggesting that angiotensin-(1-12) serves as a precursor to angiotensin II in brain pathways regulating blood pressure and autonomic function. Since angiotensin-(1-12) metabolism is independent of renin [4], this peptide may provide an alternate pathway for central angiotensin peptide formation.

SUMMARY Due to its key role in blood pressure regulation, the RAS has been an intense subject of research and an important therapeutic target for cardiovascular disease. Independent of blood pressure lowering effects, blockade of angiotensin II activity with ACE inhibitors or AT1 receptor antagonists reduces sympathetic activity and restores baroreflex function, in part mediated by increased levels of angiotensin-(1-7). These observations illustrate the importance of interactions between the RAS and autonomic nervous system in central regulation of blood pressure. A wider influence of the RAS on other facets of autonomic function, including energy metabolism, body weight regulation and glucose homeostasis at peripheral and central nervous system sites is emerging. The discovery of new RAS components, including ACE2, angiotensin-(1-7) and angiotensin-(1-12), will improve our understanding of these interactions that contribute broadly to autonomic regulation and may open new avenues for pharmacologic targeting of this system.

Acknowledgement

References [1] Fyhrquist F, Saijonmaa O. Renin-angiotensin system revisited. J Intern Med 2008;264:224–36. [2] Bader M. Tissue renin-angiotensin-aldosterone systems: Targets for pharmacological therapy. Annu Rev Pharmacol Toxicol 2010;50:439–65. [3] Trask AJ, Ferrario CM. Angiotensin-(1-7): pharmacology and new perspectives in cardiovascular treatments. Cardiovasc Drug Rev 2007;25:162–74. [4] Varagic J, Trask AJ, Jessup JA, Chappell MC, Ferrario CM. New angiotensins. J Mol Med 2008;86:663–71. [5] Xia H, Lazartigues E. Angiotensin-converting enzyme 2: central regulator for cardiovascular function. Curr Hypertens Rep 2010;12:170–5. [6] Nagata S, Kato J, Sasaki K, Minamino N, Eto T, Kitamura K. Isolation and identification of proangiotensin-12, a possible component of the renin-angiotensin system. Biochem Biophys Res Commun 2006;350:1026–31. [7] McKinley MJ, Albiston AL, Allen AM, Mathai ML, May CN, McAllen RM, et al. The brain renin-angiotensin system: location and physiological roles. Int J Biochem Cell Biol 2003;35:901–18. [8] Averill DB, Diz DI. Angiotensin peptides and baroreflex control of sympathetic outflow: pathways and mechanisms of the medulla oblongata. Brain Res Bull 2000;51:119–28. [9] Grobe JL, Xu D, Sigmund CD. An intracellular renin-angiotensin system in neurons: fact, hypothesis, or fantasy. Physiology (Bethesda) 2008;23:187–93. [10] Phillips MI. Functions of angiotensin in the central nervous system. Annu Rev Physiol 1987;49:413–35. [11] Sakima A, Averill DB, Kasper SO, Jackson L, Ganten D, Ferrario CM, et al. Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen. Am J Physiol Heart Circ Physiol 2007;292:H1412–H1419. [12] Isa K, Garcia-Espinosa MA, Arnold AC, Pirro NT, Tommasi EN, Ganten D, et al. Chronic immunoneutralization of brain angiotensin-(1-12) lowers blood pressure in transgenic (mRen2)27 hypertensive rats. Am J Physiol Regul Integr Comp Physiol 2009;297:R111–5. [13] Arnold AC, Isa K, Shaltout HA, Nautiyal M, Ferrario CM, Chappell MC, Diz DI. Angiotensin-(1-12) requires angiotensin converting enzyme and AT1 receptors for cardiovascular actions within the solitary tract nucleus. Am J Physiol Heart Circ Physiol 2010;299:H763–71.

Funding: NIH HL-51952.


C H A P T E R

24 Aldosterone and the Mineralocorticoid Receptor J. Howard Pratt Aldosterone, the only true mineralocorticoid, is secreted by the zona glomerulosa, a thin layer of cells at the periphery of the adrenal cortex. Aldosterone’s principal functions are to contain extracellular potassium (K) concentration within narrow limits and to maintain adequate extracellular volume by guarding against sodium (Na) loss. The two stimuli that regulate its secretion are therefore K (only small increments are required to significantly augment the levels of aldosterone) and angiotensin II (which increases in response to the need for volume expansion and retention of additional Na). Although not as well-established, there is compelling evidence that the adrenergic nervous system influences aldosterone secretion. Rays of adrenal medullary cells extending out to the zona glomerulosa have been described in the rat adrenal, and catecholamines have been shown to stimulate aldosterone secretion in vitro. More recently, variants in the β-adrenergic receptor were found to associate with low renin hypertension. Taken together, the findings suggest the possibility of a significant contribution by the adrenergic nervous system to hypertensive states resulting from overproduction of aldosterone [1].

K channel. The amount of K secreted is a principal determinant of extracellular K concentration. Thus, by increasing the number of ENaCs residing on the apical surface, aldosterone achieves a needed level of Na reabsorption, together with maintenance of K homeostasis.

Effects of Aldosterone That Target Distal Nephron

Excess Aldosterone and the Development of Hypertension

The functions of aldosterone begin with its occupancy of the nuclear receptor known as the mineralocorticoid receptor (MR). The ligand coupled to its receptor becomes a functioning transcription factor, and thus there is genomic delay in the onset of its actions. MR is expressed in a variety of tissues including the heart and brain, but the major site for its expression is the kidney’s distal nephron. Here, the principal target is the epithelial Na channel or ENaC located in cortical collecting duct [2]. Aldosterone induces translation for the protein serum glucocorticoid kinase type I (Sgk-1) [3]. The ultimate effect is to render an ubiquitin ligase (Nedd4-2) incapable of removing ENaC from the apical surface of the cell [4]. The Na that reaches the cell interior via ENaC exits the cell at the basolateral surface via the Na,K-ATPase; the exchange of Na for K leads to secretion of K into the tubular lumen via a


Specificity of MR for Aldosterone; Role of 11-β-Hydroxysteroid Dehydrogenase Type II (11BHSD2) Cortisol is also a ligand for MR and its circulating levels are much higher than those of aldosterone. However, under normal circumstances cortisol is converted to cortisone by 11BHSD2, an enzyme expressed in the vicinity of MR. Cortisone is biologically inactive; it has no binding affinity to MR. A deficiency in 11BHSD2 can result from a rare genetic disorder (apparent mineralocorticoid excess), from eating licorice (glycyrrhizynic acid in licorice inhibits 11BHSD2) or from Cushing’s syndrome (typically due to ectopic ACTH secretion where extremely high cortisol levels overwhelm the enzyme). In each case, there can be hypertension and typically hypokalemia.

Autonomous hypersecretion of aldosterone (primary aldosteronism) not uncommonly results in severe hypertension due to excessive reabsorption of Na in the distal nephron. The ensuing volume expansion suppresses the renin-angiotensin axis. The diagnosis is established when the plasma level of aldosterone is increased while the level of plasma renin activity (PRA) is low. An additional diagnostic approach is to utilize the ratio of plasma aldosterone to PRA (aldosterone/renin ratio). (Fig. 24.1) There are two types of primary aldosteronism (Fig. 24.2): that which is caused by a solitary adrenal adenoma (frequently 1 cm in diameter), also known as Conn’s syndrome [5]; and, the most common form, bilateral adrenal hyperplasia. The prevalence of primary aldosteronism is now estimated to be 10% of patients with hypertension

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24. AldosTERonE And THE MInERAloCoRTICoId RECEPToR

Two types of primary aldosteronism

Solitary adenoma MR ENaC (α - β - γ subunits)

Right

Left

Bilateral adrenal hyperplasia

FIGURE 24.2 The great majority of cases of primary aldosteronApical

Basolateral

FIGURE 24.1 Schematic depiction of mechanism for how aldosterone increases Na uptake by ENaC in principal cells of collecting duct. ENaC residing on the apical surface consists of three subunits (alpha, beta, gamma). ENaC is removed from the cell surface by the ubiquitin ligase Nedd4-2. Aldosterone binds to MR in the cell’s interior resulting in a transcription factor that increases expression of Sgk-1. The latter phosphorylates Nedd4-2, and by so doing, inactivates it to where it can no longer displace ENaC from the apical surface. The result is a prolonged residency time with greater Na reabsorption.

[6]. The prevalence increases with age and is exceptionally high in patients with resistant hypertension [7] (patients with uncontrolled hypertension on three or more antihypertensive drugs). Although it was initially thought that hypokalemia was a requisite feature, it is now well accepted that most patients with primary aldosteronism have a normal serum K concentration. Treatment approaches depend on whether the source of excess aldosterone is from a unilateral adrenal adenoma or arises from hyperplasia of both adrenals. Once it is confirmed, often by performing an adrenal venous catheterization, that a single adenoma is causative, it can be removed using laparoscopic surgery. Medical management for bilateral disease is employed most often. There are currently two MR blockers, spironolactone and eplerenone. In addition, direct inhibitors of ENaC can be used such as amiloride. A description of medical treatment options is listed in Table 24.1.

Low-Renin Hypertension but not Primary Aldosteronism Many patients with hypertension who may require multiple antihypertensive drugs and patients who are typically older have suppressed levels of PRA. Their aldosterone levels are in the normal range and thus do not have primary aldosteronism based on established criteria. These patients more often than not respond extremely

ism are caused either by an adrenal adenoma or by bilateral adrenal hyperplasia. The former is less common and is removed with surgery, usually laparoscopically. In the instance where there is bilateral disease, medical management that mitigates the actions of aldosterone is preferred.

TABLE 24.1 Medical Treatment options for Treating AldosteroneInduced Hypertension (Primary Aldosteronism and low-Renin, normal Aldosterone, Hypertension) Drug

Mechanism

Advantages

Disadvantages

Spironolactone

Competive antagonist of MR.

1. Extremely effective in lowering blood pressure. 2. Reduced mortality rate by 30% in RALES clinical trial with CHF patients [9]. 3. Cost is trivial; only pennies a day.

Spironolactone also binds to the androgen receptor which can result in dose-dependent side effects.

Eplerenone

Competive antagonist of MR.

1. Selective for MR (no binding to androgen receptor. 2. Also shown to improve outcomes in CHF [10–11].

Although generic version available, cost is considerably more than cost of spironolactone.

Amiloride

Direct inhibitor of ENaC.

1.Effective in lowering blood pressure. 2. Few side effects. 3. Inexpensive.

A theoretical disadvantage is that it does not block aldosterone’s effects at the tissue level (e.g. the heart).

MR, mineralocorticoid receptor; ENaC, epithelial Na channel; RALES, Randomized Aldactone Evaluation Study.


AldosTERonE And THE MInERAloCoRTICoId RECEPToR

well to MR blockade. Indeed, recognition of this large body of hypertensive patients who respond to this targeting of antihypertensive therapy has been one of the recent breakthroughs in the area of hypertension.

Aldosterone as a Mediator of Tissue Injury Angiotensin II infused into an experimental animal produces injury to multiple organ systems. It is now known that the injury can be prevented by concurrent treatment with an MR antagonist such as spironolactone indicating that aldosterone and not angiotensin II conveys much of the tissue damage [8]. Thus, aldosterone has more than a single undesirable feature: too much causes not only hypertension but damage to a variety of tissues including kidney, heart, and vasculature. Some of the largest and most noteworthy clinical trials of the effectiveness of MR blockade have been in patients with congestive heart failure (Table 24.1). In summary, aldosterone plays a pivotal role in maintaining K homeostasis and extracellular volume. Its Na reabsorptive actions bolstered by today’s typically rich in Na diet is thought to participate in the pathogenesis of hypertension and in some instances to confer risk of injury to vital tissues such as heart and kidney. Treatment regimens that include MR antagonist are becoming increasingly utilized.

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[2] Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 1993;361:467–70. [3] Pearce D, Verrey F, Chen SY, Mastroberardino L, Meijer OC, Wang J, et al. Role of SGK in mineralocorticoid-regulated sodium transport. Kidney Int 2000;57:1283–9. [4] Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, et al. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na() channel cell surface expression. EMBO J 2001;20:7052–9. [5] Conn JW. Primary aldosteronism, a new clinical sydrome. J Lab Clin Med 1955;45:6–17. [6] Young Jr WF. Minireview: Primary aldosteronism–changing concepts in diagnosis and treatment. Endocrinology 2003;144:2208–13. [7] Calhoun DA, Jones D, Textor S, Goff DC, Murphy TP, Toto RD, et al. Resistant hypertension: Diagnosis, evaluation, and treatment. A scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Hypertension 2008;51:1403–19. [8] Rocha R, Chander PN, Khanna K, Zuckerman A, Stier Jr CT. Mineralocorticoid blockade reduces vascular injury in stroke-prone hypertensive rats. Hypertension 1998;31:451–8. [9] Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study investigators. N Engl J Med 1999;341:709–17. [10] Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 2003;348:1309–21. [11] Zannad F, McMurray JJ, Krum H, van Veldhuisen DJ, Swedberg K, Shi H, et al. Eplerenone in patients with systolic heart failure and mild symptoms. N Engl J Med 2011;364:11–21.

References [1] Pratt JH. The adrenergic nervous system conversing with the adrenal cortex: New implications for salt-sensitive hypertension. Hypertension 2006;48:820–1.


C H A P T E R

25 Vasopressin and Disorders of Water Homeostasis Joseph G. Verbalis VASOPRESSIN SECRETION The primary physiologic action of arginine vasopressin (AVP) is its function as a water-retaining hormone. The central sensing system (osmostat) for control of release of AVP is located in the hypothalamus anterior to the third ventricle that also includes the circumventricular organ, the organum vasculosum of the lamina terminalis (OVLT). The osmostat controls release of AVP to cause water retention, and also stimulates thirst to cause water repletion. Osmotic regulation of AVP release and thirst are usually tightly coupled, but experimental lesions and some pathologic situations in humans demonstrate that each can be regulated independently. The primary extracellular osmolyte to which the osmoreceptor responds is sodium. Under normal physiologic conditions, glucose and urea readily traverse neuron cell membranes and do not stimulate release of AVP. Basal osmolality in normal subjects lies between 280 and 295 mOsm/kg H2O, but for each individual osmolality is maintained within narrow ranges. Increases in plasma osmolality as little as 1% will stimulate the osmoreceptors to release AVP. Basal plasma levels of AVP are 0.5 to 2 pg/mL, which are sufficient to maintain urine osmolality above plasma osmolality and urine volume in the range of 2–3 L/day. When AVP levels are suppressed below 0.5 pg/ml, urine osmolality decreases to less than 100 mOsm/kg H2O and a free water diuresis ensues to levels approaching 800–1000 ml/h (18–24 L/d). Increases in plasma osmolality cause a linear increase in plasma AVP and a corresponding linear increase in urine osmolality. At a plasma osmolality of approximately 295 mOsm/kg H2O, urine osmolality is maximally concentrated to 1000–1200 mOsm/kg H2O. Thus, the entire physiologic range of urine concentration is accomplished by relatively small changes in plasma AVP of 0 to 5 pg/ml. AVP secretion is also stimulated by low blood volume and pressure. High-pressure baroreceptors are located in the aorta and carotid sinus, and low-pressure baroreceptors are located in the right and left atria. Stimuli for pressure and volume receptors are carried via the glossopharyngeal (ninth) and vagal (tenth) cranial nerves to the nucleus tractus solitarius in the brainstem. Subsequent


secondary and tertiary projections converge on the magnocellular neurons, where they provide inhibitory as well as excitatory inputs. Decreases in blood pressure or vascular volume stimulate AVP release, whereas situations that increase blood volume or left atrial pressure (e.g., negative-pressure breathing) decrease secretion of AVP. The release of AVP in response to changes in volume or pressure is less sensitive than the release in response to osmoreceptors, and generally a 10–15% reduction in blood volume or pressure is needed to stimulate release of AVP. However, once arterial pressure falls below this threshold, the stimulated response is exponential, and plasma levels of AVP achieved that are markedly greater than those achieved by osmotic stimulation. Other nonosmotic stimuli, such as nausea and intestinal traction, also act through similar nonosmotic neural pathways to release AVP.

VASOPRESSIN ACTIONS Three known receptor subtypes mediate the actions of AVP. They all are classical G protein-coupled receptors with seven transmembrane domains, and are classified according to the second messenger system to which they are coupled. The AVP V1a (V1aR) and V1b (V1bR) receptors are linked to the phosphoinositol signaling pathway via Gaq/11 GTP binding proteins that activate phospholipase C activity, with intracellular calcium acting as the second messenger. V1aR are present on vascular smooth muscle cells, hepatocytes, and platelets, and mediate the well-known pressor effects of AVP on peripheral resistance and blood pressure. V1bR are found predominately on corticotrophs cells of the anterior pituitary, where they mediate corticotrophin (ACTH) release in concert with the well-known effects of corticotrophin releasing hormone (CRH). V2R, or antidiuretic receptors, are mainly localized in the collecting duct cells of the kidney where they regulate water excretion. V2R are G protein-coupled receptors that activate adenylyl cyclase with subsequent increased intracellular cyclic AMP levels upon ligand activation. The increased cAMP initiates the movement of

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aquaporin-2 (AQP2) water channels from the cytoplasm to the apical (luminal) membrane of the collecting duct cells. Once inserted into the apical membrane, these channels allow facilitated rapid transport of water from the collecting duct lumen into the cell along osmotic gradients. The water then exits the cell through the basolateral membrane and into the kidney medullary circulation via aquaporin-3 and aquaporin-4 water channels, which are constitutively present in the basolateral membrane. This entire process is termed antidiuresis. In the absence of AVP, the AQP2 channels are re-internalized from the apical membrane into subapical vesicles. This prevents active reabsorption of water from the collecting duct lumen, resulting in diuresis. In addition to this rapid “shuttling” of the AQP2 water channels to regulate water reabsorption on a minute-to-minute basis, AVP also acts via V2R to regulate long-term stores of AQP2; i.e., increased AVP stimulates AQP2 synthesis and the absence of AVP results in decreased AQP2 synthesis. The hypertonic medullary interstitium determines the maximum concentration of the final urine, which is isotonic with the inner medulla of the kidney under conditions of maximal antidiuresis.

TABLE 25.1 Pathogenesis of Hypoosmolar Disorders DEPLETION (PRIMARY DECREASES IN TOTAL BODY SOLUTE  SECONDARY WATER RETENTION)a Renal solute loss Diuretic use Solute diuresis (glucose, mannitol) Salt wasting nephropathy Mineralocorticoid deficiency Non-renal solute loss Gastrointestinal (diarrhea, vomiting, pancreatitis, bowel obstruction) Cutaneous (sweating, burns) Blood loss DILUTION (PRIMARY INCREASES IN TOTAL BODY WATER  SECONDARY SOLUTE DEPLETION)b Impaired renal free water excretion Increased proximal reabsorption Hypothyroidism Impaired distal dilution

DISORDERS OF BODY WATER HOMEOSTASIS Disorders of body fluids are among the most commonly encountered problems in clinical medicine. This is because many different disease states can potentially disrupt the finely balanced mechanisms that control the intake and output of water and solutes. Although solute and water homeostasis are closely linked, clinical disorders of body fluids are generally divided into disorders of water homeostasis and disorders of solute homeostasis. Since body water is the primary determinant of the osmolality of the extracellular fluid (ECF), disorders of water homeostasis can be broadly divided into hypoosmolar disorders, in which there is an excess of body water relative to body solute, and hyperosmolar disorders, in which there is a deficiency of body water relative to body solute. Because sodium is the main constituent of plasma osmolality, these disorders are typically characterized by hyponatremia and hypernatremia, respectively.

HYPOOSMOLALITY Hypoosmolality indicates excess water relative to solute in the ECF; because water moves freely between the ECF and the intracellular fluid (ICF), this also indicates an excess of total body water relative to total body solute. Imbalances between body water and solute can be generated either by depletion of body solute more than body water, or by dilution of body solute from increases in body water more than body solute (Table 25.1). This is an oversimplification of complex physiology, and most

Syndrome of Inappropriate antidiuretic hormone secretion (SIADH) Glucocorticoid deficiency Combined increased proximal reabsorption and impaired distal dilution Congestive heart failure Cirrhosis Nephrotic syndrome Decreased urinary solute excretion Beer potomania Excess water intake Primary polydipsia Dilute infant formula a Virtually all disorders of solute depletion are accompanied by some degree of secondary retention of water by the kidneys in response to the resulting intravascular hypovolemia; this mechanism can lead to hypoosmolality even when the solute depletion occurs via hypotonic or isotonic body fluid losses. b Disorders of water retention primarily cause hypoosmolality in the absence of any solute losses, but in some cases of SIADH secondary solute losses occur in response to the resulting intravascular hypervolemia and this can then further aggravate the hypoosmolality (however, this pathophysiology does not likely contribute to the hyponatremia of edema-forming states such as congestive heart failure and cirrhosis, since in these cases multiple factors favoring sodium retention will result in an increased total body sodium).

hypoosmolar states include components of both solute depletion and water retention. Nonetheless, this general concept has proven to be useful because it provides a simple framework for understanding the basic etiologies of hypoosmolar disorders. Definitive identification of the etiology of hypoosmolality is not always possible at the time of presentation, but categorization according to the patient's


HyPoosmolAlITy

ECF volume status represents the first step in ascertaining the underlying cause of the disorder.

Decreased ECF Volume (Hypovolemia) Clinically detectable hypovolemia indicates some degree of solute depletion. Even isotonic or hypotonic fluid losses can cause hypoosmolality if water or hypotonic fluids are subsequently ingested or infused. A low urine sodium concentration (UNa) suggests a non-renal cause of solute depletion, whereas a high UNa suggests renal causes of solute depletion. Diuretic use is the most common cause of hypovolemic hypoosmolality. Most etiologies of solute losses causing hypovolemic hypoosmolality will be clinically apparent, although some salt-wasting nephropathies and mineralocorticoid deficiency may be difficult to diagnose during early phases of these diseases.

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TABLE 25.2 Criteria for the Diagnosis of sIADH ESSENTIAL 1. Decreased effective osmolality of the extracellular fluid (Posm  275 mOsm/kg H2O). 2. Inappropriate urinary concentration (Uosm  100 mOsm/kg H2O with normal renal function) at some level of hypoosmolality. 3. Clinical euvolemia, as defined by the absence of signs of hypovolemia (orthostasis, tachycardia, decreased skin turgor, dry mucous membranes) or hypervolemia (subcutaneous edema, ascites). 4. Elevated urinary sodium excretion while on a normal salt and water intake. 5. Normal thyroid, adrenal and renal function. SUPPLEMENTAL 6. Abnormal water load test (inability to excrete at least 80% of a 20 ml/ kg water load in 4 hours and/or failure to dilute Uosm to 100 mOsm/ kg H2O). 7. Plasma AVP level inappropriately elevated relative to plasma osmolality. 8. No significant correction of serum [Na] with volume expansion but improvement after fluid restriction.

Normal ECF Volume (Euvolemia) Virtually any disorder causing hypoosmolality can present with a volume status that appears normal by standard methods of clinical evaluation. Because clinical assessment of volume status is not very sensitive, the presence of normal or low blood urea nitrogen and uric acid concentrations are helpful laboratory correlates of relatively normal ECF volume. In these cases, a low UNa (30 mmol/L) suggests depletional hypoosmolality secondary to ECF losses with subsequent volume replacement by water or other hypotonic fluids. Such patients may appear euvolemic by the usual clinical parameters used to assess hydration status. A high UNa (30 mmol/L) generally indicates a dilutional hypoosmolality such as SIADH, the most common cause of euvolemic hypoosmolality. The clinical criteria necessary to diagnose SIADH remain as initially defined by Bartter and Schwartz in 1967 (Table 25.2). Many different disorders are associated with SIADH, which can be divided into four major etiologic groups: tumors, CNS disorders, drug effects, and pulmonary diseases.

Increased ECF Volume (Hypervolemia) Clinically detectable hypervolemia indicates whole body sodium excess, and hypoosmolality in these patients suggests a relatively decreased intravascular volume and/or pressure leading to water retention as a result of elevated plasma AVP levels and decreased distal delivery of glomerular filtrate to the kidneys. Such patients usually have a low UNa because of secondary hyperaldosteronism, but under certain conditions the UNa may be elevated (e.g., diuretic therapy). Hyponatremia generally does not occur until relatively advanced stages of congestive heart failure, cirrhosis, or the nephrotic syndrome, by which time diagnosis is usually not difficult. Renal failure can also cause retention of both sodium and water.

The clinical manifestations of hyponatremia are largely neurological, and primarily reflect brain edema resulting from osmotic water shifts into the brain. These range from nonspecific symptoms such as headache and confusion, to more severe manifestations such as decreased sensorium, coma, seizures, and death. Significant central nervous system (CNS) symptoms generally do not occur until the serum sodium concentration ([Na]) falls below 125 mmol/L, and the severity of symptoms can be roughly correlated with the degree of hypoosmolality. Individual variability is marked, and for any patient the level of serum [Na] at which symptoms will appear cannot be accurately predicted. Several factors other than the severity of the hypoosmolality also affect the degree of neurological dysfunction. The most important is the time course over which hypoosmolality develops. Rapid development of severe hypoosmolality frequently causes marked neurological symptoms, whereas gradual development over several days or weeks is often associated with relatively mild symptomatology despite profound degrees of hypoosmolality. This is because the brain counteracts osmotic swelling by extruding extracellular and intracellular solutes, including potassium and a variety of small organic molecules (amino acids, polyols and methylamines) called organic osmolytes. Since this is a time-dependent process, rapid development of hypoosmolality can result in brain edema before this adaptation occurs, but with slower development of the same degree of hypoosmolality brain cells can lose solute sufficiently rapidly to prevent cell swelling, brain edema, and neurological dysfunction. Underlying neurological disease also affects the level of hypoosmolality at which CNS symptoms appear; moderate hypoosmolality is of little concern in an otherwise healthy patient, but can cause morbidity


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in a patient with an underlying seizure disorder. Nonneurological metabolic disorders (hypoxia, hypercapnia, acidosis, hypercalcemia, etc.) similarly can affect the level of osmolality at which CNS symptoms occur.

HYPEROSMOLALITY Hyperosmolality indicates a deficiency of water relative to solute in the ECF. Because water moves freely between the ICF and ECF, this also indicates a deficiency of total body water relative to total body solute. Although hypernatremia can be caused by an excess of body sodium, the vast majority of cases are due to losses of body water in excess of body solutes, caused by either insufficient water intake or excessive water excretion. Consequently, most of the disorders causing hyperosmolality are those associated with inadequate water intake and/or deficient pituitary AVP secretion (Table 25.3). Although hyperosmolality from inadequate water intake is seen frequently in clinical practice, this is usually not due to an underlying defect in thirst but rather results from a generalized incapacity to obtain and/or ingest fluids, often stemming from a depressed sensorium. Evaluation of the patient’s ECF volume status is important as a guide to fluid replacement therapy, but is not as useful for differential diagnosis since most hyperosmolar patients will manifest some degree of hypovolemia. Rather, assessment of urinary concentrating ability provides the most useful data with regard to the type of disorder present. Using this approach, disorders of hyperosmolality can be categorized as those in which renal water conservation mechanisms are intact but are unable to compensate for inadequately replaced losses of hypotonic fluids from other sources, or those in which renal concentrating defects are a contributing factor to the deficiency of body water.

Diabetes Insipidus Diabetes insipidus (DI) can result from either inadequate AVP secretion (central or neurogenic DI) or inadequate renal response to AVP (nephrogenic DI). Central DI is caused by a variety of acquired or congenital anatomic lesions that disrupt the neurohypophysis, including pituitary surgery, tumors, trauma, hemorrhage, thrombosis, infarction, or granulomatous disease. Severe nephrogenic DI is most commonly congenital due to defects in the gene for the AVP V2R (X-linked recessive pattern of inheritance) or in the gene for the AQP2 water channel (autosomal recessive pattern of inheritance), but relief of chronic urinary obstruction or therapy with drugs such as lithium can cause an acquired form sufficient to warrant treatment. Short-lived nephrogenic DI can result from hypokalemia or hypercalcemia, but the mild concentrating defect generally does not by itself cause hypertonicity and responds to correction of the underlying disorder. Regardless of the

TABLE 25.3 Pathogenesis of Hyperosmolar Disorders WATER DEPLETION (DECREASES IN TOTAL BODY WATER IN EXCESS OF BODY SOLUTE) 1. Insufficient water intake Unavailability of water Hypodipsia (osmoreceptor dysfunction, age) Neurological deficits (cognitive dysfunction, motor impairments) 2. Hypotonic fluid loss* A. Renal: diabetes insipidus Insufficient AVP secretion (central DI, osmoreceptor dysfunction) Insufficient AVP effect (nephrogenic DI) B. Renal: other fluid loss Osmotic diuresis (hyperglycemia, mannitol) Diuretic drugs (furosemide, ethacrynic acid, thiazides) Post-obstructive diuresis Diuretic phase of acute tubular necrosis C. Non-renal fluid loss Gastrointestinal (vomiting, diarrhea, nasogastric suction) Cutaneous (sweating, burns) Pulmonary (hyperventilation) Peritoneal dialysis SOLUTE EXCESS (INCREASES IN TOTAL BODY SOLUTE IN EXCESS OF BODY WATER) 1. Sodium Excess Na administration (NaCl, NaHCO3) Sea water drowning 2. Other Hyperalimentation (intravenous, parenteral) *Most hypotonic fluid losses will not produce hyperosmolality unless insufficient free water is ingested or infused to replace the ongoing losses, so these disorders also usually involve some component of insufficient water intake.

etiology of the DI, the end result is a water diuresis due to an inability to concentrate urine appropriately. Because patients with DI do not have impaired urine Na conservation, the ECF volume is generally not markedly decreased and regulatory mechanisms for maintenance of osmotic homeostasis are primarily activated: stimulation of thirst and pituitary AVP secretion (to whatever degree the neurohypophysis is still able to secrete AVP). In cases where AVP secretion is totally absent (complete DI), patients are dependent entirely on water intake for maintenance of water balance. However, in cases where some residual capacity to secrete AVP remains (partial DI), plasma osmolality can eventually reach levels that allow moderate degrees of urinary concentration. Although untreated DI can lead to both hyperosmolality and volume depletion, until the water losses become severe, volume depletion is minimized by osmotic shifts of water from the ICF into the more osmotically concentrated ECF.

Osmoreceptor Dysfunction The primary osmoreceptors that control AVP secretion and thirst are located in the anterior hypothalamus, and lesions of this region in animals cause hyperosmolality through a combination of impaired thirst and osmotically


DIsoRDERs of WATER HomEosTAsIs AnD THE AuTonomIC nERVous sysTEm

stimulated AVP secretion. Initial reports in humans described this syndrome as “essential hypernatremia,” and subsequent studies used the term “adipsic hypernatremia” in recognition of the profound thirst deficits found in most of the patients. All of these syndromes are now grouped together as disorders of osmoreceptor function. Most of the cases reported to date have represented various degrees of osmoreceptor destruction associated with different brain lesions. In contrast to lesions causing central DI, these lesions usually occur more rostrally in the hypothalamus. For all cases of osmoreceptor dysfunction it is important to remember that afferent pathways from the brainstem to the hypothalamus generally remain intact; therefore, these patients will usually have normal AVP and renal concentrating responses to baroreceptor-mediated stimuli such as hypovolemia and hypotension. The clinical manifestations of hyperosmolality can be divided into the signs and symptoms produced by dehydration, which are largely cardiovascular, those caused by the hyperosmolality itself, which are predominantly neurological and reflect brain dehydration as a result of osmotic water shifts out of the central nervous system, and those which are secondary to excessive renal water losses in patients with DI. Cardiovascular manifestations of hypertonic dehydration include hypotension, kidney failure secondary to decreased renal perfusion, acute tubular necrosis or rhabdomyolysis, and in severe cases hypotensive shock. Neurological manifestations range from nonspecific symptoms such as irritability and decreased sensorium to more severe manifestations such as chorea, seizures, coma, focal neurological deficits, and cerebral infarction. The severity of symptoms can be roughly correlated with the degree of hyperosmolality, but individual variability is marked and for any single patient the level of serum [Na] at which symptoms will appear cannot be predicted. Similar to hypoosmolar syndromes, the length of time over which hyperosmolality develops can markedly affect clinical symptomatology. Rapid development of severe hyperosmolality is frequently associated with marked neurological symptoms, whereas gradual development over several days or weeks generally causes milder symptoms. In this case the brain counteracts osmotic shrinkage by increasing intracellular content of solutes. These include electrolytes such as potassium and organic osmolytes which previously had been called “idiogenic osmoles” (for the most part these are the same organic osmolytes that are lost from the brain during adaptation to hypoosmolality discussed previously). The net effect of this process is to protect the brain against excessive shrinkage during sustained hypertonicity. However, once the brain has adapted by increasing its solute content, rapid correction of the hyperosmolality can cause brain edema since it takes a finite time (24–48 h in animal studies) to dissipate the accumulated solutes, and until this process has been completed the brain will accumulate excess water as the plasma osmolality is normalized. This effect is most often seen in dehydrated pediatric

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patients, who can develop seizures as a result of rapid rehydration.

DISORDERS OF WATER HOMEOSTASIS AND THE AUTONOMIC NERVOUS SYSTEM The major relation between the autonomic nervous system and disorders of water retention such as hyponatremia results from effects on AVP secretion. Osmotic regulation of AVP secretion is not known to be influenced significantly by autonomic inputs to osmoreceptive cells. However, a wide variety of non-osmotic stimuli also stimulate AVP secretion, many of which originate in peripheral baroreceptors or chemoreceptors, and travel to the brainstem via vagal and glossopharyngeal afferent nerves. Prominent among these are hypovolemia, hypotension and nausea. Secretion of AVP from the neurohypophysis represents a balance between excitatory and inhibitory stimuli. In the presence of strong non-osmotic stimuli, osmotic inhibition of AVP secretion is dampened, or prevented, resulting in inappropriate AVP secretion that can lead to water retention with resulting hypoosmolality and hyponatremia. Thus, many cases of SIADH are due to afferent signaling through the autonomic nervous system. Neither hyponatremia nor hypernatremia are known to cause direct effects on autonomic efferent outputs.

Further Reading Anderson RJ, Chung H-M, Kluge R, et al. Hyponatremia: A prospective analysis of its epidemiology and the pathogenetic role of vasopressin. Ann Intern Med 1985;102:164–8. Bartter FC, Schwartz WB. The syndrome of inappropriate secretion of antidiuretic hormone. Am J Med 1967;42:790–806. Ellison DH, Berl T. Clinical practice. The syndrome of inappropriate antidiuresis. N Engl J Med 2007;356:2064–72. Ghirardello S, Malattia C, Scagnelli P, Maghnie M. Current perspective on the pathogenesis of central diabetes insipidus. J Pediatr Endocrinol Metab 2005;18:631–45. Knepper MA. Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin. Am J Physiol 1997;272:F3–F12. Schrier RW. Pathogenesis of sodium and water retention in high-output and low-output cardiac failure, nephrotic syndrome, cirrhosis and pregnancy. New Engl J Med 1988;319:1065–72 and 1127–1134. Verbalis JG. The syndrome of inappropriate antidiuretic hormone secretion and other hypoosmolar disorders. In: Schrier RW, editor. Diseases of the Kidney. Philadelphia: Lippincott Williams and Wilkins; 2001. p. 2511–48. Verbalis JG. Brain volume regulation in response to changes in osmolality. Neuroscience 2010;168:862–70. Verbalis JG, Goldsmith SR, Greenberg A, Schrier RW, Sterns RH. Hyponatremia treatment guidelines 2007: Expert panel recommendations. Am J Med 2007;120:S1–S21. Zerbe R, Stropes L, Robertson G. Vasopressin function in the syndrome of inappropriate antidiuresis. Annu Rev Med 1980;31:315–27.


C H A P T E R

26 Calcitonin Gene-Related Peptide and Adrenomedullin Donald J. DiPette, Scott C. Supowit INTRODUCTION Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) belong to a superfamily of closely related genes that also includes calcitonin and amylin. The potent vasodilator activity of CGRP (a sensory neuropeptide) and AM (acting as a circulating endocrine/paracrine factor) and their widespread distribution in peripheral tissues indicate a critical role in protecting tissues from injury, in addition to regulating systemic hemodynamics and regional organ blood flows under normal physiological and pathophysiological conditions. Considerable evidence indicates that both of these peptides possess significant protective activity against hypertension-induced heart and kidney damage, heart failure, and ischemia/reperfusion injury. These protective functions of CGRP and AM are mediated not only by their potent vasodilator activity, but also through the inhibition of oxidative stress, inflammation, and necrosis/apoptosis. The effects of CGRP and AM on these three mechanisms will be the focus of this chapter.

SYNTHESIS AND LOCALIZATION OF CGRP AND AM There are two forms of the 37 amino acid CGRP, α and β, which differ in only two amino acids in rats and three in humans. α-CGRP is derived from the tissue specific splicing of the calcitonin/CGRP gene. Whereas calcitonin is produced mainly in the C cells of the thyroid, CGRP synthesis is limited almost exclusively to specific regions of the central and peripheral nervous systems. The β-CGRP gene does not produce calcitonin and is also synthesized primarily in neuronal tissues. α-CGRP is prevalent in the central nervous system and in the peripheral sensory neural network. β-CGRP is also prevalent in the central nervous system, but peripherally is common in intestinal neurons. However, the biological activities of both peptides are similar in most vascular beds. CGRP and its receptors are widely distributed in the nervous and cardiovascular systems. In the periphery, prominent sites of


CGRP synthesis are the dorsal root ganglion (DRG). These structures contain the cell bodies of sensory nerves that terminate peripherally on blood vessels and all other tissues innervated by sensory nerves and centrally in laminae I/II of the dorsal horn of the spinal cord. A dense perivascular CGRP neural network is seen around the blood vessels in all vascular beds. In these vessels CGRP containing nerves are found at the junction of the adventitia and the media passing into the muscle layer. It is thought that circulating CGRP is largely derived from these perivascular nerve terminals and represents a spillover phenomenon related to the release of these peptides to promote vasodilation or other tissue functions. Receptors for CGRP have been identified in the media and intima of resistance vessels as well as the endothelial layer. Adrenomedullin (AM) is a 52 amino acid peptide that was originally isolated from pheochromocytoma cells. Since that time it has been shown to be produced in a number of different cells and tissues that are relevant to the cardiovascular system including vascular smooth muscle (VSMCs) and endothelial cells. A number of cytokines, growth factors, and hormones have been reported to increase AM expression including TNF-α and -β, IL-1α and -β, dexamethasone, cortisol, retinoic acid, thyroid hormone, and shear stress.

RELEASE OF CGRP FROM SENSORY NERVE TERMINALS CGRP-rich nerve fibers are components of the primary afferent nervous system, comprising principally capsaicin-sensitive C- and Aδ-fiber nerves that respond to chemical, thermal, and mechanical stimuli. Although these nerves have traditionally been thought to “sense” stimuli in the periphery and transmit the information centrally, there was early evidence that they also have an efferent function. It is clear that DRG neuron-derived peptides are released at peripheral sensory nerve terminals in the absence of afferent nerve stimulation. The continuous release of peptides from DRG neurons may reflect a paracrine function implying that these neurons participate in

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the continuous regulation of blood flows and other tissue activities. Sensory nerve terminals can either increase or decrease the release of CGRP in response to factors including protons, nerve growth factor, vascular wall tension, bradykinin/prostaglandins, endothelin, the sympathetic nervous system and the renin-angiotensin-aldosterone system.

CGRP AND AM SIGNALING The CGRP receptor is unique in that CGRP and AM signal through the common receptor CLR (calcitonin-like receptor). Ligand specificity is determined by co-expression of either of two chaperone proteins, receptor activity modifying proteins: RAMP1 for CGRP and RAMP2 for AM. Another RAMP (RAMP3) has also been postulated to confer AM specificity to the CLR. It now appears that a functional CGRP (or AM) receptor must include 3 proteins in a complex: the ligand binding, membrane spanning peptide, CLR, the chaperone peptide, RAMP1 or RAMP2, as well as a third peptide, the receptor component protein, RCP, that couples the receptor to the cellular signal transduction pathway. The CGRP/AM receptor is coupled to G-proteins with the CGRP/AM receptor-mediated stimulation of cAMP, downstream of Gαs, being the primary and best understood signal transduction pathway for CGRP and AM. There are, however, reports that the CGRP/AM receptor can activate other G proteins thereby providing a mechanism for fine-tuning CGRP and/or AM signaling in different cell types and tissues. Other reports indicate that CGRP and AM are capable of activating K-ATP channels of vascular smooth muscle. There is additional evidence that the vasodilator and other responses evoked by CGRP and AM are mediated, in part, by NO release, which in turn stimulates cGMP production, and that various vascular beds differ in their dependence on the endothelium for the dilator response to CGRP. Therefore, the biological activities can be mediated via endothelium-dependent and -independent mechanisms.

ATTENUATION OF OXIDATIVE STRESS BY CGRP AND AM In disease states such as hypertension, heart failure, and ischemia/reperfusion injury, overproduction of reactive oxygen (ROS) species lowers antioxidant defenses and alters signaling pathways resulting in endothelial and vascular dysfunction. Oxidative stress leads to the activation of transcription factors such as NFκ-B resulting in the up-regulation of inflammatory response genes. Multiple studies of vascular inflammation have focused on a critical role for NADPH oxidase. Endothelial, VSCMs, and adventitial cells contain NADPH oxidase. The activation of this enzyme is dependent on multiple stimuli including Ang II, norepinephrine, high glucose, shear stress, and a

number of inflammatory cytokines. Increased NADPH oxidase activity, and subsequent ROS production, plays a central role in a number of cardiovascular disease states. Indirect evidence for an antioxidant role for CGRP is provided by evidence that AM binds and activates the same receptor as CGRP. The mechanisms underlying this activity vary depending on cell types and experimental conditions. In addition, we, and other investigators, have reported that AM can attenuate Ang II-evoked ROS generation in VSMCs and ECs primarily through the inhibition of NADPH oxidase. We have recently demonstrated, for the first time, virtually identical results with CGRP. It has also been demonstrated that AM can inhibit ROS production via thiol redox systems and that CGRP can inhibit mitochondrial generated ROS in cardiac myocytes.

ATTENUATION OF INFLAMMATION BY CGRP AND AM Sensory nerves have long been considered to be involved in the development of inflammation. However, more recent studies indicate that chemical ablation of sensory nerves and genetic deletion of the sensory neuropeptide α-CGRP or the TRPV receptor, a primary activator of sensory nerve fibers, results in a marked increase in the severity of inflammation in hypertension-induced end organ damage and ischemia/reperfusion injury in the heart, liver, and gut. Likewise, AM has been implicated in both the progression and remission of the inflammatory response with the weight of the evidence indicating a protective, anti-inflammatory role for AM in a number of cardiovascular disease states. The enhanced inflammatory response in the aforementioned pathological conditions exhibits increased levels of pro-inflammatory markers and cytokines as well as tissue accumulation of neutrophils. Attenuation of inflammatory responses in the liver and the gut by sensory nerve activation appears to be due, at least in part, to a CGRP-induced increase in the endothelial production of prostacyclin, a potent anti-inflammatory agent. In vitro evidence indicates that CGRP can markedly stimulate prostacyclin production and release in endothelial cells. CGRP can significantly increase intracellular Ca2 in several different cell types including epithelial cells. In one study it was reported that this increase in intracellular Ca2 was mediated by the activation of phospholipase C (PLC). It was demonstrated that CGRP activates adenylate cyclase and releases prostacyclin from human umbilical vein endothelial cells, however, a mechanism for prostacyclin release was not established. As described previously it is well established that CGRP and can significantly induce NO production in vascular endothelial cells. It has also been reported that in endothelial cells endogenous and exogenous NO is a potent activator of cyclooygenase 1 (COX 1), a key enzyme for prostacyclin synthesis. AM treatment of bovine aortic endothelial cells induced a marked increase in cAMP accumulation and intracellular


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ConClusIon

Ca2 through independent pathways. The intracellular free Ca2 increase resulted from PLC activation and inositol 1,4,5-triphosphate formation. This increase in free Ca2+ resulted in an activation of endothelial NO synthase (eNOS). Similarly, studies involving the AM-evoked endothelium-dependent vasorelaxation in rat aorta indicated that AM induced the PI3K/Akt pathway that was implicated in the production of NO, which in turn induced endothelium-dependent vasodilation.

INHIBITION OF APOPTOSIS BY CGRP AND AM In cardiovascular disease states such as hypertensioninduced end organ damage, heart failure, and ischemia/ reperfusion injury, there are two types of cell death, necrosis and apoptosis. Activation of pro-survival kinase cascades has been hypothesized to attenuate cell death via anti-apoptosis mechanisms. The PI3K-Akt and Erk 1/2 kinase cascades activate the cardio-protective pathway. Several lines of evidence suggest that both CGRP and AM have significant anti-apoptotic activity in vivo and in vitro (isolated rat vascular smooth muscle cells and isolated rat cardiomyocytes) in the context of cardiovascular disease states and that this is correlated with the activation of PI3K-Akt and Erk 1/2 kinase pathways. The PI3K-AKT and ERK 1/2 kinase cascades are activated by a number of receptors including G-protein coupled receptors. These pathways participate in the regulation of proliferation, differentiation, and survival. The mechanism of the pro-survival activities of these pathways is mediated, in part, by the inhibition, via phosphorylation, of the pro-apoptotic proteins BAD, BAX, BIM, inactivation of capsases, and inhibition of Bcl-2. CGRP has also been implicated in the attenuation of ischemia/reperfusion-evoked cardiac mitochondrial dysfunction through inhibition of GSK 3β via its phosphorylation by the PI3k-Akt and ERK 1/2 kinase cascades. Both CGRP and AM also appear to be protective against necrosis in cardiovascular diseases, however, it is beyond the scope of this chapter to address this issue.

CONCLUSION The CGRP/AM system represents a unique homeostatic mechanism that is relevant to normal cardiovascular function and the pathophysiology of several cardiovascular disease states. There are two related vasodilator peptides where one is a sensory neuropeptide (CGRP) and the other acts as a circulating hormone (AM). Both proteins signal through the same receptor, which has a unique composition compared to other G-protein coupled receptors. While both CGRP and AM contribute to the progression of the inflammatory response and tissue damage in some contexts, in a number of cardiovascular pathological conditions CGRP and/or AM play a compensatory protective role via the attenuation of oxidative stress, inflammation, and necrosis/apoptosis.

Further Reading Bowers MC, Katki KA, Rao A, Koehler M, Spiekerman A, DiPette DJ, et al. Role of calcitonin gene-related peptide in hypertension-induced renal damage. Hypertension 2005;46:51–7. Brain SD, Grant AD. Vascular actions of calcitonin gene-related peptide and adrenomedullin. Physiol Rev 2004;84:903–34. Dickerson I. The CGRP-receptor component protein: a regulator for CLR signaling. In: Hay DL, Dickerson IM, editors. The calcitonin generelated peptide family: form, function, and future perspectives. New York, NY: Springer  Business Media; 2010. p. 59–73. Huang R, Ma H, Karve A, DiPette DJ, Bowers MC, Supowit SC, et al. Deletion of the mouse α-calcitonin gene-related peptide gene increases the vulnerability of the heart to ischemia/reperfusion injury. Am J Physiol. Heart Circ Physiol 2008;294:1291–7. Okajima K, Harada N. Regulation of inflammatory responses by sensory neurons: Molecular mechanisms and possible therapeutic applications. Cur Med Chem 2006;13:2241–51. Prado MA, Evans-Bain B, Dickerson IM. Receptor component protein (RCP): a member of a multi-protein complex required for G-proteincoupled signal transduction. Biochem Soc Trans 2002;30:460–4. Supowit SC, Rao A, Bowers MC, Zhao H, Fink G, Patel P, et al. Calcitonin gene-related peptide protects against hypertension-induced heart and kidney damage. Hypertension 2005;45:109–14. Walker CS, Conner AC, Poyner DR, Hay DL. Regulation of signal transduction by calcitonin gene-related peptide receptors. Trends in Pharm Sci 2010;31:476–83. Yanagawa B, Nagaya N. Adrenomedullin: molecular mechanisms and its role in cardiac disease. Amino Acids 2007;32:157–64.


C H A P T E R

27 Leptin Signaling and Energy Homeostasis Kamal Rahmouni INTRODUCTION Historically, the importance of the central nervous system in the regulation of energy homeostasis derived from the clinical observation of excessive subcutaneous fat in patients with pituitary tumors. The role of the hypothalamus in body energy storage was later established using discrete lesions or surgical transection of neural pathways [1]. For years, it was postulated that to control body fat stores, the brain must receive afferent input in proportion to the current level of body fat. The identification in 1994 of leptin [2], the ob gene product, provided powerful support for the concept of a feedback loop between the periphery and the brain for energy homeostasis (Fig. 27.1). Leptin, a 167-amino acid protein secreted by adipocytes, circulates in proportion to the adipose tissue mass. This hormone relays a satiety signal to the hypothalamus after entering the brain by a saturable specific transport mechanism. Leptin gene expression and secretion are increased by overfeeding, high fat diet, insulin and glucocorticoids and decreased by fasting and sympathetic nerve activation. The severe obesity and the hyperphagia caused by the absence of leptin or its receptor in rodents and humans make it clear that this hormone is fundamental for the control of body weight and food intake. Leptin promotes weight loss by reducing appetite and by increasing energy expenditure through stimulation of sympathetic nerve activity (Fig. 27.1). The effect of leptin on the sympathetic nervous system is an important aspect in the regulation of energy homeostasis and several other physiological functions. Leptin is also involved in regulation of glucose metabolism, sexual maturation and reproduction, the hypothalamic-pituitary-adrenal system, thyroid and growth hormone axes, angiogenesis and lipolysis, hematopoiesis, immune or proinflammatory responses, and bone remodeling (Fig. 27.1). Leptin also contributes to the regulation of cardiovascular function and appears to be involved in the pathophysiology of obesity-associated hypertension [3].

LEPTIN RECEPTOR The leptin receptor is a single transmembrane protein belonging to the cytokine-receptor super-family. Due to alternative splicing of the mRNA, at least six leptin


receptor isoforms have been identified (designated Ob-Ra to Ob-Rf). Five isoforms (Ob-Ra to Ob-Rd and Ob-Rf) differ in the length of their intracellular domain, while Ob-Re, which lacks the transmembrane domain, is a soluble form of the receptor [4]. The Ob-Rb form encodes the full receptor, including the long intracellular domain, which contains all the motifs necessary to stimulate the intracellular machinery involved in leptin signaling. The high levels of the short intracellular domain forms in the choroid plexus may act to transport leptin across the blood–brain barrier [4].

INTRACELLULAR MECHANISMS ASSOCIATED WITH THE LEPTIN RECEPTOR The divergent signaling capacities of the leptin receptor, ObRb, mediate the stimulation of various intracellular pathways that are important for leptin control of physiological processes (Fig. 27.2). Leptin binding to ObRb triggers the activation of the receptor-associated Janus kinase (Jak) 2 tyrosine kinase. Once activated, Jak2 phosphorylates other tyrosine residues within the ObRb including tyrosine (Tyr)1138, Tyr1077 and Tyr985, each of which mediates the activation of distinct downstream signaling pathways [5]. ObRb activation also promotes the activation of phosphatidylinositol 3 kinase, although this appears to be cell-type specific and the mechanisms underlying this regulation remain unclear. Whereas phosphorylated Tyr1138 of ObRb recruits and activates signal transducer and activator of transcription STAT3, the phosphorylation of Tyr1077 promotes the tyrosine phosphorylation and activation of STAT5. Activated STAT3 and STAT5 translocate to the nucleus to modulate gene transcriptional with important implications for the regulation of metabolism and body energy balance. For instance, knock-in mice that have a mutation at Tyr1138 of ObRb, which disrupts leptininduced STAT3 signaling, are severely obese and hyperphagic, but in contrast to the mice lacking leptin or ObRb these mice remain fertile and are less diabetic [5]. On the other hand, phosphorylation of Tyr985 creates a binding site for the COOH-terminal SH2 domain of the tyrosine phosphatase, PTPN11 (also known as SHP2), leading to the activation of extracellular signal-related kinase (ERK)

131


132

27. LEPTIn SIgnALIng And EnERgy HomEoSTASIS

-Insulin secretion and sensitivity -Kidney function -Endothelium function -Hematopoiesis -Immune system -Angiogenesis -Thermogenesis -Reproduction -Wound healing

↑ SNA

↑ Leptin ↓ Food intake

↑ Energy expenditure -Blood pressure -Neuroendocrine function -Osteogenesis -Reproduction -Insulin secretion -Glucose homeostasis

Fat tissue

FIGURE 27.1 Role of leptin in the negative loop regulating body weight. Leptin is secreted by adipocytes and circulate in the blood in concentration proportional to fat mass content. Action of leptin on its receptor in the brain inhibits food intake and increases energy expenditure through stimulation of sympathetic nerve activity (SNA). This leads to a decrease in adipose tissue mass and body weight. In addition, leptin is involved in the regulation of several physiological processes either directly or through its action in the brain.

ObRb

Tyr

P

IRS IRS

Tyr985

P

Tyr1138

P

Shp2 Shp2

P Tyr1077

STAT5 STAT5

STAT3 STAT3

ERK ERK

PI3K PI3K

FIGURE 27.2 Molecular mechanisms involved in leptin receptor (ObRb) signaling. In the central nervous system, there are four primary intracellular signaling pathways that emanate from ObRb: signal transducer and activator of transcription STAT3 and STAT5 proteins, phosphoinositol-3 kinase (PI3K) and the extracellular signal-related kinase (ERK). Each pathway is activated by phosphorylation of specific tyrosine residues in ObRb.

signaling pathway. While Tyr985 mediates most ERK stimulation during ObRb signaling, tyrosine phosphorylation sites on Jak2 appear to account for a fraction of ERK activation by leptin independently from ObRb phosphorylation. Tyr985 also binds suppressors of cytokine signaling-3 which act as a negative regulator to inhibit STAT3 signaling.

SITES OF LEPTIN ACTION IN THE BRAIN The arcuate nucleus of the hypothalamus is considered a major site for the regulation of physiological processes by leptin [5–7]. Supporting this view, the arcuate nucleus contains the highest concentration of ObRb and


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is the most responsive brain nucleus to leptin in terms of activation of intracellular signaling pathways associated with the leptin receptor (e.g. STAT3). The relevance of leptin signaling in the arcuate nucleus was further supported by lesioning studies which demonstrated a lack of feeding response to leptin after the arcuate nucleus had been destroyed. In addition, restoration of the expression of ObRb in the arcuate nucleus of leptin receptor-deficient Koletsky rats or in mice that have a leptin receptor null allele leads to decreased food intake and body weight. These findings indicate that leptin receptor signaling in the arcuate nucleus is critical to the effects of leptin on energy homeostasis. However, recent evidence demonstrating ObRb expression and leptin actions in several other brain regions has led to the concept of a distributed brain network of leptin action [8].

INTERACTION OF LEPTIN AND NEUROPEPTIDES IN THE HYPOTHALAMUS After activation of leptin receptors in the central nervous system, the signal is transduced by a series of integrated neuronal pathways that regulate endocrine and autonomic function [1,6]. Although the arcuate nucleus contains several neuronal populations, two classes of neurons have been well characterized with regard to leptin action: proopiomelanocortin (POMC) neurons (which also express cocaine- and amphetamine-related transcripts (CART) that are activated by leptin; and neuropeptide Y (NPY) neurons (which also express agouti-related protein (AgRP) that are inhibited by leptin (Fig. 27.3).

Melanocortin System There is strong evidence that many of leptin’s actions are mediated by stimulation of the melanocortin system [6,7,9]. The melanocortins are peptides that are processed from the polypeptide precursor POMC, which is produced by neurons in the arcuate nucleus of the hypothalamus and the nucleus of the tractus solitarius. POMC neurons are known to express the leptin receptor, and leptin binding leads to stimulation of neuronal firing activity and increase the gene expression of POMC and CART genes. This result in higher secretion of alpha-melanocyte stimulating hormone (α-MSH), which in turn binds to a number of a family of melanocortin receptors. Five melanocortin receptors (MC-1R to MC-5R) have been identified. The MC-3R and MC-4R are highly expressed in the central nervous system [9]. The critical role for MC-4R in energy balance was demonstrated by target disruption of the MC-4R gene inducing hyperphagia and obesity in mice [9,10]. Antagonism of a central melanocortin receptor is also important in the regulation of energy homeostasis [7,9]. This concept emerged with the discovery of production within the hypothalamic neuron of a potent and selective

Leptin

⊕

NPY neuron

POMC neuron

α −MSH

NPY

AgRP

-

⊕

MC3/4-R

NPY-R

Energy balance FIGURE 27.3 Schematic illustration of the leptin-sensitive neuronal populations in the hypothalamic arcuate nucleus; those activated (catabolic pathway represented by proopiomelanocortin (POMC) neurons) and those inhibited (anabolic pathway represented by the neuropeptide Y (NPY) neurons which also express agouti related protein, AgRP). In POMC neurons, leptin increases neuronal firing and POMC gene expression promoting the secretion of alpha-melanocyte stimulating hormone (α-MSH), an agonist of the melanocortin 3 and 4 receptors (MC3/4-R) located in the second order neurons. Conversely, in NPY neurons, leptin inhibits the neuronal firing rate and decreases the expression and secretion of NPY and AgRP (antagonist of the MC3/4-R), promoting MC3/4-R activation and NPY-R inhibition, respectively. Leptin suppression of the NPY anabolic pathway and stimulation of the POMC catabolic pathway reduce food intake and promote thermogenesis (resulting in a decrease in body weight).

antagonist of MC-3R and MC-4R. This molecule known as agouti related peptide (AgRP) is expressed only in the arcuate nucleus of the hypothalamus by the same neurons that express NPY. The expression levels of AgRP are upregulated by fasting and by leptin deficiency.

Neuropeptide Y NPY, a 36-amino acid peptide, is the most potent orexigenic (promote increased energy intake) peptide activated by decreases in leptin [9]. In the hypothalamus, NPY is synthesized by neurons of the arcuate nucleus and secreted from their terminals in the paraventricular nucleus and lateral hypothalamus. Injection of NPY into the cerebral ventricles or direct hypothalamic administration increases food intake and promotes obesity. In NPY neurons, leptin inhibits neuronal firing and decreases the expression of NPY and AgRP genes. Accordingly, levels of NPY are dramatically increased in the hypothalamus of leptin-deficient mice. Moreover, knockout of the NPY gene reduces the obesity and other endocrine alterations resulting from chronic leptin deficiency in ob/ob mice by about 50% [9].


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27. LEPTIn SIgnALIng And EnERgy HomEoSTASIS

Other Mediators

CONCLUSION

The complicated nature of leptin signaling pathways may be suggested from the essentially normal phenotype of NPY knockout mice [9], despite the potent stimulatory effects of NPY on food intake and body weight. This suggests that there are complementary and/or overlapping effector systems that compensate for the absence of NPY. Subsequently, other candidate molecules that can mediate the effects of leptin have been identified. For example, leptin-dependent sympathetic activation to brown adipose tissue appears to be mediated by corticotrophin releasing factor as the sympathoexcitatory effect of leptin to this tissue was substantially inhibited by pretreatment with the corticotrophin releasing factor receptor antagonist [10]. Several hypothalamic neuropeptides, monoamines, and other transmitter substances have emerged as candidate mediators of leptin action in the central nervous system (Table 27.1). TABLE 27.1 Example of neuropeptide and monoamine Candidate mediators in the Transduction of Leptin Action in the Central nervous System Catabolic Molecules

Anabolic Molecules

Proopiomelanocortin (POMC) and Neuropeptide Y (NPY) derived peptides Corticotrophin releasing factor (CRF)

Agouti related peptide (AgRP)

Cocaine-and amphitamineregulated transcript (CART)

Melanin-concentrating hormone (MCH)

Urocortin

Hypocritin 1 and 2/Orexin A and B

Neurotensin

Galanin

Interleukin 1β

Noradrenaline

Glucagon like peptide 1 Oxytocin Neurotensin

Energy balance is a highly regulated phenomenon, and the importance of the central nervous system in this regulation of energy homeostasis is well-established. The centers of regulation of food intake and body weight are distributed throughout the central nervous system with the hypothalamus playing a major role. The discovery of leptin has illuminated this field of neuroscience. This hormone constitutes the signal from adipose tissue that acts in the brain to complete the feedback loop that regulates appetite and energy expenditure. The identification of the leptin receptor and its sites of action in the brain have resulted in the striking progress in dissecting the brain circuitries that regulate energy homeostasis. Despite the lack of many pieces of this puzzle, the network of the brain pathways that control energy balance is rapidly being defined.

References [1] Elmquist JK, Elias CF, Saper CB. From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 1999;22:221–32. [2] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–32. [3] Rahmouni K. Obesity, sympathetic overdrive and hypertension: the leptin connection. Hypertension 2010;55:844–5. [4] Tartaglia LA. The leptin receptor. J Biol Chem 1997;272:6093–6. [5] Myers Jr MG. Deconstructing leptin: from signals to circuits. Diabetes 2010;59:2708–14. [6] Morton GJ, Cummings DE, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight. Nature 2006;443:289–95. [7] Flier JS. Obesity wars: molecular progress confronts an expanding epidemic. Cell 2004;116:337–50. [8] Grill HJ. Distributed neural control of energy balance: Contributions from hindbrain and hypothalamus. Obesity 2006;14:216–21. [9] Inui A. Transgenic approach to the study of body weight regulation. Pharmacol Rev 2000;52:35–61. [10] Rahmouni K, Haynes WG. Leptin and the cardiovascular system. Recent Prog Horm Res 2004;59:225–44.

Serotonin Dopamine


C H A P T E R

28 The Endothelin System Ernesto L. Schiffrin INTRODUCTION: ENDOTHELIN SYSTEM COMPONENTS The 21 amino acid peptide endothelin (ET) was isolated and cloned in 1985 by Yanagisawa et al. [1] ET-1, -2 and -3 are isopeptides with different functions [2], and there are also larger 31 and 32-amino acid peptides. ET-1 is the most abundant ET produced in blood vessels and the kidney, whereas ET-3 is mainly a neuropeptide. In endothelial and other cells, furin and other enzymes act on proETs to generate 38–39 amino acid peptides (big ETs) that are converted into mature 21-amino acid ETs by zinc-dependent endoproteases, the endothelin-converting enzymes (ECE-1 and 2). ECEs cleave big ET-1 at the Val21-Trp22 bond, yielding ET-1. ECE-1, of which there are four differentially spliced isoforms encoded by a single gene resulting from four alternative promoters, is present in endothelial cells. The four isoforms of ECE-1 differ by their N-terminal amino-acid, which is responsible for their cellular localization. ECE-1a, c and d are extracellular, whereas ECE-1b is an intracellular enzyme which heterodimerizes with other ECE-1 isoforms and regulates their activity. ECE-2 in smooth muscle cells converts big ET-1 to ET-1 in the vicinity of ET receptors, thus protecting it from degradation. Other enzymes that generate ETs include matrix metalloproteinase-2 that cleaves the Gly32-Leu33 bond to generate ET-1[1–32], chymase from mast cells that cleaves big ET-1 at the Tyr31-Gly32 peptide bond, yielding ET-1[1–31], and neutral endopeptidase, but their physiological importance is unclear. ET production is modulated by inhibitors such as shear stress (in blood vessels) and nitric oxide (NO), and stimulators (epinephrine, thrombin, angiotensin II (Ang II), vasopressin, cytokines, insulin, growth factors (TGF-β1) and hypoxia). Leptin stimulates ET-1 generation by endothelial cells, which may be a mechanism involved in vascular injury in the metabolic syndrome and in type 2 diabetes mellitus. ET-1 induces potent vasoconstriction, inflammation and cell growth by acting on ETA and ETB receptors present in the vascular wall on smooth muscle cells, whereas endothelial cells only possess ETB receptors which stimulate release of NO and prostacyclin, mediating vasodilation [2]. ETA or ETB receptors appear to predominate in the


adrenal gland depending on the species [3]. ETA receptors predominate in the kidney. ETB receptors are the predominant receptor subtype in the brain and peripheral nervous system.

EFFECTS OF PREPROET-1 OR ET RECEPTOR GENE DELETION Inactivation in mice of the ET-1 gene or the ETA receptor gene results in minor blood pressure elevation [4]. This occurs as a consequence of abnormal craniofacial development that interferes with breathing and raises blood pressure through anoxia. The aorta exhibits developmental abnormalities as well, with the phenotype resembling the Pierre Robin syndrome. ET-3 is the cognate ligand of ETB receptors and acts mainly on neural or neurally derived tissues. It regulates migration of neural crest cells, and mutations or gene inactivation of ETB receptors induce aganglionic megacolon and pigmentary abnormalities. Hereditary and sporadic human aganglionic megacolon (Hirschprung’s disease) result in some cases from mutations of the ETB receptor gene. Heterozygous ETB receptor knockout mice have slightly elevated blood pressure, which supports the hypothesis that the physiological action of ETB receptors is vasodilatory.

MECHANISM OF ACTION OF ET ET receptors stimulate phospholipase C, inositol trisphosphate generation and calcium release, leading to calmodulin activation, diacylglycerol production and protein kinase C stimulation [2]. The ras-raf-mitogen activated kinase (MAPK) cascade and non-receptor tyrosine kinases also participate in the intracellular signaling pathways activated by ET receptor stimulation. Reactive oxygen species (ROS) generation by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, mitochondria, and uncoupled NO synthase contribute to intracellular signaling via growth factor receptor transactivation and MAPK activation. ETA receptors also induce cell growth and apoptosis through NFκB activation. ETB receptors may also have apoptotic effects.

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136

28. THE EndoTHElIn SySTEm

PreproET-1 N

C Furinor PC7 N

ET-1[1-32] MMP-2

Big ET-1(38-39 aa)

RENAL EFFECTS OF ET-1

C ECE-1 M t Met

Leu

Ser Ser

Cys

Ser Cys

Asp

N

ET‐1(1‐21)

Lys Glu Cys

Val

Tyr

Phe Cys

ETA receptors

His

Leu Asp

ETB receptors

Ile

mice have enhanced vascular lipid biosynthetic enzyme gene expression, and demonstrate increased atherosclerosis when crossed with apoE knockout mice [11].

Ile

Trp C

ET-3

FIGURE 28.1 Biosynthesis and molecular structure of endothelin-1 (ET-1). PreproET-1 is cleaved by furin or protein convertase (PC)7 to generate the 38 or 39 amino acid (aa) big ET-1. The latter is cleaved by endothelin convertase-1 (ECE-1a, b, c, d) to generate the mature 21-aa ET-1, which can bind with high affinity to both ETA and ETB receptors. ET-2 and ET-3 exhibit 2 and 5 aa differences respectively. ET-3 is the cognate ligand of ETB receptors. ET-1 [1-31] is an additional peptide of the system produced in the vasculature and the airway that functions as a vasoconstrictor of tracheal and vascular smooth muscle and could be involved in allergic inflammation ET-1 [1-32] is generated in the vascular wall by the action of matrix metalloproteinase-2 (MMP-2).

PATHOPHYSIOLOGY OF THE ENDOTHELIN SYSTEM IN EXPERIMENTAL MODELS The endothelin system plays a pathophysiological role in hypertension [5–7], atherosclerosis, coronary artery disease, heart failure, subarachnoid hemorrhage and cerebral vasospasm, diabetes, primary pulmonary hypertension (the only approved indication of ET antagonists), pulmonary fibrosis, scleroderma, diabetic and non-diabetic renal disease, renal failure, hepatorenal syndrome, glaucoma, prostate cancer and its metastasis, and may also be involved in pheochromocytoma. In salt-dependent models of experimental hypertension such as DOCA-salt hypertension or Dahl salt-sensitive rats, and in severe hypertension such as stroke-prone spontaneously hypertensive rats (SHRsp), in particular when salt-loaded or treated with the nitric oxide synthase inhibitor L-NAME, enhanced production of ET-1 [8] induces hypertrophic remodeling of large and small arteries rather than the usual “eutrophic” remodeling without true vascular hypertrophy more often found in essential hypertension and in SHR, which regresses after treatment with ET antagonists [6–9]. In a mouse that overexpresses human preproET-1 in the endothelium by use of the endothelium-specific promoter Tie-2, small artery hypertrophic remodeling, vascular inflammation and endothelial dysfunction occurs despite the fact that blood pressure is not elevated, demonstrating the ability of ET-1 to induce blood pressure-independent vascular remodeling [10]. These

Although salt loading stimulates ET-1 production, activation of renal ETB receptors inhibits sodium reabsorption [12]. In Ang II-infused mice, the dual ETA/ETB receptor blocker bosentan partially prevented activation of the procollagen gene and rats overexpressing human angiotensinogen and human renin which develop malignant hypertension, exhibited reduced renal and myocardial damage after treatment with bosentan. In salt-loaded SHRsp, increased expression of ET-1 was associated with enhanced generation in the kidney of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I expression and matrix metalloproteinase (MMP)-2 activity, and were reduced by a selective ETA antagonist. ETs are implicated in both the development and progression of chronic kidney disease (CKD) [13]. The major pathological effects in CKD are ETA receptor-mediated. In a recent study, selective ETA receptor antagonism slightly reduced blood pressure, and to greater degree proteinuria and arterial stiffness in renal patients suggesting that the reduction in proteinuria and arterial stiffness is partly independent of blood pressure, and that selective ETA receptor antagonism may confer cardiovascular and renal benefits in patients with CKD [14,15].

CARDIAC EFFECTS OF ET-1 TGF-β1 expression and collagen deposition in the heart of DOCA-salt hypertensive rats are increased, and are prevented by ETA blockade. ETA receptor antagonism also blocked the expression of inflammatory mediators (NFκB and adhesion molecules) and the anti-apoptotic molecule X inhibitor of apoptosis peptide (xIAP) [16]. ETA receptor blockade prevented aldosterone-induced cardiac and vascular fibrosis, which suggests that ET-1 mediates in part effects of aldosterone on the heart and blood vessels [17]. Patients with essential hypertension, primary aldosteronism or renovascular hypertension have enhanced ultrasound backscatter signals resulting from tissue heterogeneity in the myocardium, which correlates with plasma aldosterone and immunoreactive ET, suggesting that as in experimental animal models, aldosterone and ET-1 contribute to myocardial fibrosis in human hypertension. However, studies using ETA receptor antagonists in heart failure patients have not demonstrated efficacy in chronic studies, although acute short-term trials suggested beneficial effects.

ET-1 IN ESSENTIAL HYPERTENSION In primary human hypertension plasma concentrations of immunoreactive ET are normal in Caucasians,


EndoTHElInS And THE nERvouS SySTEm

SFO Hypothalamus AP

PVN VLM PP

Sympathetic Outflow Baroreceptors

Vasopressin

Kidney

Blood vessels

NTS

Sympathetic ganglia

Heart

Adrenal medulla

FIGURE 28.2 Role of endothelins (ETs) in the brain and sympathetic nervous system. ETs produced in rostral areas of the brain including the subfornical organ (SFO) act on the hypothalamus to stimulate secretion of vasopressin and on the brainstem to stimulate cardiovascular regulatory centers. The latter, in the ventrolateral medulla (VLM) area postrema (AP) and nucleus tractus solitariius (NTS) modulated by baroreceptor input, stimulate sympathetic outflow, which via sympathetic ganglia, modulates heart rate, vascular tone, kidney blood flow and renin secretion as well as water and sodium handling, and catecholamine release by the adrenal medulla. ETA receptors are involved at most stages, except some in which ETB receptors play a role.

but elevated in African-Americans, who have a volume expanded low-renin form of hypertension and in whom an increase in vascular smooth muscle vasoconstrictor ETB receptors has been documented. High plasma ET levels may be related to subclinical renal dysfunction and smoking rather than hypertension. However, vascular mRNA levels of preproET are increased in stage 2 hypertension of the JNC 7 classification [7]. ETA receptor antagonists caused greater vasodilatation in the forearm of hypertensive than normotensive subjects, suggesting that ETA receptors play an important role in ET-1-dependent vascular tone in essential hypertension [18,19]. The ETB blocker BQ-788 constricted forearm resistance arteries in normotensive subjects, which suggests that ETB receptors are vasodilator in normotensive subjects, whereas the forearm in hypertensive subjects was vasodilated, suggesting that ETB receptors are vasoconstrictor in hypertensive patients. Whereas in normotensive subjects forearm blood flow response to the ETA receptor blocker BQ-123 was similar in white and black subjects, ETA receptor antagonism was a more potent vasodilator in blacks than in whites among hypertensive individuals although ET-1 induced equipotent vasoconstriction, which suggests that increased ETA-mediated vasoconstriction

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may contribute to blood pressure elevation in hypertensive blacks. ET-1 at concentrations of 1011 mol/L may potentiate other vasoconstrictors (e.g. phenylephrine or serotonin), a mechanism which is under the influence of the EDN1 K198N polymorphism in the coding region of the preproET-1 gene, and could be enhanced in hypertension, contributing to blood pressure elevation [20]. Clinical trials in human hypertension with the ETA/ETB antagonist bosentan [21] and the ETA antagonist darusentan resulted in reduced blood pressure. However, ETA antagonists may induce liver damage and liver enzyme elevation, which has resulted in halting of human hypertension studies with ETA antagonists.

MOLECULAR GENETICS OF THE ENDOTHELIN SYSTEM A polymorphism (EDN1 K198N) in the coding region of the prepro-ET-1 gene has been associated with hypertension in overweight individuals [22], and with increased vasoreactivity [20]. ECE1 C-388A is a polymorphism present in the 5-regulatory region of the ECE-1b gene. It results in enhanced expression of ECE-1b, with increased generation of ET-1. Its presence was reported in untreated hypertensive German women in whom the A allele had effects on daytime and night-time BP, and in women in the French epidemiological study Étude du Vieillissement Artériel (EVA). However, the EDN1 K198N polymorphism of preproET-1 was not associated with BP in either men or women in this study, but interacted with the ECE1 C-338A variant to influence systolic and mean BP levels in women. Stimulation of ET by androgens could explain the absence of effect in males.

ENDOTHELINS AND THE NERVOUS SYSTEM ETs are found in rostral portions of the brain, in the hypothalamus (paraventricular nuclei, colocalized with vasopressin containing neurons) and the dorsal motor nucleus of the vagus and medulla oblongata as well as brainstem areas [23] known to regulate cardiovascular function such as the area postrema, the ventrolateral medulla and the nucleus tractus solitarius. ETs cans stimulate the central and peripheral sympathetic nervous system and act on the carotid bodies and on cervical superior and nodose ganglia, influencing hemodynamic regulation by the baroreflex and by chemoreflexes [24–29]. Also, ETs released by postganglionic sympathetic neurons modulate catecholamine release and vascular tone. They also stimulate catecholamine release from adrenal glands via ETA and possibly ETB receptors [30]. ET release has been implicated in vasospasm in the cerebral circulation, and particularly in the vasospasm


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of subarachnoid hemorrhage (SAH). In the latter condition, high concentrations of immunoreactive ET have been reported in cerebrospinal fluid (CSF). Although in experimental models of SAH endothelin antagonists have demonstrated some efficacy in combatting vasospasm, ET antagonists are not indicated in SAH in humans. Recently, the interactions between the sympathetic nervous system and endogenous endothelin in patients with essential hypertension have been studied by recording muscle sympathetic nerve activity during infusion of an ETA antagonist [31]. Endogenous ET-1 appeared to have a sympatho-excitatory effect both in normotensive and hypertensive subjects through ETA receptors, contributing to basal sympathetic vasomotor tone. Essential hypertensive subjects demonstrated an increased susceptibility to the sympatho-excitatory effect of endogenous ET-1.

CONCLUSION ET-1 is a potent vasoconstrictor that also promotes cardiac, vascular and renal inflammation, hypertrophy and fibrosis. ET receptor antagonists could prevent some of the complications of hypertension, atherosclerosis and diabetes, and it is possible that they could achieve blood pressure-independent cardiovascular protection. However, because of side effects, their potential usefulness in hypertension, heart failure, atherosclerosis, CKD, diabetes and other diseases cannot be currently exploited. The only approved indication to date of ETA receptor blockade is primary pulmonary hypertension. Whether the role of ETs on the central and sympathetic nervous system will allow their therapeutic use in humans in the future is uncertain.

Acknowledgements The work of the author was supported by grant 37917 from the Canadian Institutes of Health Research (CIHR), a Canada Research Chair (CRC) from the CIHR/ Government of Canada CRC Program, and a Canada Fund for Innovation grant.

References [1] Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5. [2] Schiffrin EL, Touyz RM. Vascular biology of endothelin. J Cardiovasc Pharmacol 1998;32:S2–S13. [3] Davenport AP, Hoskins SL, Kuc RE, Plumpton C. Differential distribution of endothelin peptides and receptors in human adrenal gland. Histochem J 1996;28:779–89. [4] Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T, et al. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development 1998;125:813–24. [5] Larivière R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension 1993;21:294–300.

[6] Schiffrin EL. Endothelin: Potential role in hypertension and vascular hypertrophy. Hypertension 1995;25:1135–43. [7] Schiffrin EL, Deng LY, Sventek P, Day R. Enhanced expression of endothelin-1 gene in resistance arteries in severe human essential hypertension. J Hypertens 1997;15:57–63. [8] Goel A, Su B, Flavahan S, Lowenstein CJ, Berkowitz DE, Flavahan NA. Increased endothelial exocytosis and generation of endothelin-1 contributes to constriction of aged arteries. Circ Res 2010;107:242–51. [9] Li JS, Larivière R, Schiffrin EL. Effect of a nonselective endothelin antagonist on vascular remodeling in deoxycorticosterone acetatesalt hypertensive rats. Evidence for a role of endothelin in vascular hypertrophy. Hypertension 1994;24:183–8. [10] Amiri F, Virdis A, Neves MF, Iglarz M, Seidah NG, Touyz RM, et al. Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction. Circulation 2004;110:2233–40. [11] Simeone SMC, Li Melissa W, Paradis P, Schiffrin EL. Vascular gene expression in mice overexpressing human endothelin-1 targeted to the endothelium. Physiol Genomics 2011;43:148–60. [12] Kohan DE, Rossi NF, Inscho EW, Pollock DM. Regulation of blood pressure and salt homeostasis by endothelin. Physiol Rev 2011;91:1–77. [13] Saleh MA, Boesen EI, Pollock JS, Savin VJ, Pollock DM. Endothelin-1 increases glomerular permeability and inflammation independent of blood pressure in the rat. Hypertension 2010;56:942–9. [14] Dhaun N, MacIntyre IM, Melville V, Lilitkarntakul P, Johnston NR, Goddard J, et al. Blood pressure-independent reduction in proteinuria and arterial stiffness after acute endothelin-A receptor antagonism in chronic kidney disease. Hypertension 2009;54:113–9. [15] Wenzel RR, Littke T, Kuranoff S, Jurgens C, Bruck H, Ritz E, et al. Avosentan reduces albumin excretion in diabetics with macroalbuminuria. J Amer Soc Nephrol 2009;20:655–64. [16] Ammarguellat FZ, Gannon PO, Amiri F, Schiffrin EL. Fibrosis, matrix metalloproteinases, and inflammation in the heart of DOCAsalt hypertensive rats: role of ET(A) receptors. Hypertension 2002;39:679–84. [17] Pu Q, Neves MF, Virdis A, Touyz RM, Schiffrin EL. Endothelin antagonism on aldosterone-induced oxidative stress and vascular remodeling. Hypertension 2003;42:49–55. [18] Cardillo C, Kilcoyne CM, Waclawiw M, Cannon RO, Panza JA. Role of endothelin in the increased vascular tone of patients with essential hypertension. Hypertension 1999;33:753–8. [19] Haynes WG, Hand MF, Johnstone HA, Padfield PL, Webb DJ. Direct and sympathetically mediated venoconstriction in essential hypertension. Enhanced responses to endothelin-1. J Clin Invest 1994;94:1359–64. [20] Iglarz M, Benessiano J, Philip I, Vuillaumier-Barrot S, Lasocki S, Hvass U, et al. Preproendothelin-1 gene polymorphism is related to a change in vascular reactivity in the human mammary artery in vitro. Hypertension 2002;39:209–13. [21] Krum H, Viskoper RJ, Lacourcière Y, Budde M, Charlon V. The effect of an endothelin-receptor antagonist, bosentan, on blood pressure in patients with essential hypertension. Bosentan Hypertension Investigators. N Engl J Med 1998;338:784–90. [22] Tiret L, Poirier O, Hallet V, McDonagh TA, Morrison C, McMurray JJV, et al. The Lys198Asn polymorphism in the endothelin-1 gene is associated with blood pressure in overweight people. Hypertension 1999;33:1169–74. [23] Giaid A, Gibson SJ, Herrero MT, Gentleman S, Legon S, Yanagisawa M, et al. Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurons of the human brain. Histochemistry 1991;95:303–14. [24] Dai X, Galligan JJ, Watts SW, Fink GD, Kreulen DL. Increased .O2– production and upregulation of ETB receptors by sympathetic neurons in DOCA-salt hypertensive rats. Hypertension 2004;43:1048–54.


ConCluSIon

[25] Dai SM, Shan ZZ, Miao CY, Yin M, Su DF. Hemodynamic responses to endothelin-1 and endothelin antagonists microinjected into the nucleus tractus solitarius in rats. J Cardiovasc Pharmacol 1997;30:475–80. [26] Itoh S, van den Buuse M. Sensitization of baroreceptor reflex by central endothelin in conscious rats. Amer J Physiol 1991;260:H1106–H1112. [27] Kopp UC, Grisk O, Cicha MZ, Smith LA, Steinbach A, Schluter T, et al. Dietary sodium modulates the interaction between efferent renal sympathetic nerve activity and afferent renal nerve activity: role of endothelin. Amer J Physiol Regul Integr Comp Physiol 2009;297:R337–51.

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[28] Lau YE, Galligan JJ, Kreulen DL, Fink GD. Activation of ETB receptors increases superoxide levels in sympathetic ganglia in vivo. Amer J Physiol Regul Integr Comp Physiol 2006;290:R90–5. [29] Mosqueda-Garcia R, Inagami T, Appalsamy M, Sugiura M, Robertson RM. Endothelin as a neuropeptide: cardiovascular effects in the brainstem of normotensive rats. Circ Res 1993;72:20–35. [30] Yamaguchi N. Role of ET(A) and ET(B) receptors in endothelin-1induced adrenal catecholamine secretion in vivo. Amer J Physiol 1997;272:R1290–R1297. [31] Bruno RM, Sudano I, Ghiadoni L, Masi L, Taddei S. Interactions between sympathetic nervous system and endogenous endothelin in patients with essential hypertension. Hypertension 2011;57:79–84.


C H A P T E R

29 Pharmacology of the Nucleous Tractus Solitarii Ching-Jiunn Tseng, Pei-Wen Cheng, Che-Se Tung The nucleus tractus solitarii (NTS) is the primary integrative center for cardiovascular control and other autonomic functions in the central nervous system (CNS). The NTS not only integrates covergent information but itself is the site of substantial modulation. Several neurotransmitters or neuromodulators are involved in cardiovascular regulation in the NTS. This section presents some of the recent findings and their molecular mechanisms in the NTS.

NUCLEUS TRACTUS SOLITARII The NTS is located in the dorsal aspect of the medulla oblongata, and receives visceroceptive information from cardiopulmonary and gastrointestinal sites. It is a bilateral structure, that when extending caudally, comes medial and close to the walls of the fourth ventricle. At the level of the posterior tip of the area postrema, the neuronal groups within the NTS fuse medially and form the commisural nucleus of the NTS. This part of the NTS lies dorsal to the central canal and the dorsal motor nucleus of the vagus. The caudal limit of the NTS is located at the level of the pyramidal decussation [1]. The NTS has long been identified as a site where the first synape of the baroreceptor reflex is located. Therefore the NTS, as well as other key central nuclei in the hypothalamus and other forebrain regions, have important roles in mediating cardiovascular responses to acute stresses. From the NTS, pathways project to cholinergic parasympathetic neurons located in the dorsal vagal motor nucleus and the nucleus ambiguus, as well as to a group of inhibitory neurons in the caudal ventrolateral medulla (CVLM), which, in turn, project to tonically active sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM). A rise in arterial blood pressure in the periphery is detected by baroreceptors located in the carotid arteries and aortic arch and these activate neurons in the NTS via the branches of the vagus and glossopharyngeal nerves. The activation of NTS neurons stimulates inhibitory neurons in the CVLM, which attenuate the activity of the RVLM neurons, leading to


a reduction in sympathetic activity and a fall in blood pressure [2]. Hypertension can be due to central interruption of baroreflexes in the NTS since electrolytic lesions of blood flow was similar to that produced by sinoaortic denervation [3]. An analogous condition has also been found in the clinic: a very rare case of continuous hypertension and tachycardia after excision of a cerebellar hemangioblastoma at the dorsal medulla oblongata showed a small injury at the dorsocaudal medulla that was located at the caudal site of the NTS by postoperative MR imaging [4]. The NTS is richly innervated by neurons containing a number of potential neurotransmitters and neuromodulators, such as glutamate, noradrenaline, adrenaline, acetylcholine, serotonin, angiotensin II (Ang II), vasopressin, β-endorphin, enkephalins, neuropeptide Y, adenosine, nitric oxide (NO), insulin, etc. Among these neuromodulators related to central cardiovascular regulation, this chapter addresses the cardiovascular effects of NO, insulin, adenosine and Ang II and their molecular signaling pathways in the NTS.

CARDIOVASCULAR EFFECTS OF NO IN THE NTS NO, synthesized from the semiessential amino acid L-arginine by nitric oxide synthase (NOS), is a remarkable regulatory molecule and plays an important role in physiological functions. By using immunohistochemistry, NADPH-diaphorase staining and autoradiography, neuronal NOS (nNOS) was found at a high concentration in regions of the brain stem, especially in the NTS and rostral ventrolateral medulla. Unilateral microinjection of L-arginine into the NTS produced prominent doserelated depressor and bradycardic effects and reduced renal sympathetic nerve activity [5]. In addition, 4 to 6 hours after intravenous injection of bacterial endotoxinlipopolysaccharide, there was a time-related potentiation of the L-arginine-induced depressor and bradycardic effects in the NTS. In the investigation of the mechanisms of action of NO in the NTS, evidence suggests that

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the cardiovascular effects produced by L-arginine in the NTS are inhibited by pharmacological interventions that block NO production and the cGMP-PKG signaling pathway within the nucleus [5]. Furthermore, the interactions among NO and other important neurotransmitters have been investigated in the NTS. NO, adenosine and glutamate have reciprocal inhibition in the release of other transmitters [6,7].

CARDIOVASCULAR EFFECTS OF ADENOSINE IN THE NTS The endogenous nucleoside adenosine has been studied for its potential role as a neuromodulator in a number of autonomic functions. The highest density of adenosine uptake sites within the CNS has been observed in the NTS. The natural precursor of adenosine in the CNS is ATP, which is dephosphorylated and rapidly becomes adenosine via the activity of ectonucleotidases to synaptic release. Among four adenosine receptor subtypes, A2A receptor is expressed at high levels in limited regions of the brain that are primarily linked to adenylyl cyclase activation. In the NTS, activation of A2A receptors has been demonstrated to elicit dose-related decreases in blood pressure (BP). These studies suggest that the cardiovascular modulatory effects of adenosine in the NTS are mediated predominantly by the A2A receptor. Based on the finding, adenosine has been shown to play an important role in central cardiovascular control. The depressor and bradycardic effects of adenosine in the NTS are attenuated by a NOS inhibitor [6]. Endothelial NOS (eNOS), originally identified in vascular endothelium, is expressed in several nonendothelial cell types, including neurons of various rat brain regions. In addition, eNOS-generated NO in the NTS plays a role in the control of baroreflex gain and arterial pressure [8]. Suppression of the endogenous eNOS by overexpression of dominant-negative eNOS in bilateral NTS increased spontaneous baroreceptor reflex gain in conscious Wistar rats and decreased BP in mature spontaneously hypertensive rats [8]. Signaling molecules that regulate eNOS activity include phosphatidylinositol 3-kinase (PI3K)/Akt, cyclic nucleotide-dependent kinases (protein kinase A and protein kinase G), protein kinase C, CaMKII and ribosomal protein S6 kinase (RSK). Experimental studies demonstrate that adenosine may phosphorylate eNOS through activation of MEK/ERK cascades in the NTS [9]. This result is further supported by reports that inhibition of ERK1/2 activation attenuated the eNOS phosphorylation that was induced by estrogen and vascular endothelial growth factor [9]. Although it has been reported for years that ERKs could phosphorylate and activate eNOS, the exact signaling mechanisms that couple the activated ERK1/2 to eNOS activation remained uncertain. Evidence indicates that there is a novel adenosine-ERKeNOS signaling pathway related to the production of NO in the regulation of BP and heart rate in the NTS [9].

CARDIOVASCULAR EFFECTS OF INSULIN IN THE NTS Insulin receptors are unevenly distributed throughout the brain, with a particularly high density in the choroid plexus, cerebral cortex, olfactory bulb, hippocampus, cerebellum, and hypothalamus, which indicates the significant role of insulin in the CNS. Recent evidence has demonstrated that the peripheral and central influence of insulin on cardiovascular regulation is due to effects on the sympathetic nervous system (SNS). However, the significance of the insulin-mediated system in the control of blood pressure is not well-understood. It is well-known that the binding of insulin with its receptors results in activation of the insulin receptor (IR) tyrosine kinase. The activated kinase then phosphorylates tyrosine residues of IR substrates (IRSs). IRSs are adaptor proteins that are linked to the activation of two main signaling pathways including PI3K-Akt/protein kinase B (PKB) pathway, which is responsible for most of the metabolic actions of insulin, and the Ras-mitogen-activated protein kinase (MAPK) pathway, which regulates expression of some genes and cooperates with the PI3K pathway to control cell growth and differentiation [10]. In addition, several studies have shown that enhancing insulin sensitivity with insulin sensitizers may improve insulin resistance and limit the development of adverse cardiovascular consequences. These observations suggest that signaling defects at the IR or post-receptor levels can lead to insulin resistance and may be associated with cardiovascular diseases, including hypertension We reported a novel insulin-PI3K-Akt-NOS signaling pathway related to the production of NO in the regulation of BP and HR in the NTS of normotensive rats [11]. Conversely, there is an additional signal transduction pathway involved in insulin-mediated cardiovascular effects in the NTS of insulin resistance rats that is NOSdependent but independent of the protein kinase Akt [12].

CARDIOVASCULAR EFFECTS OF ANGIOTENSIN II IN THE NTS Ang II is a powerful vasoconstrictor. Hyperactivity of Ang II has been shown to play a major role in hypertension. However, the underlying molecular mechanisms are still not fully understood. These pathological and physiological actions of Ang II are mediated through its type 1 receptor (AT1R). It is wellknown that the expression of the AT1R is 2 to 4-fold higher in the neurons from spontaneously hypertensive rat (SHR) compared with those from WKY rat. In normotensive rats, the Ang II-AT1R-PKC-eNOS signaling pathway relates to production of NO in the regulation of BP and HR. Several lines of evidence demonstrate that reactive oxygen species (ROS) play a role in central autonomic networks that are involved in Ang II-mediated signaling [13]. For instance,


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FIGURE 29.1 The proposed adenosine, insulin and Ang II signaling pathway in the regulation of blood pressure in the NTS.

inhibition of Ang II activity by losartan (AT1R antagonist) significantly increased the expression of extracellular signal-regulated kinase (ERK)1/2, RSK, and also increased nNOS phosphorylation, which is involved in Ang II- ROSmediated modulation of BP in the NTS of SHR [14]. Thus the ERK1/2-RSK-nNOS signaling pathway may play a significant role in Ang II-mediated central BP regulation [14].

CONCLUSION The sympathetic nervous system has moved toward center stage in cardiovascular medicine. Recently studies have shown that sympathetic hypereractivity participates in the development, maintenance and progression of elevated blood pressure. The CNS–mean arterial pressure (CNS-MAP) set-point theory has recently been proposed. It has been hypothesized that hypertension occurs as the result of a primary shift of the CNS-MAP set point to a higher operating pressure, which results in increased sympathetic nerve activity. The NTS, located at the dorsal part of the brainstem, is recognized to be an important integral center in the CNS. Ubiquitously distributed neuromodulators such as adenosine, insulin and Ang II, were found to participate in sympathetic activity regulation in the NTS. Several

studies have investigated the signaling mechanism of neuromodulator with regard to cardiovascular modulation in the NTS and found that the adenosine-ERK-eNOS, insulin-PI3K-Akt-NOS and Ang II-ERK1/2-RSK-nNOS signaling pathways participate in central cardiovascular control (Fig. 29.1). Further investigation of the molecular mechanisms involved in sympathetic nervous activity modulation might elucidate the pathogenesis of the CNS-MAP set-point shift and sympathetic overactivity in essential hypertension. These observations should be of help in further understanding of the pathogenesis and optimal treatment of blood pressure disorders.

References [1] Mosqueda-Garcia R. Central autonomic regulation. In: Robertson D, Low PA, Polinsky RJ, editors. Primer on the autonomic neuvous system. U.S.A: Academic Press; 1996. p. 3–12. [2] Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci 2006;7:335–46. [3] Doba N, Reis DJ. Role of central and peripheral adrenergic mechanisms in neurogenic hypertension produced by brainstem lesions in rat. Circ Res 1974;34:293–301. [4] Ideguchi M, Kajiwara K, Yoshikawa K, Kato S, Ishihara H, Fujii M, et al. Continuous hypertension and tachycardia after resection of a hemangioblastoma behind the dorsal medulla oblongata: relationship to sympathetic overactivity at the neurogenic vasomotor center. J Neurosurg 2010;113:369–73.


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[5] Tseng CJ, Liu HY, Lin HC, Ger LP, Tung CS, Yen MH. Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 1996;27:36–42. [6] Lo WC, Jan CR, Wu SN, Tseng CJ. Cardiovascular effects of nitric oxide and adenosine in the nucleus tractus solitarii of rats. Hypertension 1998;32:1034–8. [7] Lin HC, Wan FJ, Kang BH, Wu CC, Tseng CJ. Systemic administration of lipopolysaccharide induces release of nitric oxide and glutamate and c-fos expression in the nucleus tractus solitarii of rats. Hypertension 1999;33:1218–24. [8] Waki H, Murphy D, Yao ST, Kasparov S, Paton JF. Endothelial NO synthase activity in nucleus tractus solitarii contributes to hypertension in spontaneously hypertensive rats. Hypertension 2006;48:644–50. [9] Ho WY, Lu PJ, Hsiao M, Hwang HR, Tseng YC, Yen MH, et al. Adenosine modulates cardiovascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthase in the nucleus tractus solitarii of rats. Circulation 2008;117:773–80.

[10] Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 2006;7:85–96. [11] Huang HN, Lu PJ, Lo WC, Lin CH, Hsiao M, Tseng CJ. In situ Akt phosphorylation in the nucleus tractus solitarii is involved in central control of blood pressure and heart rate. Circulation 2004;110:2476–83. [12] Hsiao M, Lu PJ, Huang HN, Lo WC, Ho WY, Lai TC, et al. Defective phosphatidylinositol 3-kinase signaling in central control of cardiovascular effects in the nucleus tractus solitarii of spontaneously hypertensive rats. Hypertens Res 2008;31:1209–18. [13] Sun C, Zubcevic J, Polson JW, Potts JT, Diez-Freire C, Zhang Q, et al. Shift to an involvement of phosphatidylinositol 3-kinase in angiotensin II actions on nucleus tractus solitarii neurons of the spontaneously hypertensive rat. Circ Res 2009;105:1248–55. [14] Cheng WH, Lu PJ, Ho WY, Tung CS, Cheng PW, Hsiao M, et al. Angiotensin II inhibits neuronal nitric oxide synthase activation through the ERK1/2-RSK signaling pathway to modulate central control of blood pressure. Circ Res 2010;106:788–95.


P A R T III

AUTONOMIC PHYSIOLOGY



C H A P T E R

30 Entrainment of Sympathetic Rhythms Michael P. Gilbey SYMPATHETIC RHYTHM The term “sympathetic rhythm” describes the mo ment to moment waxing and waning in amplitude (sig nal strength) of population activity recorded from whole sympathetic nerves that contain thousands of fibers. Such rhythms are commonly observed in the discharges of pre and postganglionic nerves regulating the heart and blood vessels. Rhythm is frequently only an emergent property of population activity; i.e., the discharges of single neurons sampled from the population may not necessarily dem onstrate rhythmicity, however, the tendency for subpopu lations of neurons to discharge action potentials almost coincidentally but intermittently gives rise to rhythmic ity in aggregate activity. Although Adrian and colleagues described rhythmic activity in the first published record ings of mammalian sympathetic nerves in 1932, underly ing mechanism(s) and possible functional significance are still uncertain [1–3].

Cardiac- and Respiratory-related Rhythms The most common sympathetic rhythms are cardiac (pulse) and respiratoryrelated. Concerning respiratory rhythm, two components can be distinguished; one as sociated with central respiratory activity and another dependent upon afferent activity related to pulmonary ventilation (e.g., from arterial baroreceptors and/or pul monary stretch receptors) [4,5]. Mechanisms Underlying Rhythms Two major hypotheses have been proposed to account for cardiac and respiratoryrelated rhythms in sympa thetic discharges. Phasic Inputs Generate Rhythms The classic view holds that these rhythms are imposed upon sympathetic discharge by “external” inputs. In the case of the cardiacrelated rhythm, an increase in barore ceptor discharge during systole is considered to inhibit tonic excitatory drive to sympathetic nerves and thereby give rise to waxing and waning of sympathetic discharges [3]. A similar mechanism is proposed for rhythms related


to pulmonary ventilation cycles where activation of lung stretch and/or baroreceptor afferents, for example, may cause periodic inhibition of activity (independent of the central respiratory rhythm generating network) [4,4a,5]. Concerning central respiratoryrelated rhythms it is sug gested that elements within the central respiratory net work provide excitatory and/or inhibitory inputs to central networks providing tonic drive to sympathetic nerves [4,5]. Additionally, Richter and Spyer have argued for the existence of a common cardiorespiratory network [6], and see Chapter 31. Entrainment of Rhythms The observation of a nonrespiratory and noncardiac related sympathetic rhythm (“10 Hz” rhythm: Green and Hoffman, 1967) provided the first indication that sympa thetic rhythms might not arise exclusively from phasic inputs to tonic sympathetic tone generating networks. Some 8 years later Taylor and Gebber observed that sym pathetic rhythms with a frequency similar to heart rate persisted following baroreceptor denervation [1,3]. As a result the idea developed that cardiacrelated rhythms in sympathetic nerve discharge might be a consequence of the entrainment of central oscillator(s) within the brain stem (i.e., that central networks driving sympathetic out flow may be intrinsically capable of generating their own rhythms). In this scheme, phasic baroreceptor input acts as a forcing input that can entrain a central oscillator. Consequently, in the absence of, or with reduced barore ceptor activity, there is a continuous phase shift between cardiac cycle and sympathetic rhythm resulting from lack of entrainment [1–3]. Additionally, there is evidence to support the hypothesis that respiratoryrelated rhythms in sympathetic discharges arise from oscillator(s) other than those within the central respiratory networks [7]. First, in vagotomized anesthetized animals, a “slow” rhythm in the frequency range of the central respiratory rhythm has been observed during central apnea (indicated by absence of rhythmic phrenic discharge). Second, “slow” rhythms in the discharges of pairs of sympathetic nerves at the fre quency of central respiratory drive have been observed to remain correlated after mathematical removal of the com ponent of these signals common to phrenic nerve activity (an indicator of central respiratory drive); i.e., “theoretical

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removal of central respiratory drive”. Third, locking ratios other than 1:1 can be observed between rhythmic phrenic and sympathetic discharge (e.g. 2:1, 3:1, 2:3). It is also apparent that afferents activated during pulmonary ventilation can act as an entraining force [2]. However, the observations concerning the entrainment of respira tory rhythm must be mindful of findings that indicate great complexity in central respiratory rhythm generating networks and their differential influence on the various somatic motor outflows driving respiratory muscles.

How Many Central Oscillators? Gebber and colleagues have provided evidence that separate oscillators, capable of coupling, may drive activ ity to different sympathetic nerves: they noted from paired nerve recordings, that rhythmic discharges of similar fre quency could be phaselocked, but this was not obliga torily; their variation in amplitude was not necessarily proportionate [7]. In addition, the observations of Gilbey and colleagues have raised the intriguing possibility that activity of sympathetic neurons regulating the same tar get may be influenced by a family of weakly coupled or uncoupled oscillators and that their degree of synchro nization is influenced by inputs related to lung inflation, central respiratory drive, various afferents (e.g., somatic and baroreceptor) and possibly arousal state [2,8].

The Spinal Cord and Sympathetic Rhythms Traditionally, supraspinal sites have been the focus of interest regarding sympathetic rhythm generation [1,5,6]. However, recently it has been demonstrated, both in situ and in isolated slices, that sympathetic rhythms can be generated within the spinal cord [2,9]. Therefore, it appears that sympathetic rhythms generated within the spinal cord may be entrained by inputs arising both from peripheral afferents and supraspinal networks; e.g., respi ratory [2]. Intrathecal application of 5hydroxytryptamine (5HT) induced a cutaneous vasomotor sympathetic rhythm in a spinalized rat preparation similar to that seen in an intact preparation [2] and spontaneous rhythmic activity was recorded from the intermediolateral cell columns (IML: the location of the cell bodies of sympathetic pregangli onic neurons) of thoracic spinal cord slices taken from neonatal rats [9]. In a spinal cord slice preparation, rhyth mic activity could be induced or enhanced by 5HT recep tor agonists and reduced by gap junction blockers. These neurophysiological observations are strengthened by the findings that the IML receives a dense 5HTcontaining innervation and membrane localized Cx36 immunoreac tivity, indicative of gap junctions, has been observed in sympathetic preganglionic neurons. These findings point to a potentially important func tion of spinal cord circuitry both for the generation and

entrainment of sympathetic rhythms. Furthermore, they demonstrate the potential for the neurochemical modula tion of sympathetic rhythmogenesis both at the single cell and neural network level. Consequently, aberrant sympa thetic rhythm generation and/or entrainment induced by changes in CNS function may result in peripheral disease (e.g., cardiovascular: see Chapter 31).

Functional Significance Whatever mechanism(s) lie behind the generation of sympathetic rhythms, their characteristic phasic nature in dicates coordination of neuronal discharges. Whereas it is clear why coordinated phasic discharges are required in locomotor and respiratory motor control, the need for pat terning and synchronization in sympathetic motor control of heart and blood vessels is not readily apparent. With regard to neuroeffector transmission, many rhythms will be filteredout (i.e., a sympathetic rhythm above 1 Hz will not lead to a 1 Hz vasomotion) as the time constant for response is ~2 seconds [1–3]. It has been suggested that coordination may be par ticularly easy to achieve between oscillating neural networks. Gebber and colleagues have suggested, based upon experimental observation, that coupled sympa thetic oscillators may be important in the generation of differential patterns of sympathetic activity to blood ves sels of muscle, skin and viscera associated with behav ioural alerting [1]. Furthermore, the observations on entrainment of sympathetic rhythms indicate that when appropriate sympathetic and respiratory networks may “bind” together to form a highly coordinated “supernet work” [2]. There are also many indications that pattern and syn chrony coding are used in addition to rate coding in vari ous nervous system functions [2]. At the level of the single neuron, pattern of firing appears important in determin ing probability of transmitter release, synaptic plasticity, types of transmitter released/coreleased and receptors activated. In these ways, firing pattern probably influ ences ganglionic and neuroeffector transmission [2,3,10]. Synchrony may be important as it favours summation, which raises the efficacy of transmission and can also have longerterm influences on synaptic and neuroeffec tor function. Synchrony therefore may enhance ganglionic transmission by increasing the probability of summa tion of weak inputs and improve neuroeffector control by coordinate activation of postjunctional receptors [2,3]. Consequently, if as suggested by Gilbey and coworkers [2] a population of neurons regulating a single target is influenced by a family of oscillators their dynamic and graded synchronization through variable entrainment could lead to the modulation of target organ function. In this manner aberrant entrainment of sympathetic rhythms might lead to peripheral pathology consequent to inappro priate sympathetic activity.

III. AUTONOMIC PHYSIOLOGY

SYMPATHETIC RHYTHM

References [1] Barman SM, Gebber GL. “Rapid” rhythmic discharges of sympa thetic nerves: Sources, mechanisms of generation, and physiological relevance. J Biol Rhythm 2000;15:365–79. [2] Gilbey MP. Sympathetic rhythms and nervous integration. Clin Exp Pharmacol Physiol 2007;34:356–61. [3] Malpas SC. The rhythmicity of sympathetic nerve activity. Prog Neurobiol 1998;56:65–96. [4] Habler HJ, Janig W, Michaelis M. Respiratory modulation in the activity of sympathetic neurons. Prog Neurobiol 1994;43:567–606. [4a] Huang C, Marina N, Gilbey MP. Impact of lung inflation cycle fre quency on rat muscle and skin sympathetic activity recorded using suction electrodes. Auton Neurosci 2009;150:70–5. [5] Koepchen HP, Klussendorf D, Sommer D. Neurophysiological background of central neural cardiovascularrespiratory coordina tion: Basic remarks and experimental approach. J Auton Nerv Syst 1981;3:335–68.

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[6] Richter DW, Spyer KM, Gilbey MP, Lawson EE, Bainton CR, Wilhelm Z. On the existence of a common cardiorespiratory network. In: Koepchen HP, Huopaniemi T, editors. In cardiorespiratory and motor coordination. Berlin: Springer Verlag; 1991. p. 118–30. [7] Zhong S, Zhou SY, Gebber GL, Barman SM. Coupled oscillators account for the slow rhythms in sympathetic nerve discharge and phrenic nerve activity. Am J Physiol 1997;272:R1314–R1324. [8] Staras K, Change HS, Gilbey MP. Resetting of sympathetic rhythm by somatic afferents causes postreflex coordination of sympathetic activity in rat. J Physiol London 2001;533:537–45. [9] Pierce ML, Deuchars J, Deuchars SA. Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice. Neuroscience 2010;170:827–38. [10] Karila P, Horn JP. Secondary nicotinic synapses on sympathetic B neurons and their putative role in ganglionic amplification of activ ity. J Neurosci 2000;20:908–18.


C H A P T E R

31 Cross-talk Between Body Systems: Respiratory-Cardiovascular Coupling in Health and Disease Julian F.R. Paton, Anthony E. Pickering

INTRODUCTION Although the common perception of the pulse is one of a stable and constant beat, periodic heart rate variability has been observed in fish, amphibians, reptiles and mammals suggesting that its appearance during evolution was not coincidental and that it confers a survival advantage. Much of this periodic heart rate fluctuation is linked to the phases of respiration. Classically, in man, heart rate increases on inhalation and falls during exhalation (so called respiratory sinus arrhythmia; RSA) and is most prominent in the young and well trained athletes. Why RSA exists is not wholly clear, yet we know that its loss is a powerful predictor of morbidity and mortality from cardiovascular disease [1]. The latter indicates a major protective role for RSA in cardiovascular health, which is a good reason to discuss its potential function. Blood pressure also expresses a respiratory oscillation, often called the high frequency oscillation or HFO. The functional significance of the HFO is if anything even less clear than that of RSA but both oscillations demonstrate the strong cross-talk between the circulatory and respiratory systems. This chapter focuses on the central neural inter-connectivity between the brainstem respiratory pattern generator and neural networks governing sympathetic and parasympathetic activity. The putative functional roles of RSA and HFO in health are discussed; additionally their plasticity will be illustrated with examples of alterations in coupling strength and patterns between respiration and cardiovascular autonomic motor outputs. The importance of these changes is discussed in the context of disease states and how this may provide novel insights into our mechanistic understanding of cardiovascular pathologies.


CARDIOVASCULAR AUTONOMIC ACTIVITY: COUPLING TO THE RESPIRATORY PATTERN GENERATOR Anrep et al. [2] described RSA for the first time and provided mechanistic insight into its causes. These included a baroreceptor reflex-mediated modulation of heart rate, triggered by respiratory induced changes in venous return (secondary to oscillations in intrathoracic pressure) and abdominal thoracic pumping, and consequent blood pressure waves in time with the respiratory cycle. When this peripheral change in venous return was negated, RSA still persisted leaving the heart rate modulation as being due either to pulmonary stretch receptor feedback following lung inflation and/or direct coupling to central respiratory drive. Both mechanisms were found to play a role. Since those seminal studies, it has been found that cardiac sympathetic motor activity exhibited prominent bursting during late-inspiration [3] whereas cardiac vagal activity fired preferentially during early expiration (postinspiration) [4]. Recordings from in situ perfused preparations (without either pulmonary stretch receptor activation or respiratory related changes in venous return) show prominent respiratory sinus arrhythmia, confirming the important role of central coupling between the respiratory and cardiac parasympathetic preganglionic neurons (Fig. 31.1) [5]. The burst of cardiac vagal activity seems to originate centrally at the level of the preganglionic neurons in the nucleus ambiguus that are inhibited during inspiration but excited during post-inspiration [4]. The magnitude of RSA is principally a function of this cardiac vagal tone or the excitability state of the preganglionic cardiac vagal motoneurons [4]. Differences in the cardiac vagal tone explain the variable degree of RSA between individuals.

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In terms of vasomotor sympathetic neurons, recordings at either the pre- or post-ganglionic levels show respiratory related busting during late-inspiration and early expiration contributing to the HFO [6–8]. The respiratory-related alterations in venous return/cardiac output described above also contribute to the respiratory modulation of arterial pressure. However, the fluctuations persist in the working heart brainstem preparation indicating again there is an important central neural component to the coupling [8]. These data provided unequivocal evidence that manifestation of both RSA and HFO was dependent, in part, on a central respiratory modulation of both limbs of the autonomic nervous system. An argument supporting the concept that the cardiovascular and respiratory systems are actually a single system (even within the brain) comes

Respiration Phrenic nerve

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FIGURE 31.1 Respiratory sinus arrhythmia is in part due to central nervous coupling between the brainstem respiratory rhythm generator and autonomic motor outflows. Schematic depicting respiratory sinus arrhythmia expressed here as a rise in heart rate during central inspiratory drive (i.e. phrenic nerve discharge) and a bradycardia coincident with the start of expiration. These data were obtained in the absence of lung inflation; the latter removes any influence from pulmonary stretch receptor feedback and baroreceptor reflex activation secondary to respiratory induced changes in venous return. This indicates that cross-talk between the central respiratory pattern generator with sympathetic and cardiac vagal neural circuitry within the brainstem contributes significantly to the arrhythmia.

WKY

WHY IS THE CIRCULATORY SYSTEM RESPIRATORY-MODULATED? The exact function/s of RSA and HFO are debated with no unified consensus and require further investigation.

RSA Four suggestions are: 1. The most likely role is to provide close matching of ventilation with pulmonary perfusion. It has been experimentally demonstrated that oxygenation of blood is optimized if increases in cardiac output are timed to occur when fresh air arrives within the alveoli [9], which is the case with RSA as heart rate rises during inspiration. However, no respiratory related alterations in arterial tension of oxygen are detectable but there could be in the pulmonary veins. In contrast, “reverse” RSA increased ventilatory dead space and intrapulmonary shunting [9]. 2. Based on the fact that a red blood cell spends the same amount of time in an alveolar capillary per cardiac cycle (in human), it has been proposed that RSA enhances cardio-respiratory efficiency and prevents “wasted” heart beats by decreasing heart rate/cardiac output on exhalation [9]. 3. The bradycardia of RSA assists in stabilizing blood pressure and counter acts the respiratory related increase in cardiac output that results from increased venous return. 4. It is plausible that slowing of heart rate during expiration enhances coronary blood flow by prolonging diastole. A constant frequency of cardiac contraction (B)

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from their tight central nervous coupling reflecting the commonality of function.

7

**

6

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FIGURE 31.2 Enhanced respiratory-sympathetic (SNA) coupling in the spontaneously hypertensive rat (SHR). (A) comparison of phrenic nerve activity (PNA) triggered sympathetic activity between normotensive (Wistar Kyoto, WKY) and SHR from the in situ arterially perfused rat preparation. In the SHR there was a greater strength in coupling as central inspiratory drive was comparable between rat strains. Note, the increased coupling produced a larger wave in arterial perfusion pressure (PP). (B) in conscious rats blood pressure was measured using radio-telemetry and showed a high frequency (HF) component in the systolic blood pressure (SBP) that was greater in the SHR than the normotensive control rat. Data from ref 8.


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CARDIovASCulAR DISEASE: oPPoSITE EffECTS on RSA AnD Hfo

(i.e. loss of RSA) could limit coronary blood flow at any given blood pressure as may occur in heart failure and hypertension. The loss of this mechanism may contribute to the increased susceptibility of patients with these diseases to left ventricular tachycardia and sudden cardiac death. Of these four possibilities direct evidence only exists to support the first hypothesis.

CARDIOVASCULAR DISEASE: OPPOSITE EFFECTS ON RSA AND HFO

HFO There is no definitive explanation for the functional role of HFO. This may be related to the experimental difficulty in removing and reconstituting the sympathetic vasomotor drive as has been elegantly done for vagal tone and RSA [9]. Rhythmic contraction and relaxation of vascular smooth muscle of arterioles (i.e., vasomotion) is a physiological process thought to aid blood flow through tissues by reducing resistance [10]. Such a process may become more important when metabolic demand increases such as during exercise. It is likely that the increased central respiratory drive that accompanies exercise may augment HFO to optimise vascular conductance in skeletal muscle to both increase oxygenation and remove end products. There is no reason why such a mechanism cannot also apply to the lungs to optimize ventilation perfusion matching, although this has not been directly shown to our knowledge. HFO may also improve capillary exchange to ensure both low interstitial pressure and constant para-capillary fluid flux [10]. In diabetic patients with neuropathy the amplitude of flow-motion was reduced compounding issues with perfusion. However, in diabetics without neuropathy no such reduction in

(A)

vasomotion was seen indicating a high dependence on sympathetic innervation [10]. In hypertensive animals and humans, vasomotion is increased and this positively correlates with increased blood pressure [10]. Whether hypertension triggers more vasomotion or whether excessive vasomotion drives the hypertension is unclear but discussed below (see HFO, below.)

RSA Loss of vagal tone in cardiovascular diseases can be clearly demonstrated by the diminished change in heart rate on administration a vagolytic drug like atropine and also by the loss of RSA [11]. The cause of this absence of cardiac vagal tone and RSA may have a common origin – depression of the cardiac vagal limb of the baroreceptor reflex. The latter normally provides a major excitatory synaptic drive to cardiac vagal motoneurons [4]. At which point this depression occurs (afferent, brainstem, efferent) is not known. Depressed synaptic transmission at the level of the cardiac vagal ganglion in heart failure dogs was reported [12] and this could be restored by the administration of acetycholinesterase inhibitors. Whether this involves a pre- and/or postsynaptic effect and the precise mechanism(s) involved remain unknown. Interestingly it has been shown that reductions in nitric oxide within the cardiac ganglia dis-faciliate vagal transmission, which can be restored by over expression of neuronal nitric oxide synthase [13]. However, transmission failure at the neuroeffector junction cannot be ruled out. A central component

central apnea

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∆ PP with return of PNA (mmHg)

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Tone developed by respiratorysympathetic coupling

FIGURE 31.3 In hypertension the increased vascular resistance is driven, in part, by the enhanced respiratory-sympathetic coupling. (A) Reinstating breathing after suppressing central respiratory drive (with hypocapnia) allows an assessment of the contribution of respiratorysympathetic coupling to vasomotor tone. As phrenic nerve activity (PNA) returns this couples to sympathetic nerve activity (SNA) driving up arterial perfusion pressure (PP). Notice the immediate saw-toothed increase in PP when PNA returns with summation of the PP oscillations driving pressure up. (B) Graph indicates that the respiratory-sympathetic coupling contributed more to PP (and vascular resistance) in the SHR than the normotensive Wistar Kyoto rat (WKY). All data from the arterially perfused in situ preparation. From ref 8.


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to this vagal dysfunction is suggested from a model of intermittent hypoxia induced cardiovascular pathology exhibiting decreased excitability of cardiac vagal preganglionic neurons [14].

the stimulus coincides with the onset of expiration [20]. This may aid in reducing arterial pressure transiently by limiting venous return.

NEW HORIZONS

HFO In contrast to a decline in RSA, HFO are augmented in hypertension, at least in the spontaneously hypertensive (SH) rat (Fig. 31.3) [8]. The amplitude of respiratory-related sympathetic bursting was augmented in SH compared to normotensive rats and showed a phase shift peaking in late inspiration rather than early expiration [8]; the significance of the latter is unknown but agrees with earlier observations in the SHR [15]. The increased coupling of respiratory-sympathetic modulation was without significant change in central inspiratory drive [8]. This supporting the notion that the coupling strength was augmented. The resultant increase in coupling strength produced greater HFO in arterial pressure in the SH rat. These waves summated raising vascular resistance and arterial pressure was greater in the SH than normotensive rat (i.e. 15 vs. 5 mmHg) (Fig. 31.2) [8]. Alteration in coupling strength and pattern of coupling have also been seen in the angiotensin II/high salt induced hypertensive rat [16], the Goldblatt hypertensive rat (Oliveira-Sales EB, Campos RR and Paton, JFR – unpublished) and rats treated with chronic intermittent hypoxia (CIH) [17]. In the latter case, CIH produced hypertension associated with increased respiratory modulation of sympathetic activity produced by the unique appearance of an extra respiratory-related burst in sympathetic activity occurring during late expiration. This was subsequently found to correlate with abdominal motor discharge, which was not present in control animals, suggesting that CIH treated rats expire actively (i.e. forced expiration) and that this rhythm drives the additional sympathetic burst contributing to the hypertension [17]. The importance of increased blood pressure variability in mediating the end organ damage is increasingly appreciated in animal models such as the SHR [18] and also in man [19]. This respiratory-sympathetic coupling therefore represents an interesting alternative potential target for treatment.

CARDIOVASCULAR MODULATION OF RESPIRATORY ACTIVITY This review would be incomplete without mentioning the emerging data supporting the reciprocity of cardiovascular-respiratory coupling. Brainstem neurons receptive to pulmonary stretch receptor inputs (essential for inspiratory phase termination) receive convergent inputs from baroreceptors. This now appears important for mediating baroreceptor reflex depression of breathing [20]. Prolongation of the expiratory pause can be evoked by baroreceptor stimulation but this is most effective when

There are some key studies that need to be performed. Given its protective role, the site(s) and mechanisms of blockade of cardiac vagal transmission must be identified in cardiovascular disease to allow novel, targeted therapy to be devised. Regarding HFO we need a better comprehension of its functional role from basic experimental investigations and the factors that govern its plasticity and amplification. Similarly we need to know whether such amplified coupling exist in human patients with heart failure and/or hypertension. Our pilot data support that this may be the case (Fisher J, Pickering AE & Paton JFR – unpublished). If so, an understanding of the changes in breathing rate and/ or pattern may provide new insight into methods to control excessive sympathetic activity in cardiovascular diseases. Changes in the sensitivity of central and peripheral chemoreception are potentially most relevant. Breathing rate and pattern may be a sensitive prognostic indicator for cardiovascular disease allowing earlier intervention and prevention treatment. All said, cardiovascular-respiratory coupling is in take-off mode for translation to the clinic.

References [1] La Rovere MT, et al. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. ATRAMI (Autonomic Tone and Reflexes After Myocardial Infarction) Investigators. Lancet 1998;351:478–84. [2] Anrep GV, Pascual W, Rössler R. Respiratory variations of the heart rate. II. The central mechanism of the sinus arrhythmia and the inter-relationship between central and reflex mechanism. Proc Roy Soc Lond 1936;119(Series B):218–30. [3] Paton JF, Boscan P, Pickering AE, Nalivaiko E. The yin and yang of cardiac autonomic control: vago-sympathetic interactions revisited. Brain Res Rev 2005;49:555–65. [4] McAllen RM, Spyer KM. The baroreceptor input to cardiac vagal motoneurons. J Physiol 1978;282:365–74. [5] Paton JFR. A working heart-brainstem preparation. J Neurosci Meth 1996;65:63–8. [6] Adrian ED, Bronk DW, Phillips G. Discharges in mammalian sympathetic nerves. J Physiol 1932;74:115–33. [7] Habler HJ, Janig W, Michaelis M. Respiratory modulation in the activity of sympathetic neurons. Prog Neurobiol 1994;43:567–606. [8] Simms AE, Paton JF, Pickering AE, Allen AM. Amplified respiratory– sympathetic coupling in the spontaneously hypertensive rat: does it contribute to hypertension? J Physiol 2009;587:597–610. [9] Hayano J, Yasuma F, Okada A, Mukai S, Fujinami T. Respiratory sinus arrhythmia. A phenomenon improving pulmonary gas exchange and circulatory efficiency. Circ 1996;94:842–7. [10] Nilsson H, Aalkjaer C. Vasomotion: mechanisms and physiological importance. Mol. Interv. 2003;3:79–89. 51 [11] Polson JW, McCallion N, Waki H, Thorne G, Tooley MA, Paton JFR, et al. Evidence for cardiovascular autonomic dysfunction in neonates with coarctation of the aorta. Circ 2006;113:2844–50. [12] Bibevski S, Dunlap ME. Ganglionic mechanisms contribute to diminished vagal control in heart failure. Circ 1999;99:2958–63.


nEw HoRIzonS

[13] Wang L, Li D, Dawson TA, Paterson DJ. Long-term effect of neuronal nitric oxide synthase over-expression on cardiac neurotransmission mediated by a lentiviral vector. J Physiol 2009;587:3629–37. [14] Yan B, Soukhova-O'Hare GK, Li L, Lin Y, Gozal D, Wead WB, et al. Attenuation of heart rate control and neural degeneration in nucleus ambiguus following chronic intermittent hypoxia in young adult Fischer 344 rats. Neuroscience 2008;153:709–20. [15] Czyzyk-Krzeska MF, Trzebski A. Respiratory-related discharge pattern of sympathetic nerve activity in the spontaneously hypertensive rat. J Physiol 1990;426:355–68. [16] Toney GM, Pedrino GR, Fink GD, Osborn JW. Does enhanced respiratory-sympathetic coupling contribute to peripheral neural mechanisms of angiotensin II-salt hypertension? Exp Physiol 2010;95:587–94.

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[17] Zoccal DB, Simms AE, Bonagamba LH, Braga VA, Pickering AE, Machado BH, et al. Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity. J Physiol 2008;586:3253–65. [18] Miao CY, Xie HH, Zhan LS, Su DF. Blood pressure variability is more important than blood pressure level in determination of endorgan damage in rats. J Hypertens 2006;24:1125–35. [19] Tatasciore A, Renda G, Zimarino M, Soccio M, Bilo G, Parati G, et al. Awake systolic blood pressure variability correlates with target-organ damage in hypertensive subjects. Hypertension 2007;50:325–32. [20] Baekey DM, Molkov YI, Paton JFR, Rybak IA, Dick TE. Effect of baroreceptor stimulation on the respiratory pattern: Insights into respiratory-sympathetic interactions. Resp Physiol Neurobiol 2010;174:135–45.


C H A P T E R

32 Circadian Rhythms and Autonomic Function Diego A. Golombek Nested deep within the brain, a master clock guides most of our temporal regulation of physiology and behavior. Indeed, mammalian diurnal rhythms are generated by the hypothalamic suprachiasmatic nuclei (SCN) and finely tuned to environmental periodicities, the most important being the light–dark cycle. Light reaches the clock through a direct retinohypothalamic tract (RHT), primarily through glutamatergic innervation, and its action is probably regulated by a variety of other neurotransmitters. The SCN continue to work precisely under constant conditions, originating circadian (i.e., with a period of about 24 h) rhythms in free-running conditions. This simple scheme can be better described by considering diverse entrainment agents (zeitgebers) which include food availability, arousal and temperature. In addition. a variety of so-called peripheral oscillators are able to generate autonomous circadian cycles in vitro (e.g., the liver, fibroblasts, diverse brain regions, lung, retina, etc.), although their influence is probably restricted to their local environment. Photic entrainment is achieved primarily by stimulation of retinal photoreceptors that include the classical rod and cone signal transduction, but also through melanopsin, a photopigment present in retinal ganglion cells involved in non-visual responses. Within the SCN, rhythms are generated in circadian pacemaker cells by a complex of molecular feedback loops that positively and negatively regulate the transcription of core genes (e.g. period, cryptochrome, bmal1) of the circadian clock. The transcription-translation loop that generates molecular oscillations of clock genes is remarkably conserved among species and even distant groups, suggesting a possibly monophyletic origin of such mechanism. Forward genetic approaches have unraveled several genetic components isolated from distinct circadian phenotypes, advancing our knowledge of the molecular circadian clock. In the mid-1990s the first mammalian clock gene (CLOCK) was discovered and led to the description of the loop in which CLOCK and BMAL heretodimerize and promote the transcription of PER (period) and CRY (cryptochrome) genes, whose proteic products in turn heterodimerize, translocate to the nucleus and negatively regulate the activity of CLOCK/BMAL. Deletions or


mutations in these genes or in their post-transcriptional regulation (such as phosphorylation) result in arrhythmicity or in profound changes in the circadian structure. In addition, genome-wide identification of clock-controlled genes, as well as their protein products and their interactions, provides important information for the understanding of the systemic behavior of circadian cogs and wheels within and between cells. In addition, post-translational modifications of clock proteins are deeply related to circadian regulation, as well as the recent description of epigenetic mechanisms that are controlled by the clock and also regulate the transcription of clock genes.

SCN OUTPUT AND AUTONOMIC CONTROL The output of the SCN includes both humoral (i.e., by vasopressin release) and neural mechanisms, which are directly related to the autonomic nervous system and, in turn, to the circadian regulation of body functions. The best known example of SCN output is probably the regulation of melatonin synthesis by the pineal gland. Pineal melatonin content exhibits a clear diurnal and circadian rhythm which is regulated by light in a daily and seasonal fashion. The SCN regulates pineal function by innervation of paraventricular (PVN) neurons which project to pre-ganglionic neurons in the intermediolateral nucleus of the thoracic spinal cord, which in turn project to the noradrenergic neurons of the superior cervical ganglion (SCG) (Fig. 32.1). The noradreneregic unnervation of the pineal is crucial in the activation and light-response of the enzymes responsible for melatonin synthesis and release. Melatonin (and, as stated below, corticosterone) might be considered the humoral hand of the circadian clock that might help in setting the pace and phase of diverse physiological variables. This SCN control of pineal melatonin can be extrapolated to other autonomic controls of hormonal secretion. For example, tracing experiments revealed a SCNPVN-sympathetic nervous system-adrenal gland axis that controls corticosterone release, by means of a diurnal

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FIGURE 32.1 The circadian clock located in the hypothalamic suprachiasmatic nuclei (SCN) sends temporal information to the body through humoral and neural pathways interacting with the autonomic nervous system. The SCN is entrained by photic stimulation through the retinohypothalamic tract (RHT) which receives information from retinal photoreceptors (PRs) and intrinsically photoreceptive ganglion cells (iPRGCs). Besides targeting neuroendocrine neurons directly (depicted here as “endocrine cells”, such as GnRH- or CRH-releasing cells), the SCN synapses with diverse hypothalamic nuclei such as the medial preoptic nucleus or the dorsomedial nucleus, as well as thalamic projections (all of which are here grouped as “other nuclei”). However, the best known efferent target of the SCN are the paraventricular nuclei (PVN), which integrate sympathetic and parasympathetic signals from the circadian system and connect with relay stations in the brainstem and the spinal cord. Parasympathetic projections to the dorsal motor nucleus of the vagus (DMV) and sympathetic projections to preganglionic neurons of the intermediolateral column of the spinal cord (IML). Autonomic circadian control of peripheral organs follows these pathways. As an example, noradrenergic innervation from the IML, through the sympathetic superior cervical ganglion, to the pineal gland (P) is shown.

change in ACTH responsiveness. In a similar fashion, the SCN regulates glucose metabolism by a circadian modulation of insulin sensitivity and hepatic glucose production. In addition, the SCN relays neural information to the pancreas, the liver and the adipose tissue, the heart and the kidney (among others) by sympathetic/parasympathetic pathways. Metabolism is linked to the circadian system not only through autonomic innervation but also because the core clock genes, expressed in several tissues throughout the body, are directly related to cellular metabolic pathways. Besides the humoral and neural regulation of autonomic pathways, the SCN can be modulated by feedback from the periphery, by means of hormones (e.g., melatonin), immune factors or information conveyed by circumventricular organs such as the arquate nuclei. In addition, the master clock in the SCN might use autonomic signaling in order to keep peripheral clock oscillations synchronized, as suggested by experiments in which manipulations of autonomic connections to peripheral organs such as the liver reset clock gene expression. Disruption of the normal synchrony between central and peripheral circadian oscillators may result in severe pathological outcomes, as suggested by recent reports that indicate that abnormalities in circadian sympathetic homeostasis results in tumor developments in mice.

CIRCADIAN AND SLEEP-CONTROL OF THE AUTONOMIC NERVOUS SYSTEM The light–dark cycle influences autonomic activity, and it has been demonstrated that this regulation depends on the SCN, since lesions of the nuclei eliminate photic modulation. Clever tracing experiments performed by the group of Kalsbeek and Buijs in the Netherlands demonstrated that control of parasympathetic and sympathetic pre-autonomic neurons is completely separated and independent along the circadian pathway from the hypothalamus to the brainstem. In this way, the SCN and the circadian system are able to modulate both subdivisions of the autonomic nervous system independently, thus resulting in the most adequate responses depending on the time of day and other environmental and internal temporal cues. An imbalanced temporal regulation of sympathetic and parasympathetic activity might lead to profound changes in metabolic physiology and, eventually, to metabolic syndrome-like disease. Indeed, in experimental forced desynchronization clear endogenous circadian regulation of autonomic activity has been demonstrated (assessed by, for example, R-R interval variation in the electrocardiogram), by independent control of sympathetic and vagal innervation of the heart. However, besides it endogenous circadian control, autonomic activity also exhibits profound changes throughout


CARdIAC EvEnTs And CIRCAdIAn RHyTHms

the sleep–wake cycle. For example, sympathetic activation is reduced significantly in the transition from wakefulness to slow-wave sleep stage 4 (accompanied by a decrease in arterial blood pressure and bradycardia) determining the parasympathetic predominance typical of this stage – although it goes back to awake levels during REM sleep. Indeed, sleep styles affect autonomic regulation of several variables, including blood pressure and cardiac function: among other changes, short sleep duration correlates with the development of hypertension (as well as diabetes). It is also well-known that cardiovascular incidents do not occur at random throughout the day, but tend to be more common in the morning, suggesting that at the rest-activity transition the SCN is crucial in regulating the sympathetic/parasympathetic balance to the heart, and any changes in clock function put the system at risk. Treatments that increase the amplitude of circadian rhythms (such as exercise, diet, chronobiotics, etc.) might be useful to prevent such clock-dependent risks.

CARDIAC EVENTS AND CIRCADIAN RHYTHMS As an example of circadian autonomic regulation, we shall mention that most (if not all) cardiovascular variables exhibit strong diurnal and/or circadian rhythms, including blood pressure, heart rate, heart rate variability and many others. As a consequence, cardiovascular events also tend to occur at specific times of day, with higher frequencies in the morning and lower risks during sleep which relate to rhythmic changes in human susceptibility to such events. Besides the general pattern of higher blood pressure and heart values during the day, there are marked interindividual differences in temporal allocation of physiological variables (i.e., chronotypes), with extreme morning (“larks”) and evening types (“owls”) exhibiting a difference of several hours in the maxima of temperature, blood pressure and heart rate. Forced desynchronization protocols have demonstrated a mixed contribution from both endogenous circadian factors and sleep in the regulation of diurnal autonomic variations such as changes in heart rate and heart rate variability. Although less studied, seasonal variations in cardiac function have also been reported, including pathological consequences. As for pathological situations, it has long been known that there is a significant peak of acute myocardial infarction in the morning hours (and sometimes a secondary peak in the afternoon), as well as for ischemic and hemorrhagic stroke. Moreover, an alteration of circadian cardiac regulation in coronary disease has been demonstrated.

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Circadian desynchronization of autonomic (sympathetic/ parasympathetic) regulation of the heart correlates closely with heart disease. Finally, extreme environments (e.g., space missions, Antarctic expeditions) can also induce an autonomic/circadian disruption with accompanying neurovegetative changes. In summary, the mammalian circadian system is deeply interrelated with the autonomic nervous system. The two main output channels for the circadian clock are through humoral release or sympathetic/parasympathetic relay stations that convey temporal information throughout the body. The SCN sends neural projections to other hypothalamic nuclei and from there to the brainstem or spinal cord autonomic centers that control physiological activity. In addition, neurovegetative mechanisms exhibit a double modulation by the circadian system (and, in turn, are synchronized to environmental synchronizers) and by arousal/sleep-related mechanisms in a homeostaticlike process. In this sense, autonomic regulation should not only be regarded as a spatial/functional control of physiology and behavior but also in terms of a strong temporal modulation that helps the body to adequately devote resources and energy throughout the day.

Further Reading Buijs RM, Kalsbeek A. Hypothalamic integration of central and peripheral clocks. Nat Rev Neurosci 2001;2:521–6. Golombek DA, Rosenstein RE. Physiology of circadian entrainment. Physiol Rev 2010;90:1063–102. Kalsbeek A, Bruinstroop E, Yi CX, Klieverik LP, La Fleur SE, Fliers E. Hypothalamic control of energy metabolism via the autonomic nervous system. Ann NY Acad Sci 2010;1212:114–29. Kalsbeek A, Fliers E, Hofman MA, Swaab DF, Buijs RM. Vasopressin and the output of the hypothalamic biological clock. J Neuroendocrinol 2010;22:362–72. Kalsbeek A, Kreier F, Fliers E, Sauerwein HP, Romijn JA, Buijs RM. Circadian control of metabolism by the suprachiasmatic nuclei. Endocrinology 2007;148:5635–9. Lee S, Donehower LA, Herron AJ, Moore DD, Fu L. Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice. PLoS One 2010;5(6):e10995. Maywood ES, O’Neill JS, Reddy AB, Chesham JE, Prosser HM, Kyriacou CP, et al. Genetic and molecular analysis of the central and peripheral circadian clockwork of mice. Cold Spring Harb Symp Quant Biol 2007;72:85–94. Scheer FA, Kalsbeek A, Buijs RM. Cardiovascular control by the suprachiasmatic nucleus: neural and neuroendocrine mechanisms in human and rat. Biol Chem 2003;384:697–709. Schibler U. Circadian time keeping: the daily ups and downs of genes, cells, and organisms. Prog Brain Res 2006;153:271–82. Takahashi JS, Hong HK, Ko CH, McDearmon EL. The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 2008;9:764–75.


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33 Baroreceptor Reflexes Mark W. Chapleau Changes in blood pressure (BP) and/or blood volume are “sensed” within specific compartments of the cardiovascular system by “baroreceptors” (Fig. 33.1). Baroreceptors are mechanosensitive nerve endings that are activated by vascular and/or cardiac distension during increases in intraluminal BP. The activity of arterial baroreceptors innervating large arteries (primarily aortic arch and carotid sinuses) is increased when arterial BP rises, and decreased when BP falls. The changes in baroreceptor activity evoke rapid reflex adjustments that buffer or oppose the changes in arterial BP in a negative-feedback manner. Cardiopulmonary baroreceptors innervate the heart, vena cava, and pulmonary vasculature. Since the activity of cardiopulmonary baroreceptors correlates with intrathoracic (central) blood volume, these nerve endings are often referred to as “volume receptors” or “low-pressure” baroreceptors. The reflex adjustments triggered by changes in cardiopulmonary baroreceptor activity regulate blood volume in addition to influencing BP.

NEURAL PATHWAYS AND EFFECTOR MECHANISMS Arterial Baroreflex The neural pathways and effector mechanisms involved in baroreflex control of the circulation are summarized in Figures 33.1 and 33.2. The cell bodies (somata) of carotid sinus and aortic arch baroreceptor neurons are located in petrosal and nodose ganglia, respectively. The corresponding afferent baroreceptor activity is transmitted to the nucleus tractus solitarius (NTS) in the medullary brain stem via carotid sinus and glossopharyngeal nerves, and aortic depressor and vagus nerves, respectively. The baroreceptor inputs are integrated and relayed through a network of central nervous system (CNS) neurons controlling efferent parasympathetic nerve activity (paraSNA), sympathetic nerve activity (SNA), and release of the vasoconstrictor and antidiuretic peptide vasopressin (AVP) from the posterior pituitary gland (Figs 33.1 and 33.2). The multiple effector mechanisms by which these systems buffer increases in BP are depicted in Figure 33.2. The effector mechanisms operate in the opposite direction when arterial BP and baroreceptor activity are reduced.


BP-sensitive sensory nerves also innervate the juncture of the right carotid and right subclavian arteries (Fig. 33.1) and the coronary arteries. These nerves travel in the aortic depressor and vagus nerves with cell bodies located in the nodose ganglia. Activation of the coronary artery baroreflex modulates peripheral SNA and vascular resistance, but unlike carotid sinus and aortic arch baroreflexes, has little or no affect on HR.

Cardiopulmonary Baroreflex The cardiopulmonary region is innervated by multiple types of mechanosensitive and chemosensitive sensory nerves that affect autonomic and cardiovascular functions in a variety of ways (see Chapter 35). We focus here on vagal afferent neurons with cell bodies in the nodose ganglia and nerve endings in the heart, vena cava, and pulmonary vasculature that are sensitive to changes in central blood volume (Fig. 33.1). The electrophysiological properties of these sensory neurons, the CNS pathways engaged by their activation, and their influence on efferent effectors are similar to that of arterial baroreceptor neurons (see Figs 33.1 and 33.2), but not identical. While changes in cardiopulmonary baroreceptor activity during changes in central blood volume evoke powerful reflex changes in peripheral SNA, vascular resistance, and release of renin and AVP; the reflex has little affect on HR. The changes in SNA and vascular resistance contribute to orthostatic adjustments. Renal actions of SNA, the renin-angiotensinaldosterone system, and AVP leading to changes in Na and water reabsorption play a major role in regulation of blood volume.

DETERMINANTS OF AFFERENT BARORECEPTOR ACTIVITY Rate Sensitivity of Baroreceptors Baroreceptor activity is dependent not only on the mean level of BP, but also on the direction and rate of change in BP. Consequently, baroreceptor activity will increase or decrease to a greater extent when the change in BP occurs more rapidly leading to a more effective reflex compensation. Similarly, baroreceptor activity is higher

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FIGURE 33.1 Location of baroreceptors and neural pathways mediating baroreflex responses. (A) Arterial baroreceptor afferents innervate carotid sinuses, aortic arch and the right carotid artery-right subclavian artery juncture. Cardiopulmonary baroreceptors innervate veno-atrial juncture, atria, ventricles and pulmonary vasculature. The baroreflexes modulate paraSNA and SNA to numerous organ systems and vasopressin (AVP) release. Key targets involved in cardiovascular regulation are illustrated. (B) Major nuclei involved in baroreflex control. Increases in BP and baroreceptor activity activate excitatory neural projections from nucleus tractus solitarius (NTS) to preganglionic parasympathetic neurons in nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMNX) resulting in increases in paraSNA and decreases in HR. Activation of excitatory projections from NTS to caudal ventrolateral medulla (CVLM) causes subsequent inhibition of premotor sympathetic neurons in rostral ventrolateral medulla (RVLM) that project to preganglionic sympathetic neurons in the intermediolateral (IML) column of the thoracolumbar spinal cord. Increased baroreceptor activity also inhibits secretion of AVP from magnocellular neurons in paraventricular nucleus (PVN) and supraoptic nucleus (SON) of hypothalamus. Other CNS regions interact with these areas to modulate baroreflexes.

FIGURE 33.2 Effector mechanisms mediating reflex responses to increases in baroreceptor activity. Increases in arterial BP and baroreceptor activity increase paraSNA, decrease SNA, and inhibit release of AVP leading to an array of cardiovascular, hormonal and renal responses. Decreases in arterial BP evoke directionally opposite reflex responses. A-V node, atrio-ventricular node; Ang II, angiotensin II; H2O, water.


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during the systolic phase of the arterial pressure pulse and lower or absent during diastole. The phasic discharge of afferent activity facilitates reflex inhibition of SNA.

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and autocrine/paracrine factors including norepinephrine, prostacyclin, serotonin, nitric oxide, and reactive oxygen species modulate baroreceptor sensitivity through actions on these ion channels and membrane pumps.

Large Artery Compliance Baroreceptors are not directly sensitive to BP, but rather are sensitive to mechanical deformation of the nerve endings during distension of the arterial wall. Therefore, large artery compliance (specifically of the carotid sinuses and aortic arch) is a major determinant of the baroreceptor sensitivity to changes in BP. Decreased arterial compliance contributes to decreased baroreceptor sensitivity in atherosclerosis, hypertension, and aging.

Neuronal Mechanisms Mediating Sensory Transduction The prevailing view is that baroreceptors are activated by the opening of mechanosensitive ion channels in the sensory terminals. The resulting depolarization, if of sufficient magnitude, will trigger action potential discharge upon opening of voltage-gated Na and K channels. The action potentials are propagated towards the CNS at frequencies related to the magnitude of deformation and depolarization of the sensory terminals. Evidence suggests that members of the epithelial sodium channel (ENaC) superfamily including acid-sensing ion channel 2 (ASIC2) are components of the mechanosensitive ion channel complex. Transient receptor potential (TRP) channels have also been implicated in baroreceptor sensory transduction and/ or signaling, perhaps functioning as a mechanosensor. A variety of voltage- and ligand-gated ion channels and membrane pumps modulate the membrane potential and excitability of baroreceptors including Kv1, Kv4, BK and KCNQ (M-type) K channels; tetrodotoxin-insensitive, voltage-gated Na channels; hyperpolarization-activated cyclic nucleotide-gated (HCN) channels; serotonin 5HT3 receptor/ channels; and the Na/K–ATPase. Several neurohumoral

BAROREFLEX ADAPTATION AND RESETTING IN ACUTE HYPERTENSION Baroreceptor activity increases with a rise in arterial BP but declines over time if the acute hypertension is maintained. Furthermore, “post-excitatory depression” (PED) of baroreceptor activity occurs when BP decreases rapidly after a period of increased BP. Different mechanisms have been implicated in these two phenomena with opening of 4-aminopyridine sensitive K channels contributing to adaptation and activation of the Na/K–ATPase causing PED. Baroreceptor adaptation and PED contribute to acute resetting of the baroreceptor pressure-activity relationship to higher mean pressures in hypertension. The baroreceptor function curve is shifted in a parallel manner with little or no change in baroreceptor sensitivity (slope), and is usually accompanied by resetting of the arterial BP-HR relation (Fig. 33.3). Central mechanisms may exacerbate or oppose resetting of the baroreflex function curve. While baroreflex resetting compromises the ability to counter the sustained hypertension, it helps preserve the ability to buffer acute fluctuations in BP at the new higher prevailing level of BP.

DECREASED BAROREFLEX SENSITIVITY IN DISEASE Control of HR vs. SNA and BP, and Underlying Mechanisms Baroreflex sensitivity (BRS) for control of HR is consistently decreased in numerous pathological states including chronic hypertension, coronary artery disease,

FIGURE 33.3 Baroreceptor and baroreflex resetting during acute hypertension. An increase in mean arterial BP increases baroreceptor activity (left) and reflexively decreases HR (right). During a sustained increase in BP, baroreceptor activity decreases or adapts over time (left) and HR increases at the same level of BP (right). The baroreceptor and baroreflex function curves are shifted (reset) to higher BP during acute hypertension with preservation of the slope of the curves (sensitivity) (dashed lines).


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post-myocardial infarction, heart failure, diabetes mellitus, and obesity, and with aging. Afferent, central, and efferent mechanisms contribute to varying degrees to decreased BRS in these diseases. Chronic structural changes such as decreased large artery compliance and cardiac hypertrophy impair the afferent sensitivity of arterial and cardiopulmonary baroreceptors. In addition, neurohumoral activation and oxidative stress impair baroreflex function. For example, increased circulating levels of angiotensin II (Ang II) reset the baroreflex function curve to a higher mean level of arterial BP. This resetting is mediated by actions of Ang II on circumventricular organs that lack a blood–brain barrier (e.g., area postrema) and is independent of the rise in BP. Furthermore, Ang II acts at multiple central and peripheral sites in the nervous system to increase SNA, and decrease paraSNA and BRS. Aldosterone inhibits BRS by reducing afferent baroreceptor activity and by central actions. Factors released from activated platelets and reactive oxygen species decrease baroreceptor afferent sensitivity. Oxidative stress in the CNS contributes to increased SNA and BP in animal models of hypertension. Antioxidant therapies improve BRS in hypertension, heart failure, and in aging. In contrast to control of HR, the effects of cardiovascular disease and aging on arterial baroreflex control of SNA and BP are controversial with reports of both impaired and preserved BRS. Differences in results between studies can be explained in part by differences in: (i) the methods used to quantify changes in SNA and evaluate BRS; (ii) engagement of arterial vs. cardiopulmonary baroreflexes; (iii) control of SNA to different peripheral targets; (iv) severity of disease; and (v) experimental conditions (e.g., use of anesthesia). In most conditions, decreased BRS for parasympathetic control of HR usually precedes and/ or exceeds the decrease in BRS for sympathetic control. Structural damage to baroreceptor afferents, usually resulting from surgery or radiation, can cause “baroreflex failure” with loss of cardiovagal tone and severe, episodic periods of sympathetic-mediated hypertension (see Chapter 72). A similar phenotype is observed in patients with familial dysautonomia, a rare genetic disease with severe developmental sensory nerve defects (see Chapter 103).

Genetic Determinants of BRS Decreased BRS may be secondary to underlying cardiovascular disease or may precede and contribute to disease. BRS for control of HR is impaired in normotensive subjects with a family history of hypertension, and heritability of BRS has been confirmed in twin studies. Polymorphisms in several genes have been reported to be associated with BRS (see Chapter 34). Therefore, BRS screening in high risk patients identified by disease and/ or by the presence of specific polymorphisms may be advisable. The ability to measure BRS noninvasively from spontaneous fluctuations in systolic BP and pulse interval makes this clinical application feasible.

BRS: A DETERMINANT OF CARDIOVASCULAR RISK AND THERAPEUTIC TARGET Decreased BRS and Cardiovascular Risk Increased BP variability causes target organ damage, e.g., endothelial dysfunction, vascular and cardiac hypertrophy, kidney disease and cerebral vascular dysfunction. These insults lead to myocardial infarction, stroke, and heart and kidney failure. By minimizing BP variability and restraining SNA and BP, the arterial and cardiopulmonary baroreflexes reduce target organ damage. In addition to regulating BP, baroreflexes exert a major influence on the electrical properties of the heart through modulation of cardiac SNA and paraSNA. Myocardial infarction, heart failure, and diabetes are associated with decreased BRS for control of HR, cardiac arrhythmias, and sudden cardiac death. The decrease in BRS predicts occurrence of arrhythmias and mortality in patients suffering from these diseases suggesting a causal relationship.

BRS is a Therapeutic Target The strong inverse relationship between BRS and cardiovascular risk encourages targeting therapy to improve BRS. Baroreflexes may contribute to the benefit of standard antihypertensive therapies. For example, lowering of BP of hypertensive patients by pharmacological or dietary interventions rapidly resets the baroreflex function curve to lower mean arterial BPs. The baroreflex resetting helps stabilize BP at the new lower prevailing level of BP. Reversal of vascular and cardiac stiffening and hypertrophy with longer periods of antihypertensive treatment increases BRS. Antagonists of the renin-angiotensinaldosterone system and antioxidants increase BRS independent of BP lowering, thus providing further reductions in cardiovascular risk. Recent findings have rejuvenated the concept of specific therapeutic targeting of baroreflex pathways in cardiovascular disease. Cholinesterase inhibitors promote increases in cardiovagal tone and BRS by increasing the concentration of the neurotransmitter acetylcholine at cholinergic synapses and sinoatrial node, and amplify cholinergic signaling in left ventricle. Administration of cholinesterase inhibitors and chronic electrical stimulation of the vagus nerve each result in novel downstream anti-inflammatory effects and increased survival post-myocardial infarction. A recent study examining effects of chronic vagus nerve stimulation in patients with heart failure has provided promising results. Chronic electrical stimulation of carotid sinus baroreceptors in dog models of hypertension and in patients with drug-resistant hypertension have demonstrated long-term efficacy in lowering BP and reducing target organ damage. Chronic carotid sinus baroreceptor stimulation has also been shown to improve cardiac function, decrease arrhythmias, and prolong survival in


BRs: A DETERmINANT of CARDIoVAsCulAR RIsk AND THERAPEuTIC TARgET

dog models of heart failure. Clearly, favorable effects of increasing baroreceptor activity extend far beyond shortterm control of BP.

Further Reading Brooks VL, Sved AF. Pressure to change? Re-evaluating the role of baroreceptors in the long-term control of arterial pressure. Am J Physiol Regul Integr Comp Physiol 2005;288:R815–8. Chapleau MW, Li Z, Meyrelles SS, Ma X, Abboud FM. Mechanisms determining sensitivity of baroreceptor afferents in health and disease. Ann NY Acad Sci 2001;940:1–19. Chapleau MW, Lu Y, Abboud FM. Mechanosensitive ion channels in blood pressure-sensing baroreceptor neurons. Hamill OP, editor. Current topics in membranes, Vol 59. : Elsevier Science; 2007. p. 541–67. Chapleau MW, Sabharwal R. Methods of assessing vagus nerve activity and reflexes. Heart Fail Rev 2011;16:109–27. Glazebrook PA, Ramirez AN, Schild JH, Shieh C-C, Doan T, Wible BA, et al. Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons. J Physiol 2002;541.2:467–82. Glazebrook PA, Schilling WP, Kunze DL. TRPC channels as signal transducers. Pflugers Arch 2005;451:125–30. Guyenet PG. The sympathetic control of blood pressure. Nature Rev Neurosci 2006;7:335–46. Hainsworth R. Reflexes from the heart. Physiol Rev 1991;71(3):617–58. Handa T, Katare RG, Kakinuma Y, Arikawa M, Ando M, Sasaguri S, et al. Anti-Alzheimer’s drug, donepezil, markedly improves long-term survival after chronic heart failure in mice. J Cardiac Fail 2009;15:805–11.

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Korner PI. Baroreceptor resetting and other determinants of baroreflex properties in hypertension. Clin Exp Pharmacol Physiol Suppl 1989;15:45–64. La Rovere MT, Bigger Jr. JT, Marcus FI, Mortara A, Schwartz PJ for the ATRAMI investigators. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. The Lancet 1998;351:478–84. Lu Y, Ma X, Sabharwal R, Snitsarev V, Morgan D, Rahmouni K, et al. The ion channel ASIC2 is required for baroreceptor and autonomic control of the circulation. Neuron 2009;64:885–97. Monahan KD, Eskurza I, Seals DR. Ascorbic acid increases cardiovagal baroreflex sensitivity in healthy older men. Am J Physiol Heart Circ Physiol 2004;286:H2113–H2117. Parati G, Di Rienzo M, Mancia G. How to measure baroreflex sensitivity: from the cardiovascular laboratory to daily life. J Hypertens 2000;18:7–19. Schwartz PJ, De Ferrari GM, Sanzo A, Landolina M, Rordorf R, Raineri C, et al. Long term vagal stimulation in patients with advanced heart failure: First experience in man. Eur J Heart Fail 2008;10:884–91. Sun H, Li D-P, Chen S-R, Hittelman WN, Pan H-L. Sensing of blood pressure increase by transient receptor potential vanilloid 1 receptors on baroreceptors. J Pharmacol Exp Ther 2009;331:851–9. Taylor JG, Bisognano JD. Baroreflex stimulation in antihypertensive treatment. Curr Hypertens Rep 2010;12:176–81. Wladyka CL, Feng B, Glazebrook PA, Schild JH, Kunze DL. The KCNQ/ M-current modulates arterial baroreceptor function at the sensory terminal in rats. J Physiol 2008;586.3:795–802. Wright C, Drinkhill MJ, Hainsworth R. Reflex effects of independent stimulation of coronary and left ventricular mechanoreceptors in anaesthetized dogs. J Physiol 2000;528.2:349–58.


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34 Genetic Determinants of Baroreflex Function Italo Biaggioni The baroreflex is the archetypical mechanism of autonomic cardiovascular regulation. Its neural pathways, physiological relevance, and pathological consequences of its failure are described in detail in Chapters 33 and 72 and will not be repeated here. The importance of the baroreflex in a given subject can be assessed by the gain of the system, i.e., the magnitude of the reciprocal change in heart rate or sympathetic activity in response to alterations in blood pressure. The slope of the relationship between blood pressure and heart rate or sympathetic activity is used as a measure of baroreflex sensitivity (BRS). There is substantial inter-individual variability in these parameters. This is not surprising given the complex neural pathways involved and the biological measurements used in its estimation. Furthermore, most of the methods used to assess baroreflex function cannot isolate the predominant contribution of carotid sinus arterial baroreceptors to that of other arterial and venous afferents. In this chapter we will review the evidence suggesting that a significant component of the variability in baroreflex function is genetically determined. Less is known about the determinants of this genetic influence. A few studies have identified discrete polymorphisms associated with BRS. In general these studies have included relatively small number of subjects and results have not always been replicated. In some cases, the predicted functional consequences of these polymorphisms in the gene products provide a plausible biological explanation of their putative effect in BRS, but a direct causal relationship has been difficult to document. Earlier studies showed that baroreflex function was decreased in normotensive offspring of hypertensive parents, and that decreased baroreflex gain was a predictive factor for the subsequent development of hypertension [1]. More definitive proof of genetic influences on baroreflex function came from studies in twins showing significant correlation for BRS in monozygotic but not dizygotic twins [2]. It has been difficult to ascertain the role of individual genes in the modulation of complex phenotypes like the baroreflex. The baroreflex is the result of the interplay between afferent fibers located in vascular structures


responsive to stretch, the integration of these afferent signals in brainstem nuclei, efferent fibers carrying sympathetic and parasympathetic outflow and coupling of these fibers with cardiac and vascular structures (Chapter 33). It is likely that different genes influence each of these individual components of the baroreflex. A case in point is the observation that normotensive children with Williams syndrome have a significant reduction in BRS [3]. This syndrome is caused by the microdeletion of the long arm of chromosome 7 encompassing the locus of the elastin gene. Elastin is the main component of the extracellular matrix of arteries, and affected patients develop an arteriopathy with intimal thickening. It is plausible, therefore, that this pathology affects pressure transduction at the level of baroreceptor afferents resulting in the observed reduction in BRS. However, in humans it is not feasible to test this hypothesis by measuring afferent firing rates from the carotid sinus in response to pressure changes. The same is true for other individual components of the baroreflex arc; a gene polymorphism of deletion can theoretically affect the function of afferent structures, central brainstem pathways, or efferent neurovascular coupling. We are only able, however, to measure integrated baroreflex function and this approach will necessarily “dilute” any genetic influence on the individual components of the baroreflex. An additional level of complexity is introduced by the fact that the “baroreflex” is not a single entity, but has separate afferent inputs arising from high pressure (the carotid sinus, aortic arch and others) and low pressure (large veins) vascular structures. The efferent pathways are also differentiated into those modulating heart rate, which are predominately parasympathetic, and vascular tone, which are mostly sympathetic. It would be impossible to phenotype each of these components in the large number of patients required for genetic studies. The most commonly used method, therefore, is to estimate the relationship between systolic blood pressure and RR interval during spontaneous periods when parallel increases (or decreases) in these parameters occur. There is inherent variability in these measurements, which may also “dilute” a genetic effect.

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Despite all these limitations, a handful of studies have described associations between discrete gene polymorphisms and BRS. Polymorphisms of KCNMB1 gene coding the β1 subunit of the calcium-sensitive potassium channel BK was associated with BRS in 298 normotensive twins [4]. In particular, AA homozygous in the polymorphism of exon 4b had greater BRS than heterozygous or CC homozygous. Potassium channels (BK) are expressed in vascular smooth muscle cells and are important to calcium-mediated relaxation. They are, however, also expressed in neural tissues and the site where this polymorphism may alter BRS could not be determined from these studies. AA homozygous also had higher heart rate variability in the high frequency range, a measure of increased vagal tone. It is possible, therefore, that the increased BRS was due to the putative increase in vagal tone. A common polymorphic variant in the promoter region of the bradykinin B2 receptor gene, T-58C, was also associated with baroreflex function; greater number of T alleles was associated with increased BRS in 129 untreated hypertensive subjects, but not in 95 normotensive controls [5]. This polymorphism could account for 12% of BRS variation even after accounting for blood pressure, BMI, age and gender. In transfected cell models the  58C allele was associated with reduced B2R expression, leading the authors to suggest that decreased bradykinin actions could contribute to the reduction in BRS. In a study of Finnish subjects, a C-344T polymorphism of the aldosterone synthase gene (CYP11B2) was associated with BRS measured from the overshoot phase of the Valsalva maneuver, with the CC allele associated with decreased BRS [6]. No comparable associations were found for BRS with an insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE), or the M235T variants of angiotensinogen (AGT). The C-344T polymorphism is a common variant in the promoter region of the gene. The C allele has been found to be associated with elevated plasma levels of aldosterone. Ormezzano et al studied 17 polymorphisms of 11 genes relevant to blood pressure regulation in 146 hypertensive individuals and 105 healthy controls. Only the C  1222T polymorphism of endothelin receptor A, EDNRA, correlated with BRS in both populations after adjustment for age, gender, blood pressure and BMI [7]. Importantly, the association remained significant after accounting for the multiple number of genes tested. No other polymorphism was significantly correlated with BRS, including the CYP11B2 previously reported by Ylitalo [6]. Similarly, the C allele of the A1166C polymorphism in the AT1 receptor gene was associated with reduced BRS in 135 normotensive individuals [8]. Of note, this variant occurs in an untranslated region of the gene and is not associated with a functional change in the AT1 receptor but could be in linkage disequilibrium with other sites. Also, this appears to be one of the polymorphisms previously reported not to be associated with BRS [7].

Recently, Xing-Sheng et al evaluated the influence of nine polymorphisms in six genes, in 182 normotensive Chinese men, and found that carriers of the TT genotype of the T-786C polymorphism of the endothelial nitric oxide synthase gene had lower BRS than subjects carrying either the TC or the CC genotype [9]. It is believed that the C allele is associated with reduced expression of eNOS. The finding that subjects carrying the C allele had higher BRS would imply that eNOS-generated NO normally depresses baroreflex function. They also found that subjects with the TT genotype of the C-344T polymorphism in the aldosterone gene had higher BRS as compared to those with CC confirming the results from Ylitalo et al. [6]. Finally, subjects with the CC genotype of the T-58C polymorphism of the B2R gene had lower BRS compared to subjects with TT. These results are similar to those reported by Milan et al. in hypertensive subjects, even though the latter group of investigators found no association in normotensive subjects. Multivariable regression indicated that the three polymorphisms reported by XingSheng could explain 16% of BRS variability [9]. The measurement of BRS in virtually all of the studies described above relies on spontaneous reciprocal changes in blood pressure and heart rate analyzed with spectral analysis techniques from non-invasive measurements. This parameter estimates only the vagal modulation of heart rate by the baroreflex. Cardiovagal BRS has consistently shown to be reduced in hypertensive populations, but it is reassuring that none of the polymorphisms associated with reduced cardiovagal BRS were found to correlate with blood pressure itself, suggesting that differences in BRS reported were not secondary to blood pressure changes. It is known that the hypertensive trait has a significant genetic component, but most attempts to identify relevant genes have been disappointing; in most cases, the genes that have been identified account for only a few mmHg in blood pressure. There is less information about the hereditability of the sympathetic limb of the baroreflex modulating vascular tone. Yamada et al. studied three groups of adolescents: normotensives with a positive family history of hypertension, borderline hypertensives with a positive family history, and normotensives with a negative family history [10]. “Sympathovascular” BRS was estimated by the slope of the percent decrease in muscle sympathetic nerve activity in response to increases in systolic blood pressure induced by phenylephrine. “Sympathovascular” BRS was significantly smaller in the groups with a family history of hypertension (  8  1 %/mm Hg for both the borderline and normotensive offspring) compared to the normotensive controls (  16  1 %/mm Hg). The sympathetic contribution to blood pressure, as determined by baseline MSNA and the fall in blood pressure induced by the ganglionic blocker trimethaphan, was greater in in borderline hypertensive offspring than in normotensives with a positive family history or normotensive controls. These results indicate an impaired baroreflex capacity to dampen


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sympathetic activity resulting in greater “sympathetically driven” blood pressure in adolescents with a family history of hypertension, even before they develop hypertension. We should note that cardiovagal BRS is consistently reduced in hypertensive subjects, but baroreflex control of sympathetic tone in most studies has not been different than normotensive controls. Whereas these studies evaluated arterial high-pressure baroreceptors, Ookuwa et al. studied the function of low pressure baroreceptors located in the venous side of the circulation. Cardiopulmonary baroreflex control of forearm vascular resistance was evaluated in 12 normotensive young subjects with a family history of hypertension and compared with normotensives without a family history of hypertension [11]. Low-pressure cardiopulmonary baroreceptors were preferentially unloaded using low levels (  20 mmHg) of lower body negative pressure (LBNP). It should be noted, however, that even these low levels of LBNP can unload high-pressure baroreceptors. The reflex increase in forearm vascular resistance in response to LBNP was significantly lower in normotensive with a family history of hypertension (38  8%) than in normotensives with a negative family history (86  19%). These results suggest that cardiopulmonary baroreflexes are also impaired in subjects with a genetic predisposition to hypertension, even during the normotensive stage. In summary, there is substantial evidence indicating that a significant component of baroreflex function is genetically determined. Furthermore, reduced BRS is found in offspring of hypertensive parents, and predicts the development of hypertension. It is not clear whether reduced baroreflex buffering contributes to the development of hypertension or is mostly a trait of an underlying abnormality in autonomic regulation of blood pressure. A few polymorphisms have been associated with reduced baroreflex function, but it is not clear where in the baroreflex arc those genes are operative, nor whether there is a

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causal relationship. This, perhaps, is not surprising given the complex nature of baroreflex pathways and inherent limitations in clinical measures of its function.

References [1] Ducher M, Fauvel JP, Cerutti C. Risk profile in hypertension genesis: A five-year follow-up study. Am J Hypertens 2006;19:775–80. [2] Tank J, Jordan J, Diedrich A, Stoffels M, Franke G, Faulhaber HD, et al. Genetic influences on baroreflex function in normal twins. Hypertension 2001;37:907–10. [3] Girard A, Sidi D, Aggoun Y, Laude D, Bonnet D, Elghozi JL. Elastin mutation is associated with a reduced gain of the baroreceptor-heart rate reflex in patients with Williams syndrome. Clin Auton Res 2002;12:72–7. [4] Gollasch M, Tank J, Luft FC, Jordan J, Maass P, Krasko C, et al. The BK channel beta1 subunit gene is associated with human baroreflex and blood pressure regulation. J Hypertens 2002;20:927–33. [5] Milan A, Mulatero P, Williams TA, Carra R, Schiavone D, Martuzzi R, et al. Bradykinin B2 receptor gene (-58T/C) polymorphism influences baroreflex sensitivity in never-treated hypertensive patients. J Hypertens 2005;23:63–9. [6] Ylitalo A, Airaksinen KE, Hautanen A, Kupari M, Carson M, Virolainen J, et al. Baroreflex sensitivity and variants of the renin angiotensin system genes. J Am Coll Cardiol 2000;35:194–200. [7] Ormezzano O, Poirier O, Mallion JM, Nicaud V, Amar J, Chamontin B, et al. A polymorphism in the endothelin-A receptor gene is linked to baroreflex sensitivity. J Hypertens 2005;23:2019–26. [8] Jira M, Zavodna E, Honzikova N, Novakova Z, Vasku A, Izakovicova HL, et al. Association of A1166C polymorphism in AT(1) receptor gene with baroreflex sensitivity. Physiol Res 2010;59:517–28. [9] Xing-Sheng Y, Yong-Zhi L, Jie-Xin L, Yu-Qing G, Zhang-Huang C, Chong-Fa Z, et al. Genetic influence on baroreflex sensitivity in normotensive young men. Am J Hypertens 2010;23:655–9. [10] Yamada Y, Miyajima E, Tochikubo O, Matsukawa T, Shionoiri H, Ishii M, et al. Impaired baroreflex changes in muscle sympathetic nerve activity in adolescents who have a family history of essential hypertension. J Hypertens 1988;6:S525–8. [11] Ookuwa H, Takata S, Ogawa J, Iwase N, Ikeda T, Hattori N. Abnormal cardiopulmonary baroreflexes in normotensive young subjects with a family history of essential hypertension. J Clin Hypertens 1987;3:596–604.


C H A P T E R

35 Cardiac and Other Visceral Afferents John C. Longhurst, Liang-Wu Fu INTRODUCTION Visceral afferents convey information to the central nervous system about local changes in chemical and mechanical environments of a number of organ systems. Physiologically, the autonomic sensory nervous system provides information about the function of abdominal and thoracic organ systems, allowing for reflex responses that typically aid in the normal function of these systems. Pathophysiologically, these sensory nerves provide a warning system to alert the organism to the presence of injury or conditions that can lead to injury and cell death. Thus, they assist both in normal function and maintain homeostasis in adverse conditions. The reflex arc includes the afferent or sensory limb, central neural processing and efferent motor system innervating effector organs. This chapter focuses on afferent fibers present in vagus and sympathetic (spinal) pathways that respond to mechanical and chemical alterations in the environment, and the resulting reflex responses, using ischemia as the stimulus paradigm. Ischemia constitutes an important condition associated with cardiovascular disease. Stimulation of visceral sensory nerves leads to important cardiovascular reflex responses mediated by the autonomic nervous and humoral systems.

ANATOMICAL FRAMEWORK Finely myelinated (Aβ, Aδ) and unmyelinated (C fibers) afferent pathways innervating either unspecialized dense, diffuse or bare nerve endings, form the afferent pathway of visceral cardiovascular reflexes, Table 35.1 [1]. The nerve endings typically are located within the interstitial space and respond to mechanical and/or chemical events. Afferents ascend to the central nervous system (CNS) through mixed nerves, including the vagus or sympathetic (spinal, e.g., spinothalamic and spinoreticular) pathways. A number of nuclei in the thalamus, hypothalamus, midbrain, pons and medulla, including the nucleus tractus solitarii, caudal and rostral ventral lateral medulla, parabrachial nucleus, paraventricular nucleus, periaqueductal gray, lateral tegmental field, medullary raphé, and nucleus ambiguus, among others, process input from visceral afferents ultimately regulating sympathetic and


vagal autonomic motor fiber discharge activity directed to cardiovascular effector organs. These reflex arcs form the pathways for visceral reflexes concerned with regulation of autonomic outflow to the cardiovascular system.

AFFERENT STIMULI Visceral afferent fibers responsive to mechanical stimuli are either high or low threshold with the high threshold endings frequently serving as nociceptors [2–4]. Low threshold mechanosensitive receptors respond to changes in stress or strain and provide information relevant to digestion (gut), cardiac filling or function (cardiac venoatrial, atrial and ventricular). For example, cardiac atrial receptors, located mainly in the veno-atrial junctions, that are innervated by myelinated afferents that course through the vagus, respond to changes in balloon distension involving modest changes in volume but high tensions [5]. Many ventricular mechanosensitive C-fibers respond to changes in end-diastolic volume, and hence stretch, rather than systolic pressure, i.e., compression [6]. Sensory nerves that respond to changes in stress or strain in the gastrointestinal tract largely course through the vagus nerves and likely are concerned with transmission of information related to digestion rather than cardiovascular function [2].

ISCHEMIA Many high threshold mechanosensitive endings are triggered by chemical events and hence are bimodal in their sensitivity. Chemical stimuli activating these endings depend on the organ in which they are situated and the condition imposed [4]. Ischemia, for example, leads to the production and release of protons, kinins, serotonin, histamine, cyclooxygenase products like thromboxane, endothelin and reactive oxygen species, including hydroxyl radicals, among others (Fig. 35.1). These mediators stimulate afferent endings and frequently sensitize them to the action of other chemical mediators [7]. Other ischemia-related chemical changes associated with activation of peripheral chemoreceptors, such as hypoxia and hypercapnia, are not important stimuli of chemosensitive

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35. CARdIAC And OTHER VIsCERAl AffEREnTs

TABLE 35.1 Classification of Afferent fibers from Abdominal Viscera CrossSectional Diameter (µm)

Conduction Velocity (m/sec)

Aβ (myelinated)

6–12

Aδ (finely myelinated)

C (unmyelinated)

Fiber Type

Terminal Ending

Effective Stimulus

20–84

Pacinian corpuscle

Vibration

2–6

3–30

Unknown, bare nerve endings

Vibration, pulse pressure, contraction, distension, chemicals, noxious stimuli

0.3–1.5

0.3–2.5

Unknown, bare nerve endings

Strong mechanical stimuli, chemicals, noxious stimuli

visceral sensory nerve endings [8,9]. This differential sensitivity has led to the use of the term “chemosensitive” visceral receptors to distinguish them from the arterial chemoreceptors. It is clear that visceral organs, such as the heart, are selectively responsive to chemical events related to altered metabolism associated with deprivation of blood flow and oxygen. The sources of many mediators are parenchymal cells in the organ, for example cardiac myocytes and endothelium [10]. Reactive oxygen species likely are derived from cardiac myocytes, while the endothelium is a major source for endothelin [11,12]. Circulating precursors or enzymes activated by ischemia initiate a cascade of events leading to the production of mediators like kinins, serve as an important source along with activated blood elements, such as platelets, which release several mediators during ischemia and reperfusion. Platelets aggregate at the site of injury of arterial endothelium following rupture of an atherosclerotic plaque or following occlusion of a coronary artery [13]. Activated platelets release serotonin, histamine and

FIGURE 35.1 Diagram of mediators, their receptors and cellular messaging mechanisms involved in activation of cardiac afferents during ischemia and reperfusion. Coronary artery occlusion, for example following plaque rupture induces myocardial ischemia leading to production and release of a number of mediators including 5-hydroxytryptamine (5-HT), histamine, bradykinin (BK), endothelin (ET), reactive oxygen species (ROS), lactic acid, ATP and COX pathway products, including thromboxane A2 (TxA2) and prostaglandins (PG) from various cells. Other abbreviations: adenosine 5'-triphosphate (ATP), purinergic 2X receptor (P2X), purinergic 2Y receptor (P2Y), TxA2/prostaglandin H2 receptor (TP), histamine 1 receptor (H1), 5-HT3 receptor (5-HT3), BK2 receptor (BK2), endothelin A receptor (ETA), transient receptor potential A1 (TRPA1), protons (H), phospholipase A2 (PLA2), phospholipase C (PLC), adenyl cyclase (AC), cyclic adenosine monophosphate (cAMP), phosphatidylinositol-4,5-bisphosphate (PIP2), inositol-1,4,5triphosphate (IP3), 1,2-diacylglycerol (DAG), protein kinase A (PKA), protein kinase C (PKC), arachidonic acid (AA) and cyclooxygenase (COX).


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3

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-Antiplatelet Ab

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1

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FIGURE 35.2 Responses of cardiac ventricular afferents to repeated ischemia before and after treatment with control rabbit serum (A), exposure to a polyclonal rabbit antiplatelet antibody (B) and composite histogram of responses of eight afferents to five minutes of regional ischemia induced by occlusion of a left coronary artery branch before (a) and after (b) treatment with the antiplatelet antibody (C). Means and standard errors are shown. Panel D shows locations of the receptive fields of ischemically sensitive cardiac afferents on epicardial surface of left ventricle. Receptive fields of afferents included in this study: , Aδ afferents (n  4) and •, C-fiber afferents (n  10). * indicates nerve activity during ischemia was higher than during control (P  0.05) and † indicates response to ischemia after treatment with antibody was less than before (P  0.05). Modified from Fu, L-W and Longhurst, JC (2002) Role of activated platelets in excitation of cardiac afferents during myocardial ischemia in cats. Am J Physiol 282:H100–H109. Reproduced by permission.

thromboxane A2, each of which independently or in combination activate cardiac sympathetic afferent endings (Fig. 35.2). The action of each mediator is receptor mediated, with many, if not all of the receptors located on afferent endings. For example, bradykinin exerts its action through BK2, thromboxane through TP, histamine through H1, serotonin through 5-HT3 and endothelin through ETA receptors. Some mediators, like adenosine, remain controversial with regard to their role in stimulating afferent endings during ischemia [7,10]. Some reports, consisting mainly of reflex studies in the dog suggest that adenosine in the heart mediates autonomic reflexes that influence renal function. Other studies in cats suggest that adenosine does not stimulate ischemically sensitive cardiac afferents during ischemia [14].

AUTONOMIC REFLEX RESPONSES TO VISCERAL AFFERENT ACTIVATION Cardiovascular reflex responses to visceral afferent stimulation are either excitatory or inhibitory (Fig. 35.3). In this respect, stimulation of vagal afferents causes reflex

cardiovascular inhibition, including decreased heart rate, blood pressure, and myocardial contractility, consequent to reduced sympathetic outflow to the heart and blood vessels and increased vagal motor output to the heart [15]. Conversely, stimulation of sympathetic afferents that project centrally through sympathetic nerves and spinal pathways evokes reflex cardiovascular excitation, including increases in heart rate, blood pressure and myocardial performance, through increased sympathetic motor activity and, possibly, withdrawal of parasympathetic tone to the heart. Cardiovascular reflex responses originating from the heart consist of either reflex inhibitory or excitatory responses, or, more often, a combination of the two [16]. Thus, stimulation of the posterior-inferior and inner regions of the wall of the left ventricle leads to reflex bradyarrhythmias and hypotension, while stimulation of the anterior and superficial regions of the wall of the left ventricle leads to reflex tachyarrhythmias and hypertensive responses [5]. More commonly both vagal and sympathetic afferent pathways are stimulated concomitantly with a resulting reflex reflecting a mixed response consisting often of a small increase in blood pressure (Fig. 35.3) due to processing in the CNS, for example in the tractus solitarii and possibly the parabrachial nuclei [17].


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FIGURE 35.3 Dose-dependent responses of mean arterial pressure (MAP), heart rate (HR), and left ventricular dP/dt at 40 mmHg developed pressure (LV dP/dt40) to application of graded doses of hydrogen peroxide (H2O2) to the anterior surface of the ventricle in intact (vagal and sympathetic cardiac afferents intact, n  8), bilateral cervical vagotomized (sympathetic afferents intact, n  6) or bilateral T1–T4 ganglionectomized (vagal afferents intact, n  6) cats. Circles and brackets represent means and standard errors, respectively. * denotes significant difference comparing saline control to H2O2 (P  0.05). Modified with permission from Longhurst, JC. (2011) Regulation of autonomic function by visceral and somatic afferents. In: Central Regulation of Autonomic Functions. I. Llewellyn-Smith and A. Verberne, eds. New York: Oxford University Press.

Although stimulation of cardiac afferents can lead to reflex cardiovascular depression or excitation or a mixture of responses due to simultaneous stimulation of sensory fibers in both vagal and sympathetic pathways, stimulation of abdominal visceral afferents more commonly leads to reflex excitation since activation of afferents ascending through spinal pathways predominates. Thus, stimulation of chemosensitive, mechanosensitive and polymodal sensory endings in a number of abdominal organs activate spinal pathways that increase heart rate, myocardial contractility and arteriolar constriction in several regional circulations, including the coronary system [1,18]. Vagal afferent pathways from the abdominal region typically regulate digestive organ function, although recent studies suggest that during hypercapnic acidosis both spinal and vagal afferent activation by gastric distension leads to reflex cardiovascular depression as a result of sympathetic withdrawal and vagal activation [19]. Hence, the normal sympathoexcitatory responses evoked by abdominal visceral organ stimulation is converted to sympathoinhibition and vagal excitation by the altered baseline arterial blood gas status. These new findings may have important implications for patients undergoing abdominal surgery, when visceral afferents are stimulated either during vascular

compromise or mechanical traction. Either increases or decreases in blood pressure potentially can occur depending on the arterial blood gas and pH status. In addition to cardiovascular regulation, stimulation of visceral afferents can cause a number of other important reflex events. For example, stimulation of cardiac vagal afferents during myocardial ischemia can lead to relaxation of the stomach, the antecedent of nausea and vomiting that frequently accompanies inferior myocardial infarctions [5,10]. Stimulation of sympathetic afferents in the heart or abdominal region frequently leads to pain, e.g., angina pectoris [7]. Hence many high threshold visceral sympathetic afferents function as nociceptors [3].

PATHOLOGICAL ALTERATIONS OF VISCERAL AFFERENTS In addition to ischemia, a number of other conditions activate visceral afferents. For example, inflammation typically is associated with the production of chemical (and sometimes mechanical) changes, including increases in kinins, activation of the cyclooxygenase system and enhanced formation of reactive oxygen species. Interestingly,


175

PATHOlOgICAl AlTERATIOns Of VIsCERAl AffEREnTs

Control Stimuli Actual BK+TxA2

5

Nerve Activity (imp/s)

4

Predicted BK+TxA2

*#

3 2

*

*

1

1400

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TxA2

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(B)

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(A)

BK

C1

100

90

90

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TxA2

60

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TxA2+BK

TxA2

C2

100

70 60

50

50

40

40

40

30

30

30

20

20

20

10

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10

0

0

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80

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50

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0

0

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Time (s)

FIGURE 35.4 Bar graph summarizing average discharge activity (A) and total response over time (B) of 15 ischemically sensitive cardiac sympathetic afferents to injection of BK (1 μg), U46619 (5 μg), a thromboxane A2 (TxA2) mimetic or BK (1 μg)  U46619 (5 μg) into the left atrium (LA). Neurohistograms provide summed 2 s impulse activity of 15 cardiac afferents during administration of U46619 (C1), BK (C2) or BK  U46619 (C3). The total responses of afferent to the mediators were calculated by counting all spikes that occurred during the entire period of response when activity exceeded baseline rates by 20%. Columns and error bars represent means  standard errors. *Indicates significant difference comparing stimuli to controls (P  0.05), # significantly different from either BK or U46619 alone (P  0.05) and †denotes significant difference comparing actual BK  U46619 to predicted BK  U46619 (P  0.05). Modified from Fu L-W and Longhurst JC (2009) Bradykinin and thromboxane A2 reciprocally interact to synergistically stimulate cardiac spinal afferents during myocardial ischemia. Am J Physiol 298:H235–H244. Reproduced by permission.

some chemical mediators appear to act as primary stimuli while others may sensitize in either an additive or synergistic fashion (e.g., TxA2, Fig. 35.4) or modulate (e.g., histamine) sensory nerve endings to the action of the primary stimulus (bradykinin) [7]. Other clinical conditions, like hypertension or heart failure modify the responsiveness of nerve endings in the atria or ventricles [5]. Hypertension may do this by altering the parenchymal substrate in which the endings are located. Heart failure may do this by altering the sensitivity of the nerve ending to mechanical or chemical events.

Acknowledgments Research cited was supported by NIH grants HL066217.

References [1] Longhurst JC. Cardiovascular reflexes of gastrointestinal origin. In: Shepherd AP, Granger DN, editors. Physiology of the intestinal circulation raven. New York; 1984. p. 165–78.

[2] Longhurst J. Reflex effects from abdominal visceral afferents. In: Zucker IH, Gillmore JP, editors. Reflex control of the circulation. Caldwell, NJ: Telford Press; 1991. p. 551–77. [3] Pan H-L, Longhurst JC. Ischaemia-sensitive sympathetic afferents innervating the gastrointestinal tract function as nociceptors in cats. J Physiol (Lond) 1996;492:841–50. [4] Longhurst JC. Regulation of autonomic function by visceral and somatic afferents. In: Llewellyn-Smith I, Verberne AJM, editors. Central regulation of autonomic function (Second ed.). New York: Oxford University; 2011. [5] Longhurst JC. Cardiac receptors: Their function in health and disease. Prog Cardiovasc Dis 1984;XXVII:201–22. [6] Thoren PN. Characteristics of left ventricular receptors with nonmedullated vagal afferents. Circ Res 1977;40:415–21. [7] Fu L-W, Longhurst JC. Regulation of cardiac afferent excitability in ischemia. Handb Exp Pharmacol 2009:185–225. [8] Fu L-W, Pan H-L, Pitsillides K, Longhurst J. Hypoxia does not directly stimulate ischemically sensitive abdominal visceral afferents during ischemia. Am J Physiol 1996;271:H261–6. [9] Mark AL, Abboud FM, Heistad DD, Schmid PG, Johannsen UJ. Evidence against the presence of ventricular chemoreceptors activated by hypoxia and hypercapnia. Am J Physiol 1974;227: 273–9.


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[10] Longhurst J, Tjen-A-Looi S, Fu L-W. Cardiac sympathetic afferent activation provoked by myocardial ischemia and reperfusion: mechanisms and reflexes. Ann N Y Acad Sci 2001;940:74–95. [11] Grill HP, Zweier JL, Kuppusamy P, Weisfeldt ML, Flaherty JT. Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase. J Am Coll Cardiol 1992;20:1604–11. [12] Hans G, Schmidt BL, Strichartz G. Nociceptive sensitization by endothelin-1. Brain Res Rev 2009;60:36–42. [13] Flores NA, Sheridan DJ. The pathophysiological role of platelets during myocardial ischemia. Cardiovasc Res 1994;28:295–302. [14] Pan H-L, Longhurst J. Lack of a role of adenosine in activation of ischemically sensitive cardiac sympathetic afferents in cats. Am J Physiol 1995;269:H106–13. [15] Fu L-W, Longhurst JC. Reflex pressor response to arterial phenylbiguanide; role of abdominal sympathetic visceral afferents. Am J Physiol 1998;275:H2025–H2035.

[16] Huang H-S, Stahl G, Longhurst J. Cardiac-cardiovascular reflexes induced by hydrogen peroxide in cats. Am J Physiol 1995;268:H2114–H2124. [17] Tjen-A-Looi S, Bonham A, Longhurst J. Interactions between sympathetic and vagal cardiac afferents in nucleus tractus solitarii. Am J Physiol 1997;272:H2843–H2851. [18] Longhurst JC. Chemosensitive abdominal visceral afferents. In: Gebhart GF, editor. Proceedings: visceral pain symposium. Seattle: IASP Press; 1995. p. 99–132. [19] Tjen-A-Looi SC, Hsiao AF, Longhurst JC. Central and peripheral mechanisms underlying gastric distension inhibitory reflex responses in hypercapnic-acidotic rats. Am J Physiol 2011;300:H1003–12.


C H A P T E R

36 Autonomic Control of the Heart Rachel C. Drew, Lawrence I. Sinoway INTRODUCTION The cardiovascular system consists of the heart and a network of blood vessels that circulate blood to tissues and organs within the body. The heart works as a pump by forcing blood into the arterial circulation in order to deliver oxygen and nutrients to tissues, and remove carbon dioxide and waste products from these tissues in the venous circulation that carries blood back to the heart. This circulatory system is essential for normal homeostatic function, as well as regulation of body temperature, fluid maintenance and adjusting to altered physiological states such as exercise, and is dependent on the heart’s ability to operate properly.

THE HEART The heart is a powerful muscle that comprises of four chambers; right atrium, right ventricle, left atrium and

left ventricle (see Fig. 36.1). Blood enters the right atrium via the superior vena cava from the upper body and the inferior vena cava from the lower body. The right atrium contracts to push blood down into the right ventricle. When the right ventricle contracts, blood is forced out of the heart through the right and left pulmonary arteries towards the lungs. Oxygen-depleted blood entering this pulmonary circulation exchanges carbon dioxide for oxygen, and so oxygen-rich blood returns to the heart via the right and left pulmonary veins and into the left atrium. Contraction of the left atrium pushes blood down into the left ventricle, and left ventricular contraction forces blood out of the heart again via the aorta to the systemic circulation. Blood flow through the heart is unidirectional, which is achieved by valves positioned between the chambers and within the vessels. Blood entering the heart will pass the tricuspid valve between the right atrium and right

Aorta (to body) Pulmonary artery

Superior vena cava (from upper body)

Left pulmonary arteries (to left lung)

Right pulmonary arteries (to right lung)

Left pulmonary veins (from left lung)

Aortic valve Right pulmonary veins (from right lung)

Left atrium Mitral valve

Right atrium

Left ventricle Tricuspid valve Inferior vena cava (from lower body) Pulmonary valve Direction of blood flow Septum

Right ventricle

FIGURE 36.1 Internal structure and blood flow within the human heart. From Diseases and Conditions Index – Heart and Blood Vessel Diseases; How the Heart Works; Anatomy; http://www.nhlbi.nih.gov/health/dci/Diseases/hhw/hhw_anatomy.html; National Heart Lung Blood Institute, National Institutes of Health, US Department of Health and Human Services, 2010. Permission granted.


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ventricle, and then the pulmonary valve when being pumped out of the right ventricle and towards the lungs. When re-entering the heart, blood will pass the mitral valve between the left atrium and left ventricle, and finally the aortic valve when being forced out of the heart through the aorta to the rest of the body. The heart contracts in a repeated, rhythmic manner, yet blood is continuously delivered to tissues and organs due to the innate properties of arterial blood vessels. During ventricular contraction, the aorta and its branches distend and then during ventricular relaxation, the walls of the large arteries recoil elastically, which forces blood through the circulation continually.

the SA and AV nodes and activate them by releasing acetylcholine. This action decreases heart rate (negative chronotropy), force of atrial contraction (negative inotropy), rate of relaxation (negative lusitropy), and conduction velocity of the SA and AV nodes (negative dromotropy). As the parasympathetic nervous system is responsible for regulating functions primarily when the body is at rest, this increased vagal tone results in low heart rates of ~70 beats per minute in humans. As such, the parasympathetic nervous system is said to induce the “rest and digest” state. Sympathetic nervous control of the heart arises from the upper thoracic region of the spinal cord. Short preganglionic efferent nerve fibers, compared to long preganglionic vagal efferent fibers, enter the paravertebral chains of ganglia that are located on either side of the spinal column. These preganglionic fibers synapse with postganglionic sympathetic fibers and release acetylcholine, which binds to nicotinic receptors on the postganglionic fibers. From here, relatively long sympathetic adrenergic efferent fibers extend to the SA and AV nodes in the heart where they release the neurotransmitter norepinephrine at synapses with beta-adrenergic receptors. In contrast to parasympathetic effects, this action increases heart rate (positive chronotropy), force of ventricular contraction (positive inotropy), rate of relaxation (positive lusitropy), and conduction velocity of the SA and AV nodes (positive dromotropy). These responses are indicative of the “fight or flight” state that the sympathetic nervous system is said to elicit, and are capable of increasing heart rate up to ~200 beats per minute in humans.

AUTONOMIC NERVOUS CONTROL The autonomic nervous system can be divided into two sub-divisions, the parasympathetic nervous system and the sympathetic nervous system (see Fig. 36.2). Parasympathetic nervous control of the heart arises from vagal nuclei within the medulla oblongata in the brainstem, and efferent nervous outflow occurs via the tenth cranial nerve, known as the Vagus nerve. These long preganglionic efferent nerve fibers extend down to the heart where they synapse with small ganglia located near the sinoatrial (SA) and atrioventricular (AV) nodes in the heart. At this target organ, the neurotransmitter acetylcholine is released, which binds to nicotinic receptors and activates short postganglionic efferent nerve fibers. These postganglionic fibers synapse with muscarinic receptors in

Parasympathetic (vagus)

Hypothalamus Paravertebral Ganglia

Medulla

Heart Postganglionic Sympathetic Efferents Spinal Cord

Prevertebral Ganglia

Preganglionic Sympathetic Efferents

Blood Vessels

FIGURE 36.2 Anatomy of the autonomic nervous system and its control of the heart. From Cardiovascular Pharmacology Concepts – Autonomic Ganglia; http://cvpharmacology.com/autonomic_ganglia.htm; Richard E. Klabunde, 2010. Permission granted.


AlTEREd AuTonomIC ConTRol

The actions of the parasympathetic and sympathetic nervous systems are opposed to each other, yet they work reciprocally to bring about the necessary responses to internal and external stimuli. At rest, parasympathetic tone is predominant, which can be illustrated by observing the effects of inhibiting parasympathetic and sympathetic activation [2] (see Fig. 36.3). Atropine is a muscarinic receptor antagonist that inhibits parasympathetic activity and when administered in humans at rest, heart rate increases to ~110 beats per minute. In contrast, when propranolol, which is a beta-adrenergic receptor antagonist that inhibits sympathetic activity, is given at rest, heart rate decreases only slightly to ~50 beats per minute. When both nervous systems are blocked, heart rate remains ~100 beats per minute, which is known as the intrinsic heart rate. When an increase in heart rate from resting levels is necessary, for example during exercise, parasympathetic tone is initially withdrawn and then sympathetic tone is enhanced. Conversely, when heart rate recovers following a bout of exercise or other physiologically stressful event, sympathetic activity is first reduced and then parasympathetic activation is augmented [1].

CONTROL OF THE HEARTBEAT The heart itself is capable of generating its own electrical impulses to cause coordinated and rhythmic contractions of its chambers. The SA node, a specialized group of cardiac myocytes located in the right atrium, generates action potentials that spread throughout the atria. This causes depolarization and consequent contraction of atrial

120

HEART RATE (BEATS/MIN)

110 PROPRANOLOL

100 90

ATROPINE

80

ATROPINE

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179

muscle. This produces the normal sinus rhythm, thus the SA node is known as the “pacemaker”. These electrical impulses travel from the atria to the ventricles via the AV node, another specialized group of cardiac myocytes located in the posterior inferior region of the interatrial septum. From here, these impulses are transmitted through the bundle of His, specialized cardiac myocytes that conduct electrical activity, at the base of the ventricles along the left and right bundle branches in the interventricular septum down to the apex of the heart. These branches then divide into an extensive network called Purkinje fibers that cover the inner ventricular walls, and these impulses travel throughout this system of fibers causing depolarization and contraction of ventricular muscle. This complex yet highly coordinated sequence results in one cardiac cycle, which lasts for ~0.8 seconds. Within each cycle, there are periods of relaxation, known as diastole, and contraction, known as systole. When the whole heart is in diastole, the atria fill with blood, which then contract during atrial systole and force blood into the ventricles. The ventricles then contract during ventricular systole and eject blood into the aorta and pulmonary arteries while the atria are in diastole. The ventricles then return to diastole and the cycle begins again. In addition to the heart’s intrinsic ability to regulate its electrical activity, autonomic nervous mechanisms can also directly influence the conduction of electrical impulses throughout the heart. This action is primarily mediated via the AV node. Parasympathetic activation releases acetylcholine that binds to muscarinic receptors in the AV node, which causes slower depolarization of cardiac myocytes. This results in negative dromotropy and decreased ventricular contractility. Acetylcholine release from parasympathetic nerve fibers can also inhibit the release of norepinephrine from sympathetic nerve fibers. This neurotransmitter antagonism can also cause reduced ventricular contraction. Sympathetic activation releases norepinephrine that binds to beta-adrenergic receptors in the AV node, which causes faster depolarization of cardiac myocytes. This results in positive dromotropy and increased ventricular contractility, as the time between atrial and ventricular contraction is reduced. It is possible for the conduction velocity of action potentials to be slowed to such an extent that they completely stop, leading to AV block. In the treatment of certain medical conditions, giving drugs to induce AV block can be an effective method of stabilizing heart rate. For example, patients prone to cardiac arrhythmias can be given medication to block beta-adrenergic receptors, known as ‘beta-blockers’ [4]. This reduces the conduction velocity of electrical impulses through the AV node, thereby preventing detrimental changes in heart rhythm.

DRUG # 2

FIGURE 36.3 Effects of atropine (0.04 mg/kg total) and propranolol (0.2 mg/kg total) on heart rate in 10 nonathletes. In half of the trials, atropine was given first (top line) and in the other half, propranolol was given first (bottom line). From Katona et al. (1982) J Appl Physiol 52(6):1652–7. Permission granted.

ALTERED AUTONOMIC CONTROL Autonomic nervous control of the heart can be altered under many different physiological conditions. Exercise


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36. AuTonomIC ConTRol of THE HEART

and stress require acute changes in heart function, which are predominantly brought about by alterations in autonomic nervous control [5]. Reduced parasympathetic and augmented sympathetic nerve activity induce raises in heart rate, contractility and conduction velocity of electrical impulses, which provide the necessary increases in blood delivery to the appropriate tissues and organs that demand a greater supply. Chronic alterations in autonomic nervous control manifest in the process of aging and certain disease states. Aging is associated with heightened cardiac sympathetic and reduced parasympathetic activity, which can potentially augment the detrimental effects of any existing cardiovascular disease [3]. Several cardiovascular disease states, such as heart failure and hypertension, have also been linked to similar increases in sympathetic tone and decreases in parasympathetic activity [6–8]. These alterations can lead to a vicious cycle where the disease state is enhanced due to the autonomic changes that have occurred to compensate for the disease itself. In summary, autonomic control of the heart is integral to the regulation of heart function. It plays a major role in ensuring optimal performance of the heart, both when the body is at rest and when responding to acute or chronic physiological changes.

References [1] Arai Y, Saul JP, Albrecht P, Hartley LH, Lilly LS, Cohen RJ, et al. Modulation of cardiac autonomic activity during and immediately after exercise. Am J Physiol Heart Circ Physiol 1989;25:H132–41. [2] Katona PG, McLean M, Dighton DH, Guz A. Sympathetic and parasympathetic cardiac control in athletes and nonathletes at rest. J Appl Physiol 1982;52(6):1652–7. [3] Kaye DM, Esler MD. Autonomic control of the aging heart. Neuromol Med 2008;10:179–86. [4] Kennedy HL, Brooks MM, Barker AH, Bergstrand R, Huther ML, Beanlands DS, et al. Beta-blocker therapy in the cardiac arrhythmia suppression trial. Am J Cardiol 1994;74:674–80. [5] Robinson BF, Epstein SE, Beiser GD, Braunwald E. Control of heart rate by the autonomic nervous system: studies in man on the interrelation between baroreceptor mechanisms and exercise. Circ Res 1966;19:400–11. [6] Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, et al. Sympathetic augmentation in hypertension: role of nerve firing, norepinephrine reuptake, and angiotensin neuromodulation. Hypertension 2004;43:169–75. [7] Schwartz PJ, de Ferrari GM. Sympathetic-parasympathetic interaction in health and disease: abnormalities and relevance in heart failure. Heart Fail Rev 2011;16:107–7. [8] Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic imbalance, heart rate variability and cardiovascular disease risk factors. Int J Cardiol 2010;141:122–31.


C H A P T E R

37 Cardiac Vagal Ganglia Javier G. Castillo, David H. Adams THE VERTEBRATE NERVOUS SYSTEM

THE CARDIAC CONDUCTION SYSTEM

The vertebrate nervous system is divided into two major components: the central nervous system, consisting of the spinal cord and the brain, and the peripheral nervous system, which can be in turn subdivided into somatic and autonomic components [1]. The autonomic component of the peripheral system regulates the function of the internal organs by way of efferent nerve fibers originating from the central system [2]. In this regard, sympathetic and parasympathetic efferent nerves generate complementary motor outputs in response to sensorial impulses directed to the brain from internal organs via either mechanical or chemical receptive afferent neurons. It is important to highlight that both branches of the autonomic system use pre and postganglionic relays. Sympathetic efferent nerves containing preganglionic neurons arise segmentally from the central portion of the spine, and terminate in sympathetic ganglia that divide into bilateral chains that run parallel to the spinal cord (Fig. 37.1). In this area, they synapse with postganglionic sympathetic neurons that travel to the effector organ. In the case of the heart, these neurons are able to provide chronotropic and inotropic stimulation. The main neurotransmitters of the sympathetic nervous system are norepinephrine and acetylcholine. Parasympathetic preganglionic efferent nerves arise mainly in the brainstem and also from the spinal cord and synapse with postganglionic neurons located in the heart [3]. Acetylcholine is the main neurotransmitter, which contributes to reduction of heart rate and contractility. In addition, sensory afferent pathways with sympathetic and parasympathetic components relay information from the heart to the brain. While cardiac parasympathetic afferent pathways control sympathetic and parasympathetic output, the sympathetic afferent pathways do not have an impact on the autonomic output of the heart. These parasympathetic afferent pathways connect to efferent pathways via interneurons located within the brainstem. Sympathetic afferent nerves travel through the stellate ganglia of the sympathetic chain, and connect with motor neurons in the spinal cord.


The cardiac conduction system initiates, conducts, and controls the heart beat. It is integrated by the sinus (SA) node, the atrioventricular (AV) node, penetrating bundle, and ventricular bundle branches [4]. The sinus node is a wedge shaped structure situated at the junction of the superior vena cava with the terminal crest musculature, usually arranged about a central artery. Its cells are fascicular in nature and frequently embedded in a very fibrotic matrix. Acetylcholine positive nerves represent the main cellular subpopulation identified in the SA. The AV node is located within the atrial aspect of the atrioventricular septum, separated from the ventricle by the annulus fibrosus. The node consists of an elongated half oval with a superficial transitional layer arranged circumferentially. This transitional layer crosses through the node and terminates in the base of the tricuspid valve, while additional transitional fibers also enter the node from the floor of the coronary sinus. These fibers are arranged in parallel structures separated by fibrous tissue. The main distinguishing feature between the distal end of the AV node and the proximal penetrating bundle is the penetration of the conduction axis into the annulus fibrosus. When these fibers advance towards the ventricles, the cells reorient in a more parallel fashion and remain small in size. After reaching the central fibrous body and then the left ventricular outflow tract, the bundle subdivides into right and left bundle branches. The left bundle branch ends up descending as a sheet of cells within the septal subendocardium, whereas the right bundle branch descends intramyocardially (Fig. 37.2).

ANATOMY OF THE CARDIAC VAGAL GANGLIA The anatomy of the cardiac vagal ganglia has been consistently studied for over a century [5]. Intrinsic cardiac neurons have been reported to be located in small ganglia organized primarily over the posterior surfaces of the atria, particularly over the atrioventricular groove [6]. Only a few neurons have been associated with ventricular tissues. Recent reports have identified the presence

181


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37. CARdIAC VAGAl GAnGlIA

Brainstem

Atrial Ganglionated Plexuses There are five major atrial ganglionated plexuses in the human heart, all of them named based on their anatomical location:

Craneal Cervical Ganglia

Caudal Cervical Ganglia

Stellate Ganglia

Spinal Cord

Sinoatrial Node

1. The superior right atrial ganglionated plexus is located on the posterior surface of the right atrium adjacent to the junction of the superior vena cava and the right atrium. 2. The superior left atrial ganglionated plexus is a collection of ganglia identified on the posterior surface of the left atrium between the pulmonary veins. It is in this area where an important number of mediastinal nerves synapse. 3. The posterior right atrial ganglionated plexus lies over the posterior surface of the right atrium adjacent to the interatrial groove. 4. The posteromedial left atrial ganglionated plexus courses on the posterior medial surface of the left atrium. The subgroup of ganglia formed after the fusion of the two posterior atrial ganglionated plexuses extending anteriorly into the interatrial septum is named interatrial septal ganglionated plexus. The largest concentration of ganglia is associated with the two major ganglionated plexuses on the posterior surface of the two atria. 5. The posterolateral left atrial ganglionated plexus is the smallest among the atrial plexuses and can be identified on the posterior lateral surface of the left atrial base on the atrial side of the atrioventricular groove (Fig. 37.3).

FIGURE 37.1 The heart is innervated by preganglionic sympathetic and parasympathetic efferent nerves that originate from the spinal cord and dorsal motor nucleus of the brainstem respectively. Preganglionic (dashed) to postganglionic (solid) sympathetic transfer occurs in the sympathetic ganglion chain adjacent to the spinal cord. Postganglionic sympathetic neurons travel to the cardiac plexus, where preganglionic parasympathetic neurons synapse to postganglionic elements. From the cardiac plexus, autonomic nerves can synapse with intrinsic cardiac neurons located within the network of cardiac ganglia.

Ventricular Ganglionated Plexuses

of stained (1% solution of methylene blue) ganglia and nerves mostly in fatty areas, and only 10% of them are located adjacent to underlying muscle or between muscle fascicles [7]. Interestingly, no ganglia are usually identified in the bulk of the fat that accompanies the major coronary arteries in the atrioventricular groove. The human heart comprises an average of 450 ganglia in atrial tissues and approximately 90 ganglia in ventricular tissues. The precise anatomical configuration of every ganglionated plexus varies among individuals as does the size of ganglia within a particular region. Nerves (up to 0.2 mm in diameter) course between regional ganglia forming neural networks. In addition, interconnecting nerves in ganglionated plexuses form complete loops, which range from less than 2 mm to 1 cm in diameter.

1. The major ventricular ganglionated plexus is totally surrounded by the aortic root fat and therefore named as aortic root ganglionated plexus. This plexus is intimately related to the nerves coursing along the coronary arteries, with its left component projected towards the origin of the circumflex coronary artery. 2. At the same time, this latter component of the aortic root plexus is connected to a plexus located at the origin of the anterior descending coronary artery. Consequently, this plexus has been named anterior descending ganglionated plexus. 3. One of the smallest plexuses follows the trajectory of the posterior descending coronary artery thus called posterior descending ganglionated plexus. The last two plexuses are located adjacent to the right acute marginal

The five ventricular ganglionated plexuses may be found in the fatty areas of both ventricular bases, particularly on the superior aspects of the ventricles, the interventricular grooves, and the origins of the marginal arteries. Additionally, occasional neurons can be identified outside these areas, mostly in fat associated with branching segments of larger coronary arteries.


183

THE AuTonomIC nERVous sysTEm

1

2

Region

5

4

3

Infants

Adults

Elderly

PGP

DBH

TH

PGP

DBH

TH

PGP

DBH

TH

Sinus node (1)

++

+

+

+++

++

+

+++

+

+

Atrioventricular node (2)

++

+

+

++

+

+

+

+

+

Penetrating bundle (3)

+

+

-

++

+

+

+

+

+

Branching bundle (4)

+

+

-

++

+

+

+

+

+

Bundle branches (5)

+

-

-

+

+

+

-

-

-

FIGURE 37.2 View of the human heart illustrating the conduction system. The table shows the patterns of innervation by immunofluorescence according to developing stages. DBH: Dopamine β-hydroxylase (sympathetic activity); PGP: Protein gene product (presence of nerve fibers); TH: Tyrosine hydroxylase (sympathetic activity); –No detected nerve fibers; Scattered individual nerve fibers; Moderate number of nerve fibers, Large number of nerve fibers.

coronary artery and the left obtuse marginal coronary artery and their names are: 4. Right acute marginal ganglionated plexus; and 5. Obtuse marginal ganglionated plexus. Finally, scattered ungrouped neurons can be found embedded in fatty areas on the ventricular side of the atrioventricular groove. However, these neurons are not consistently clustered in a determined region (Fig. 37.3).

THE AUTONOMIC NERVOUS SYSTEM The integrated control of the circulatory system results from intrinsic (e.g., endothelium) and extrinsic mechanisms (e.g., autonomic nervous system). In this regard, the

autonomic nervous system mostly affects the vasomotor tone and cardiac function through the sympathetic and parasympathetic divisions. Moreover, it also has an impact on the systemic volume and the peripheral resistance by modulating the release of certain peptide hormones such as angiotensin II (AGII) or nitric oxide (NO) [8]. In fact, this neuronal control involves an accurate acquisition of inputs from the cerebral cortex and individualized sensors, the integration into particular brain regions, and the transmission of efferent activity to the heart over the sympathetic and parasympathetic pathways. Three anatomical areas of the brain have been identified as an integrated functional system that coordinates the vasomotor status of the cardiovascular system. These are: 1. The upper anterolateral medulla (vasoconstrictor).


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37. CARdIAC VAGAl GAnGlIA

2

1

5

3

7

4

6

200 Neurons

Ganglia per Heart

Superior right atrial (1)

+++

+

-

31 ± 5

Superior left atrial (2)

+++

++

+

56 ± 12

Posterior right atrial (3)

+++

++

++

194 ± 22

Posteromedial left atrial (4)

+++

++

+

161 ± 27

Posterolateral left atrial (5)

+++

+

-

16 ± 2

Aortic root

++

-

-

16 ± 2

Anterior descending

++

+

-

11.2 ± 1.1

Posterior descending (6)

+

-

-

5.2 ± 1.9

Right acute marginal

+

-

-

6.2 ± 2.8

Ganglionic Plexus Atrial Ganglionated Plexuses

Ventricular Ganglionated Plexuses

FIGURE 37.3 SuperiorObtuse view of the human of ganglionated plexuses on the surface of the atria and ventricles. The marginal (7) heart illustrating the distribution 5.2 ± 2.0 + table shows the number of neurons and ganglia in these regions. –None; Scattered; Moderate; Abundant.

2. The lower anterolateral medulla (vasodilator). 3. The nucleus tractus solitarii which integrates vasoconstrictor and vasodilator stimuli. The cerebral regions that modify the chronotropic activity of the heart are located in the thalamus, posterior and posterolateral regions of the hypothalamus, and dorsal region of the medulla. Variations of the sympathetic activity exert a very powerful control over the peripheral circulation. These efferent nerve fibers course either in specific sympathetic pathways to innervate the heart or join the paravertebral sympathetic chain and synapse in secondary

ganglia forming spinal nerves that innervate peripheral vessels. These vascular nerves terminate on small arteries, arterioles, venules, and veins and adjust vascular resistance and volume capacity and therefore the heart pacing. Cardiac nerves, which mainly arise from the stellate ganglia, innervate the atria and ventricle. Reflex sympathetic stimulation causes vasoconstriction by releasing norepinephrine (NE) from sympathetic nerve endings and epinephrine from the adrenal medulla, which stimulates α and β receptors. In this case, epinephrine provides a dual pattern of action, produces vasodilation and cardiac stimulation at low concentrations, and


THE AuTonomIC nERVous sysTEm

vasoconstriction at higher concentrations. This is a very important concept since reflex sympathetic stimulation increases cardiac output during metabolic stress, maintaining systemic pressures and heart perfusion [8]. On the other hand, although the parasympathetic nervous system plays a very insignificant role in arterial pressure regulation, it plays a crucial role in modulating the heart rate. The parasympathetic fibers of the vagus nerve provide innervation to the cardiac conduction system including the SA and AV nodes as well as the atrial myocardium, thus providing chronotropic control. When the vagus nerve is stimulated, acetylcholine reduces heart rate and contractility.

References [1] Van Stee EW. Autonomic innervation of the heart. Environ Health Perspect Oct 1978;26:151–8.

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[2] Armour JA, Murphy DA, Yuan BX, Macdonald S, Hopkins DA. Gross and microscopic anatomy of the human intrinsic cardiac nervous system. Anat Rec Feb 1997;247(2):289–98. [3] Martins J. Parasympathetic regulation of the heart. Heart Rhythm Aug 2010;7(8):1120–1. [4] Chow LT, Chow SS, Anderson RH, Gosling JA. Innervation of the human cardiac conduction system at birth. Br Heart J May 1993;69(5):430–5. [5] Chow LT, Chow SS, Anderson RH, Gosling JA. Autonomic innervation of the human cardiac conduction system: changes from infancy to senility--an immunohistochemical and histochemical analysis. Anat Rec Oct 1 2001;264(2):169–82. [6] Hildreth V, Anderson RH, Henderson DJ. Autonomic innervation of the developing heart: origins and function. Clin Anat Jan 2009;22(1):36–46. [7] Crick SJ, Wharton J, Sheppard MN, et al. Innervation of the human cardiac conduction system. A quantitative immunohistochemical and histochemical study. Circulation Apr 1994;89(4):1697–708. [8] McGrath MF, de Bold ML, de Bold AJ. The endocrine function of the heart. Trends Endocrinol Metab Dec 2005;16(10):469–77.


C H A P T E R

38 Neural Control of Blood Vessels Julian H. Lombard, Allen W. Cowley, Jr. AUTONOMIC VASOMOTOR CONTROL AND CARDIOVASCULAR HOMEOSTASIS The sympathetic and parasympathetic components of the autonomic nervous system play a crucial role in maintaining cardiovascular homeostasis and enabling the body to respond to physiological stressors. Neurogenic control, especially on the arterial side of the circulation, is generally superimposed on intrinsic tone of the vessels, although neurogenic vasoconstriction also occurs in vessels possessing little or no intrinsic tone, e.g. large veins. The effects of the autonomic nervous system on the cardiovascular system are coordinated by a combination of centrally driven autonomic nerve activity that is modulated by cardiovascular receptors (mainly the arterial baroreceptors). Neurogenic mechanisms are not only essential to maintain and regulate arterial blood pressure, but also play a crucial role in regulating the distribution of blood flow between and within individual vascular beds. As discussed below, the net effect of adrenergic constrictor mechanisms on vascular resistance and blood flow within specific vascular beds is determined not only by the frequency of sympathetic nerve traffic, but also by the modulating influences of multiple vasoactive stimuli such as hormones, autacoids, and local autoregulatory mechanisms. Although autonomic neurogenic mechanisms predominantly mediate vasoconstriction via noradrenergic nerve terminals, neurogenic vasodilation does occur in some vascular beds. Two of the primary functions of neurogenic vasodilation are in erectile tissue of the genetalia (mediated by parasympathetic nerves) and sympathetic cholinergic vasodilation of skeletal muscle arterioles in some species, although likely not in man (see below). A tonic level of sympathetically-mediated vasoconstriction in larger vascular beds, e.g., skeletal muscle and the splanchnic circulation, is essential to prevent catastrophic drops in arterial blood pressure and to regulate blood flow to match tissue needs. Due to the large volume of blood (approximately 70% of the total blood volume) on the venous side of the circulation, neurogenic regulation of the capacity of the venous side of the circulation by the sympathetic nervous system (SNS) plays a crucial role in cardiovascular homeostasis via regulation of cardiac output and ventricular filling pressure.


Sympathetically mediated vasoconstriction can decrease flow in the splanchnic circulation to ~25% of resting values. About half the blood volume in the splanchnic circulation (~15% of total blood volume) can be mobilized by passive collapse of the veins and by active venoconstriction at higher levels of SNS activation, e.g., those occurring during conditions of hemorrhagic stress. Under those conditions, sympathetically mediated constriction of precapillary arterioles can also reduce capillary pressure sufficiently to absorb interstitial fluid into the circulation when capillary pressure falls below plasma colloid osmotic pressure. In contrast to sympathetic nervous effects, parasympathetic nervous activity dilates splanchnic vessels indirectly by stimulating intestinal motility and glandular secretion, which increases intestinal metabolism. The cutaneous vascular bed is another important vascular bed for recruiting blood volume in response to increases in sympathetic nerve activity. Another important role for sympathetic regulation of cutaneous blood vessel function is in thermoregulation. In this case, vasoconstriction to restrict heat loss and vasodilatation to promote heat loss play crucial roles in regulating body temperature. Finally, the extracranial arteries in the head receive both sympathetic innervation via the superior cervical ganglion, with norepinephrine (NE) and neuropeptide Y (NPY) as transmitters or parasympathetic innervation via the sphenopalatine and otic ganglia, with vasoactive intestinal peptide (VIP), acetylcholine and nitric oxide as neurotransmitters. The primary function of the sympathetic innervation of the cerebral arteries is to shift the upper limit of cerebral autoregulation to higher pressures. By contrast, the parasympathetic nerves, which are potent dilators of the cerebral circulation, do not appear to play a significant role in regulating cerebral blood flow, although they may be involved in migraine headaches. In addition, sensory input from the trigeminovascular pathway, with calcitonin gene related peptide (CGRP) as the transmitter, may act as a protective mechanism to restore cerebral vessel tone after contractile stimuli. The latter pathway may also have a role in migraine headache. After entering the brain parenchyma, cerebral arteries lose their sympathetic and parasympathetic innervation. Within the parenchyma itself, changes in cerebral blood flow occurring in response to neuronal activity are effected

187


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38. NEuRAl CoNTRol of Blood VEssEls

Cardiac

Sympathetic Nervous System

Inotropic Effect (+) Chronotropic Effect (+)

Arteries Veins Microvessels

Targets

Norepinephrine (NE) Vascular

Transmitters

ATP Neuropeptide Y (NPY) Alpha 1 (constriction) Adrenergic (NE) Alpha 2 (constriction/dilation)

Receptors

Beta 1 and beta 2 (vasodilatation) P2 Purinergic (ATP)-constriction Y1 (NPY)-Constriction

FIGURE 38.1 Learning map summarizing cardiovascular control by the sympathetic nervous system.

via multiple mediators coordinated by the neurovascular unit, consisting of neurons, astrocytes, and the target blood vessels.

SYMPATHETIC COMPONENT OF AUTONOMIC VASOMOTOR CONTROL The sympathetic component of the autonomic nervous system plays the predominant role in regulating vascular tone and whole-body hemodynamics (see Fig. 38.1). Activation of the sympathetic nervous system usually elicits a vasoconstriction that is mediated by three main neurotransmitters (norepinephrine, ATP, and neuropeptide Y), and is roughly proportional to the level of neural activity. Stimulation of sympathetic nerve fibers in skeletal muscle can also cause a transient dilation of larger arterioles that is blocked by atropine (sympathetic cholinergic vasodilation) in some species. However, this does not appear to be the case in man, where vasodilation in anticipation of stresses such as exercise appears to be due to withdrawal of sympathetic tone and vascular relaxation mediated by β2 adrenergic receptors.

Neuroeffector Junction Figure 38.2 shows a schematic diagram of the vascular neuroeffector apparatus; and Figure 38.3 shows the dense adrenergic innervation of a small artery and the less dense adrenergic innervation of a small vein in the rat mesentery. The postganglionic autonomic nerves of the sympathetic nervous system that innervate blood vessels ramify into small bundles, which form a primary plexus located in the adventitial layer of the vessel. The terminal effector plexus is located near the medial layer where adrenergic nerve fibers approach the surface of the smooth muscle

cells and establish neuromuscular contact. These nerves end in strings of varicosities that are devoid of Schwann cell sheaths, and which release transmitter in response to action potentials in the nerves. It is now recognized that many varicosities form en passant synapses with their targets. Compared to the narrow synaptic clefts in the central nervous system, the synapses in peripheral neuroeffector units are much wider, often no closer than 100 nm from the vascular smooth muscle cells.

Sympathetic Neurotransmitters In the varicosities at the neuroeffector junction, sympathetic neurotransmitters are stored in either small or large dense-cored granular vesicles, which also contain ATP, enzymes involved in norepinephrine synthesis such as dopamine β-hydroxylase, and proteins such as chromogranin. There are three major sympathetic co-transmitters – norepinephrine, ATP and neuropeptide Y (NPY), all of which contribute to sympathetic vasoconstriction, although the relative contribution of each varies with among vascular beds and with the amount of sympathetic nerve traffic. Norepinephrine (NE) is the classical neurotransmitter released from adrenergic nerve terminals during sympathetic discharge and produces vasoconstriction by activating α1-adrenergic receptors located on vascular smooth muscle cells. Norepinephrine is found in the small dense core vesicles and seems to be primarily responsible for the moderately fast phase of adrenergic vasoconstriction. ATP is released together with norepinephrine from sympathetic nerves and produces a rapid contraction of vascular smooth muscle by activating P2 purinergic receptors, which include ligand-gated ion channels and G-protein coupled receptors. Neuropeptide Y (NPY) is also found in sympathetic nerve fibers, most likely co-localized with norepinephrine


symPATHETIC ComPoNENT of AuToNomIC VAsomoToR CoNTRol

189

Primary adventitial plexus Schwann nucleus Vasa vasorum Adventitio-medial junction

Adventitia

Terminal effector plexus

Media

Terminal effector plexus in outermost muscle lamellae

FIGURE 38.2 Arrangement of vascular neuroeffector apparatus. Postganglionic autonomic nerves ramify into small bundles forming a primary plexus, which is located in loose adventitia. Bundles give rise to varicosed fibers forming terminal effector plexus, located on surface of medial layer. (Reprinted with permission from Verity MA. Morphologic studies of the vascular neuroeffector apparatus. In: Physiology and Pharmacology of Vascular Neuroeffector Systems, JA Bevan, RF Furchgott, RA Maxwell and AA Somlyo, eds. Basel: Karger, 1971, pp. 2–12.)

compound may play a more important role with circulatory stress, as it appears to be released at the moderate to high levels of sympathetic discharge that occur with circulatory stress. NPY causes a slowly developing and persistent phase of constriction via Y1 receptors, and also potentiates vasoconstriction in response to norepinephrine.

Neurotransmitter Release and Effector Action

FIGURE 38.3 Fluorescence micrograph of a whole mount of rat mesentery showing perivascular sympathetic nerves demonstrated by Falck– Hillarp formaldehyde technique. A very dense plexus of fluorescent noradrenergic fibers supplies a small artery, whereas the corresponding vein has less well developed innervation, although still prominent. Nerve terminals are seen to accompany small vessels of arteriolar caliber. 155. (Reprinted with permission from Falck B. Observations on the possibilities of cellular localization of monoamines by a fluorescence method. Acta Physiol Scand (Suppl.) 1962;197:1–25.)

in the large dense-core vesicles. NPY is released along with norepinephrine and ATP at moderate to intense levels of sympathetic activation. The role of NPY in normal regulation of blood pressure is uncertain, but this

NE released from the adrenergic nerves binds to receptors on the VSM cells acting primarily on α1-adrenergic receptors, which cause contraction of the vascular smooth muscle via membrane depolarization with extracellular Ca2 influx and by liberation of Ca2 ions from intracellular stores via the phospholipase C/IP3 mechanism. Because not all the VSM cells are in close proximity to the adrenergic nerve varicosities, gap junctions between adjacent smooth muscle cells provide low resistance pathways, enabling electrical coupling between the cells. This allows smooth muscle cells that are not adjacent to varicosities to be activated and, in the microcirculation, allows excitation to be propagated longitudinally along the arteriole. Norepinephrine is removed from the junctional cleft primarily by active reuptake by the neurons, enzymatic degradation by catechol-O-methyl transferase, and by spillage into the circulation.

Parasympathetic Component of Autonomic Vasomotor Control The cranial and sacral nerves of the parasympathetic nervous system also regulate vascular tone, although much less is known regarding their functional roles apart


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38. NEuRAl CoNTRol of Blood VEssEls

from their role of increasing blood flow during engorgement of erectile tissue in the genitalia. It is generally accepted that cholinergic nerves contain predominantly small agranular vesicles (35–60 nm), although large granular vesicles with a dense core are often present as well. Cholinergic vasodilatation appears to be mediated via the combined action of acetylcholine, vasoactive intestinal peptide (VIP) and nitric oxide (NO). Despite its high endogenous concentration in some blood vessels, e.g. cerebral arteries, acetylcholine appears to exhibit a negligible direct effect on vascular smooth muscle tone, most likely due to a combination of a wide synaptic distance and a low synaptic concentration as a result of the activity of acetylcholinesterase. However, NO synthase and choline acetyltransferase coexist in parasympathetic ganglia and in the perivascular nerves innervating cerebral blood vessels of several species. Acetylcholine and NO are coreleased from the same nerves, and there is also evidence that acetylcholine acts as a presynaptic transmitter to modulate NO release. Taken together, those findings suggest that nitric oxide (NO) mediates the major component of parasympathetic neurogenic vasodilation in cerebral arteries. In addition to acetylcholine, parasympathetic neurons also contain vasoactive intestinal peptide (VIP) which, together with NO, contributes to vasodilation in some vascular beds, particularly the larger arteries and veins in the head and pelvis. Vasodilator neurons that synthesize VIP also contain structurally unrelated peptides, including NPY, dynorphins, enkephalin, galanin, somatostatin and calcitonin gene-related peptide (CGRP), although little is known regarding their physiological roles. Despite the existence of cholinergic vasodilation in some vascular beds, the overall contribution of the parasympathetic nervous system to the regulation of vascular tone and hemodynamics is small. Rather, the primary regulatory role of the parasympathetic nervous system in the cardiovascular system is mediated via its negative chronotropic and inotropic effects on the heart (see Fig. 38.4).

Differential Vasomotor Control Except for fight or flight responses, the sympathetic nervous system does not usually produce uniform effects on all target organs, Different postganglionic sympathetic neurons have distinct properties, and release other transmitters in addition to NE. This specific distribution of neuroactive chemicals among neurons is called chemical coding. For example, depolarization of postganglionic sympathetic neurons in the lumbar sympathetic ganglia of the guinea pig causes a brief burst of action potentials in nearly all the neurons, leading to release of ATP and NPY to cause arterial vasoconstriction; while depolarizaton of sympathetic preganglionic neurons in the inferior mesenteric ganglion causes sustained firing in about 80% of the neurons, leading to release of both NE and somatostatin, which regulates gut motility and secretion. Sympathetic neurons act in a discrete and organspecific manner, allowing blood flow in individual vascular beds to be regulated independently, depending on physiological conditions. This differential regulation involves discrete neurons in the pre-vertebral ganglia that exhibit specific electrophysiological, neurochemical, and morphological phenotypes. The final neurons in the sympathetic vasomotor pathways are small and receive fewer preganglionic inputs than the non-vasomotor neurons of the autonomic nervous system. Specific functional pools of postganglionic neurons receive convergent input from different pools of preganglionic neurons, many of which contain neuropeptides that affect the excitability of the neurons. Pools of sympathetic neurons project to specific segments of the vasculature, allowing for selective regulation of regional resistance in the proximal and distal portions of the vascular bed. In addition, the functional pools of vasomotor neurons can contain characteristic combinations of co-transmitters. Thus, vasomotor neuron pools appear to be grouped into functional pools and can be recruited as necessary to provide highly graded and specific flow within and between the vascular beds.

Heart (Major)


Chronotropic Effect (–) Intropic Effect (–)

Vasodilator Action Acetylcholine (ACh) Vascular (Minor)

Transmitters

Nitric Oxide (NO) Vasoactive Intestinal Peptide (VIP) Muscarinic Receptors (ACh)

Receptors

Guanylyl cyclase (NO) VIP (VIP)

FIGURE 38.4 Learning map summarizing cardiovascular control by the parasympathetic nervous system.


symPATHETIC ComPoNENT of AuToNomIC VAsomoToR CoNTRol

Modulation of Adrenergic Vasoconstriction There are significant regional variations in the responsiveness of blood vessels to sympathetic activity as a result of a variety of factors, including: (i) the density of α-adrenergic innervation; (ii) vascular smooth muscle sensitivity to norepinephrine; (iii) differences in α-adrenergic receptor populations between organs; (iv) differences in neuronal reuptake of norepinephrine; and (v), the structure and size of blood vessels, which affects the access of norepinephrine to the receptors, i.e. in small vessels with small junctional clefts, the actions of released norepinephrine can be more localized. An important factor in determining the ultimate effect sympathetic activity on blood flow in individual vascular beds is the modulating influence of factors that can override the vasoconstrictor effects of adrenergic activation. For example local autoregulatory mechanisms cause vasodilation in response to increases in metabolic activity particularly in the skeletal muscle, cerebral, and coronary circulations. Increases in blood flow velocity in response to shear stress on the endothelium of arterioles and small resistance arteries can also cause vasodilation by releasing nitric oxide and potentially other vasodilator compounds such as epoxygenase metabolites of the cytochrome P450 pathway of arachidonic acid metabolism. In addition, circulating hormones can either potentiate (e.g., angiotensin II) or inhibit (e.g., atrial natriuretic peptide) adrenergic vasoconstriction. Finally, the release of endogenous vasoactive substances (autacoids) such as nitric oxide, eicosanoids, histamine, kinins, adenine nucleotides, and locally produced vasodilator metabolites all can counteract sympathetic vasoconstriction and contribute to regional modulation of vascular sympathetic responses.

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Thus, the influence of the sympathetic nervous system on regional vascular beds may be affected by any of these factors, which must be carefully considered when evaluating the contribution of sympathetic nerves to the regulation of vascular tone and regional blood flow within a specific vascular bed.

Further Reading Boron WF, Boulpaep EL. Medical physiology, 2nd ed. Philadelphia: Saunders/Elsevier; 2009. Falck B. Observations on the possibilities of cellular localization of monoamines by a fluorescence method. Acta Physiol Scand 1962;197(Suppl):1–25. Franchini KG, Cowley AW. Neurogenic control of blood vessels. In: Robertson D, editor. Primer of the autonomic nervous system (2nd ed). San Diego: Elsevier Science USA; 2004. p. 139–43. Gibbins IL, Jobling P, Morris JL. Functional organization of peripheral vasomotor pathways. Acta Physiol Scand 2003;177:237–45. Hamel E. Perivascular nerves and the regulation of cerebrovascular tone. J Appl Physiol 2006;100:1059–64. Hodges GJ, Jackson DN, Mattar L, Johnson JM, Shoemaker JK. Neuropeptide Y and neurovascular control in skeletal muscle and skin. Am J Physiol Regul Integr Comp Physiol 2009;297:R546–55. Lee TJ. Nitric oxide and the cerebral vascular function. J Biomed Sci 2000;7:16–26. Joyner MJ, Halliwill JR. Sympathetic vasodilatation in human limbs. J. Physiol 2000;526(3):471–80. Koehler RC, Gebremedhin D, Harder DR. Role of astrocytes in cerebrovascular regulation. J Appl Physiol 2005;100:307–17. Verity MA. Morphologic studies of the vascular neuroeffector apparatus. In: Bevan JA, Furchgott RF, Maxwell RA, Somlyo AA, editors. Physiology and pharmacology of vascular neuroeffector systems. Basel: Karger; 1971. p. 2–12. Watts SW, Kanagy NL, Lombard JH. Receptor-Mediated Events in the Microcirculation. In: Tuma RL, Duran WN, Ley K, editors. Handbook of physiology-microcirculation. San Diego: Academic Press/Elsevier; 2008. p. 285–348.


C H A P T E R

39 Physiology of Upright Posture Wouter Wieling, Jan T. Groothuis INTRODUCTION Orthostatic stresses are common daily events for humans. They result in a shift of blood away from the chest to the distensible venous capacitance system below the diaphragm. Venous pooling is the term commonly used to describe this process. Such pooling in dependent parts results in a rapid diminution of central blood volume, i.e. the volume of blood directly available to the cardiac ventricles, which is of paramount importance for the beat-to-beat adjustment of blood pressure. Unless compensatory adjustments are promptly instituted, blood pressure falls and the subject faints within minutes. This chapter discusses these adjustments. It is concerned with a general description of available regulatory mechanisms. For details of the difference between the initial (first 30 s) cardiovascular adjustments between active and passive changes of posture, refer to Wieling et al., 2007.

REGULATORY MECHANISMS INVOLVED IN THE ADJUSTMENTS OF THE HUMAN BODY TO ORTHOSTATIC STRESS Orthostatic pooling of blood begins almost immediately upon the change from the supine to the upright posture and is estimated to total 300–800 mL; the bulk of the total change occurs within the first 5–10 s (Fig. 39.1). In humans

FIGURE 39.1 Influence of gravity on intravascular fluid shift.


orthostatic adjustments are provided by an effective set of blood pressure regulatory mechanisms. They maintain blood pressure at an appropriate level for perfusion of the vital organs, even for the brain, which is located above the heart. To achieve this, the regulatory systems increase heart rate, cardiac contractility and vascular tone to stabilize blood pressure at the level of the heart and brain. The initial reflex adjustments to orthostatic stress are mediated exclusively by neural regulatory systems. During prolonged orthostatic stress additional reflex activation of humoral regulatory systems takes place.

Arterial Baroreceptors The main sensory receptors involved in orthostatic cardiovascular reflex adjustment are the arterial baroreceptors located in the carotid sinuses and aortic arch and mechanoreceptors located in the heart and lungs (Fig. 39.2). The latter consist of a variety of stretch receptors located within the heart and the lungs. “Cardiopulmonary receptors” can be considered to function as rapid acting volume receptors, ideally suited to detect changes in the filling of the central venous circulation. Cardiopulmonary receptors act in concert with arterial baroreceptors to effect the necessary adjustments, but are not essential for the orthostatic cardiovascular adjustments. The arterial baroreceptors tonically inhibit the vasomotor centers in the brainstem. A decrease in blood pressure, as occurs on the assumption of the upright posture, removes this tonic inhibition with a resultant decrease in vagal outflow and an increase in sympathetic activity causing an increase in heart rate, cardiac contractility and vasomotor tone (Figs 39.1 and 39.2). Arterial baroreflex adjustments to orthostatic stress are fast. Decreased vagal outflow increases heart rate within one or two heartbeats. Increased sympathetic activity needs 1–3 seconds to increase heart rate, cardiac contractility and vasomotor tone. The sympathetic mediated increase in vasomotor tone is the key factor in the maintenance of blood pressure in the upright posture. Pronounced increases in heart rate are insufficient to maintain cardiac output, since the heart cannot pump blood that it does not receive. To examine the relative role of the carotid and aortic mechanoreceptors, the following points are important. First, the observation that carotid sinus receptors respond

193


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39. PHysIology of UPRIgHT PosTURE

deafferentation of the carotid baroreceptors blood pressure control in the stabilized upright posture is impaired. Remaining aortic receptors, however, limit the fall in blood pressure to 10–20 mmHg. These data seem to indicate that the carotid baroreceptors by their location are most important for orthostatic reflex adjustments and for defending the constancy of perfusion pressure of the brain during prolonged orthostatic stress. Additional data support this view. Functional abnormalities can be detected in carotid baroreflex function by neck suction in apparently healthy individuals with unexplained syncope. In addition, the decrease in orthostatic tolerance observed after spaceflight or on mobilization after prolonged bed rest is related to depressed efficiency of the carotid sinus baroreflex. Moreover, in the elderly with an excessive fall in blood pressure in the upright posture afferent baroreflex dysfunction is reported and much overlap is present between the carotid sinus syndrome and orthostatic hypotension.

Local Vasoconstrictor Mechanisms

FIGURE 39.2 Schematic drawing of the afferent and efferent pathways of the arterial baroreceptor reflex arc. Nerve fibers from the carotid and aortic join the glossopharyngeal nerve and vagus nerve respectively toward the vasomotor centre (VMC) in the brainstem. Nerve fibers from the lungs and the heart (not shown) join the vagus nerve as cardiopulmonary afferents.

more vigorously to rapid than to slow changes in pressure makes it likely that they play the major role in the initial reflex adjustments. The abnormally large initial fall and a delayed recovery of blood pressure observed in a patient with bilateral denervation of the carotid baroreceptors supports this view. However, the results of the reverse experiment, i.e. selective denervation of aortic receptors, are not available in humans. Second, neural adjustments in the stabilized fully upright posture result in an increase in diastolic blood pressure with little change in systolic blood pressure at heart level resulting in an increase in mean arterial blood pressure of about 5–10 mmHg. The aortic receptors, which are located just above heart level, sense a reduced pulse pressure, but an increased instead of a decreased mean blood pressure in the upright posture. The blood pressure sensed by the carotid baroreceptors, in contrast, drops and remains below the recumbent level since they are located about 20–25 cm above heart level. This hydrostatic effect lowers the effective pressure at the carotid baroreceptor by about 15 mmHg. A permanent state of diminution in the stimulation of the carotid baroreceptors by the fall in both mean arterial blood pressure and pulse pressure must therefore persist as long as the upright posture is maintained. Third, after surgical

Central modulation of vasomotor outflow is reinforced by local vasoconstrictor mechanisms, such as the veno-arteriolar axon reflex and a myogenic response. The veno-arteriolar axon reflex is triggered when venous pressure exceeds 25 mmHg, which results in vasoconstriction of the corresponding arteriole and is reported to elicit up to 30–45% of the total vasoconstriction in the legs in the upright posture. The myogenic response of the smooth muscle of resistance vessels in the dependent parts is triggered by an increase in transmural pressure across an arteriole. Recent studies suggest that the myogenic response can increase leg vascular resistance up to 30%. It seems to be the most important vasoconstrictor mechanism during orthostatic stress in autonomic failure.

Role of Capacitance Vessels to Orthostatic Reflex Adjustments Capacitance vessels also contribute to reducing the gravitational shift of blood. Reflex venoconstriction in the lower limbs appears of little importance. The cutaneous veins are richly innervated, but venoconstriction of these vessels is not a consistent response to the upright posture; if it occurs it is transient. The capacity of cutaneous veins to contain blood seems primarily determined by thermoregulatory and psychological stimuli. Heat markedly increases venous capacity and thus reduces orthostatic tolerance. Cold has the opposite effects. Muscle veins in human limbs have little smooth muscle and little or no sympathetic innervation and therefore, respond little, if at all, to neural stimuli. Their capacity is determined mainly by the properties of the surrounding skeletal muscle (see below). In humans, intact innervation of the splanchnic bed is of paramount importance for orthostatic tolerance. The upright posture is accompanied by constriction of


REgUlAToRy MECHAnIsMs InvolvEd In THE AdjUsTMEnTs of THE HUMAn Body To oRTHosTATIC sTREss

splanchnic resistance vessels increasing systemic vascular resistance. The increase in splanchnic vascular resistance also causes a passive expulsion of blood out of the large venous reservoir of the splanchnic bed by elastic recoil of venous vessels. Active constriction of the splanchnic capacitance vessels is potentially of great importance in mobilizing additional venous blood to maintain the cardiac filling pressures and, hence, stroke volume during orthostatic stress. The rich innervation and the great sensitivity and rapidity of the reflex responses of these vessels already to very low frequencies of sympathetic discharge appears indicative for their importance in responding to postural changes. However, because of technical and ethical constraints it has not been possible to determine whether active contraction of splanchnic veins does indeed occur.

Role of Skeletal Muscle Pump Mechanical factors play an important adjunctive role in promoting venous return in the upright posture. First, with quiet standing, the body behaves more or less as an inverted pendulum that sways about the ankles. The static increase in skeletal muscle tone involved opposes venous pooling in lower limb veins. Postural sway during quiet standing is thought to be able to compensate for otherwise poor orthostatic tolerance. The importance of static muscle contractions of the lower body in opposing gravitational pooling of venous blood has been clearly demonstrated in patients with severe orthostatic hypotension due to autonomic failure and in otherwise healthy subjects with a tendency to vasovagal fainting. Leg crossing and contraction of leg and abdominal muscles have been shown beneficial to combat orthostatic hypotension in these subjects. These maneuvers translocate venous blood pooled below the diaphragm to the chest and thereby partially restore cardiac filling pressure, stroke volume and thereby cardiac output (see Chapter 127). Second, activation of the musclevenous pump of the legs during tiptoeing or walking, in the presence of competent venous valves, pumps blood back to the heart and partially restores cardiac filling pressure. The leg-muscle pump can be considered as a “second heart”. Third, the thoraco-abdominal pump may also contribute to improve venous return; with inspiration intrathoracic pressure decreases and intra-abdominal pressure increases thereby promoting venous return. A sighing respiration often precedes an actual faint; it has been suggested that this helps to prevent syncope by enhancing the thoraco-abdominal pump and by inducing venoconstriction in the skin. However, continuous deep breathing and

195

the consequent hypocapnia cause vasoconstriction in the brain and the skin venoconstriction is only transient.

Humoral Mechanisms The activity of humoral mechanisms (renin-angiotensin system and vasopressin) is altered by postural changes. The contribution of the humoral system to circulatory orthostatic adjustments depends on the adequacy of the effective circulating blood volume, which is the component of blood volume that the volume-regulatory system responds to by initiating renal retention of water and sodium. When the effective blood volume is adequate, the humoral mechanisms are minimally involved in the initial circulatory adjustment. Activation of the humoral mechanisms becomes more important during prolonged orthostasis, particularly in combatting imminent hypotension in the volume-depleted state. During severe orthostatic stress, both activation of the renin-angiotensin system and vasopressin release are necessary for maintaining blood pressure. Under these circumstances, vasopressin may rise sharply to levels that promote reabsorption of water by the kidneys and have profound vasoconstrictor effects.

Further Reading Claydon VE, Hainsworth R. Increased postural sway in control subjects with poor orthostatic tolerance. J Am Coll Cardiol 2005;46:1309–13. Cooper VL, Hainsworth R. Effects of head-up tilting on baroreceptor control in subjects with different tolerances to orthostatic stress. Clin Sci 2002;103:21–226. Crandall CG, Shibasaki M, Wilson TE. Insufficient cutaneous vasoconstriction leading up to and during syncopal symptoms in heat stressed human. Am J Physiol (Heart Circ Physiol) 2010;299:H1168–1173. Fu Q, Witkowski S, Levine BD. Vasoconstrictor reserve and sympathetic neural control of orthostasis. Circulation 2004;110:2931–7. Groothuis JT, Thijssen DH, Lenders JW, Deinum J, Hopman MT. Leg vasoconstriction during head-up tilt in patients with autonomic failure is not abolished. J Appl Physiol 2011;110:416–22. Miller JD, Pegelow DF, Jacques AJ, et al. Skeletal muscle pump versus Respiratory muscle pump: modulation of venous return from the locomotor limb. J Physiol 2005;563:925–43. Smit AAJ, Halliwill JR, Low PA, et al. Topical Review. Pathophysiological basis of orthostatic hypotension in autonomic failure. J Physiol 1999;519:1–10. Timmers HJLM, Wieling W, Karemaker JM, et al. Denervation of carotid baro- and chemoreceptors in humans. J Physiol 2003;553:3–11. Van Heusden K, Gisolf J, Stok WJ. Mathematical modelling of gravitational effects on the circulation: importance of the time course of venous pooling and blood volume changes in the lung. Am J Physiol (Heart Circ Physiol) 2006;291:H2152–H2165. Wieling W, Krediet CT, van Dijk N, Linzer M, Tschakovsky ME. Initial orthostatic hypotension: review of a forgotten condition. Clin Sci 2007;112:157.


C H A P T E R

40 Cerebral Circulation Ronald Schondorf The cerebral circulation is tasked with selectively and specifically directing cerebral blood flow (CBF) to metabolically active regions of the brain while simultaneously responding to or in some cases defending the brain from changes in cerebral perfusion pressure (CPP), carbon dioxide (CO2) and oxygen (O2). A myriad of mechanisms are involved in this complex interplay of demands and the precise integration of these is not well understood. This chapter will touch on some of the clinically relevant factors and challenges that pertain to the control of the human cerebral circulation.

dendritic activity, or to local neuronal spiking. Lastly, increased demand from the brain’s microcirculation must be matched by increased vasodilatation of the upstream extracerebral (pial) resistance vessels. Some of this signaling may be achieved via tight gap junctions within vascular smooth muscle or the endothelium. Alternatively, increased endothelial shear in these vessels may release vasodilating substances such as nitric oxide (NO). Other mechanisms to be discovered undoubtedly exist.

REGULATION OF CEREBRAL CIRCULATION

NEUROVASCULAR COUPLING Local increases in brain activity such as occur during cognitive tasks, are reliably accompanied by parallel increases in CBF and glucose metabolism that greatly exceed the rate of oxygen consumption. The resulting decrease in paramagnetic deoxyhemoglobin can be detected as a change in brain oxygen level dependent (BOLD) signal, the basis for functional MRI. The substrate underlying this response mainly resides in the brain microcirculation beyond the Virchow Robin space. Blood flow through the intraparenchymal vessels is regulated through a combination of signals closely linked to local metabolic activity and shear stress, that originate from locally apposed astrocytes and neurons and possibly from the vascular endothelium itself [1–4]. The intrinsic neural input to the intraparenchymal vessels originates form local interneurons as well as from discrete region-specific intrinsic brain pathways that are located in part in the locus coeruleus, raphe, ventral tegmental region and nucleus basalis [3]. There are several implications that stem from this brief description of the cerebral microcirculation. First, the multiplicity of redundant mechanisms that regulate intraparenchymal CBF makes it highly unlikely that a single factor mediates vasoconstriction or vasodilatation even under well-defined conditions. Second, the complex interplay of metabolically active astrocytes and neurons underscores that caution is required when relating BOLD to a particular element of neuronal activity. Indeed BOLD is imperfectly linked either to extracellularly recorded indices of synaptic and


Basic Considerations The complex hardware (MRI or PET) used in brain mapping requires the subject to perform tasks in the supine position, thereby eliminating many other concomitant challenges to cerebral perfusion [5]. Those who treat patients with autonomic failure more often encounter the opposite end of the spectrum, routine activities of daily living that cause transient or maintained cerebral hypoperfusion and syncope. Many patients however adequately maintain cerebral perfusion despite profound orthostatic hypotension for reasons that are far from clear. The effects of the myriad of endothelial derived factors, circulating or locally released neurotransmitters, changing levels of PCO2, O2, pH, lactate and glucose as well as local cerebrovascular myogenic responses to stretch that have been identified in animals preclude any simple understanding of how cerebral perfusion is regulated or maintained in humans [6,7]. Moreover some of the mechanisms identified in animals may be species specific or alternatively not easily demonstrable in humans using techniques or interventions that are deemed permissible and ethical.

Cerebral Autoregulation Currently most of the useful clinical inferences concerning human cerebral circulatory control have been derived from observing responses to stereotyped challenges such as standing or exercise. Our understanding has been

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further enhanced by the availability of non-invasive transcranial Doppler (TCD) ultrasound and near-infrared spectroscopy (NIRS) which provide robust beat-to-beat approximations of CBF and cerebral oxygenation [6,8]. These techniques have redefined our understanding of cerebral autoregulation, one of the more important processes that defend against cerebral hypoperfusion. Cerebral autoregulation is classically defined as the intrinsic ability to maintain CBF constant (plateau portion of the autoregulation curve) over a wide range of stepwise changes in CPP. Maintenance of such a plateau, however, necessitates high feedback gains not usually encountered in biological systems [8]. Indeed the hydrostatic challenge of standing alone causes a reduction in CBF that is independent of changes in CO2 and is not offset by a siphon mechanism [9]. Whether the reduction in cardiac output during stand contributes in some fashion to the reduction in CBF is still an open question [9]. The dynamic nature of cerebral autoregulation is demonstrated by the rapid correction of CBF during relatively reproducible rapidly induced or alternatively spontaneous changes in CPP [6,8]. The presence of dynamic autoregulation can easily be appreciated by eye when CBF returns to baseline before blood pressure (BP) does, an apparent phase lead of CBF. If dynamic cerebral autoregulation is severely impaired the change in CBF essentially mirrors the change in BP. Dynamic cerebral autoregulation is optimal at frequencies 0.1 Hz and negligible at higher frequencies. A variety of time and frequency domain techniques have been employed in an attempt to describe and more importantly, to quantitate the integrity of dynamic

cerebral autoregulation [6,8]. Not surprisingly, autoregulation varies between individuals and within the same individual both minute to minute and day to day [10]. Input magnitude (BP fluctuations), changes in CO2, cerebrovascular tone and local metabolic demands of the perfused brain as well as the intrinsic non linearity of cerebral autoregulation all contribute to this variability and confound quantitation of autoregulation. For example large BP swings generated by repeat squats improve estimations of cerebral autoregulation [11]. Conversely, increased metabolic activity within the visual cortex results in impairment of posterior but not middle cerebral autoregulation [12]. In neither instance would we say that that the inherent properties of autoregulation are now different. Sufficient sample size, uniformity of population characteristics and strict standardization of test conditions and input magnitude are all necessary to detect true changes in autoregulatory efficacy between populations.

Clinical Considerations Do patients with cerebral hypoperfusion have impairment of cerebral autoregulation? Figures 40.1 and 40.2 show examples of raw tracings from one patient with neurally-mediated syncope and two with autonomic failure. In all three patients the percent decline in BP is much greater than the decline in CBF suggesting that cerebral autoregulation is intact. Some patients with autonomic failure appear to have a reduced autoregulatory capacity whereas others have an autoregulatory range that appears to be expanded. The mechanisms that could possibly

FIGURE 40.1 Raw and filtered (0.2 Hz) blood pressure (BP) and transcranial Doppler (TCD) recordings of right middle cerebral artery blood flow velocity response in a patient with neurally-mediated syncope. The small decline in CBF relative to the large decrease in BP suggests preservation of cerebral autoregulation.


REgulATIon of CEREbRAl CIRCulATIon

199

FIGURE 40.2 Raw and filtered (0.2 Hz) blood pressure (BP) and transcranial Doppler (TCD) recordings of right middle cerebral artery blood flow velocity response from two different patients with autonomic failure.

be responsible for the modification of the cerebral autoregulatory range in patients with autonomic failure are unknown. Dynamic cerebral autoregulation does not appear to be impaired in patients with neurally-mediated syncope but may be altered in patients with autonomic failure [6].

sympathetic activity in human circulatory control remains unclear, phasic increases of cerebral sympathetic activity are critical in defending against spikes in BP that may occur during many activities of daily living and during REM sleep.

References Role of Autonomic Innervation If cerebral perfusion is relatively preserved in patients with profound autonomic failure, then what is the role of the rich extrinsic autonomic innervation of the cerebral vasculature? The parasympathetic innervation of cerebral vasculature may be involved in cerebral vasodilatation but its exact role in human cerebral circulatory control remains unclear. A lively debate concerning the role of sympathetic activity on regulation of CBF has been recently published [13]. To summarize the major points ganglionic blockade or sympathodenervation does not affect resting CBF suggesting that tonic baseline sympathetic outflow to cerebral vasculature is negligible. However, changes in sympathetic activity have been implicated in the reduction of CBF to stand [9] or following beta-adrenergic blockade in subjects during high intensity exercise [13] and alphaadrenergic blockade does affect dynamic cerebral autoregulation. Lastly sympathetic stimulation does restrain the CBF increase during hypertension and recently activation of many neurons in the superior cervical ganglion of anesthetized lambs during imposed hypertension has been directly demonstrated [14]. Although the role of tonic

[1] Andresen J, Shafi NI, Bryan Jr. RM. Endothelial influences on cerebrovascular tone. J Appl Physiol 2006;100:318–27. [2] Cauli B, Hamel E. Revisiting the role of neurons in neurovascular coupling. Front Neuroenerg 2010;2:9. [3] Hamel E. Perivascular nerves and the regulation of cerebrovascular tone. J Appl Physiol 2006;100:1059–64. [4] Koehler RC, Gebremedhin D, Harder DR. Role of astrocytes in cerebrovascular regulation. J Appl Physiol 2006;100:307–17. [5] Raz A, Lieber B, Soliman F, Buhle J, Posner J, Peterson BS, et al. Ecological nuances in functional magnetic resonance imaging (FMRI): Psychological stressors, posture, and hydrostatics. NeuroImage 2005;25:1–7. [6] Singer W, Low PA, Schondorf R. Transcranial doppler evaluation in autonomic disorders. In: Low PA, Benarroch EE, editors. Clinical autonomic disorders (3rd ed.). Baltimore, Philadelphia: Lippincott, Williams & Wilkins; 2008. p. 198–218. [7] Ainslie PN, Duffin J. Integration of cerebrovascular CO2 reactivity and chemoreflex control of breathing: Mechanisms of regulation, measurement, and interpretation. Am J Physiol Regul Integr Comp Physiol 2009;296:R1473–1495. [8] Panerai RB. Transcranial doppler for evaluation of cerebral autoregulation. Clin Auton Res 2009;19:197–211. [9] Immink RV, Truijen J, Secher NH, Van Lieshout JJ. Transient influence of end-tidal carbon dioxide tension on the postural restraint in cerebral perfusion. J Appl Physiol 2009;107:816–23. [10] Brodie FG, Atkins ER, Robinson TG, Panerai RB. Reliability of dynamic cerebral autoregulation measurement using spontaneous fluctuations in blood pressure. Clin Sci (Lond) 2009;116:513–20.


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[11] Claassen JAHR, Levine BD, Zhang R. Dynamic cerebral autoregulation during repeated squat-stand maneuvers. J Appl Physiol 2009;106:153–60. [12] Nakagawa K, Serrador JM, LaRose SL, Moslehi F, Lipsitz LA, Sorond FA. Autoregulation in the posterior circulation is altered by the metabolic state of the visual cortex. Stroke 2009;40:2062–7.

[13] van Lieshout JJ, Secher NH. Point/Counterpoint: Sympathetic activity does/does not influence cerebral blood flow. J Appl Physiol 2008;105:1364–6. [14] Cassaglia PA, Griffiths RI, Walker AM. Sympathetic nerve activity in the superior cervical ganglia increases in response to imposed increases in arterial pressure. Am J Physiol Regul Integr Comp Physiol 2008;294:R1255–1261.


C H A P T E R

41 Autonomic Control of the Lower Airways Peter J. Barnes Airway nerves regulate the caliber of the airways and control airway smooth muscle tone, airway blood flow and mucus secretion.

receptors (RAR) account for 10–30% of the myelinated nerve endings in the airways. These endings are sensitive to mechanical stimulation and to protons, low chloride solutions, histamine, cigarette smoke, ozone, serotonin and prostaglandin F2α, although it is possible that some responses are secondary to the mechanical distortion produced by bronchoconstriction. C-fibers. There is a high density of unmyelinated (C-fibers) in the airways, which contain neuropeptides, including substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP). They are selectively stimulated by capsaicin and also activated by bradykinin, protons, hyperosmolar solutions and cigarette smoke. Cough. Cough is an important defense reflex that may be triggered from either laryngeal or lower airway afferents [2]. Both RAR and C-fibers mediate the cough reflex, which may be sensitized in inflammatory diseases by the release of mediators, including neurotrophins [3]. Transient receptor potential A1 (TRPA1) channels play a key role in mediating activation of cough receptors in response to many tussive agents and inflammatory mediators [4] Neurogenic inflammation. Activation of C-fibers may result in the antidromal release of neuropeptides, such as SP, NKA and CGRP (Fig. 41.2). This may increase inflammation in the airways in asthma and COPD, although the role of neurogenic inflammation is debated [5].

OVERVIEW OF AIRWAY INNERVATION Three types of airway nerve and several neurotransmitters are recognized (Table 41.1): l

l l

Parasympathetic nerves which release acetylcholine (ACh). Sympathetic nerves which release norepinephrine. Afferent (sensory nerves) whose primary transmitter is glutamate.

In addition to these classical transmitters, multiple neuropeptides have now been localized to airway nerves and may have potent effects on airway function. Several neural mechanisms are involved in the regulation of airway caliber, and abnormalities in neural control may contribute to airway narrowing in diseases, such as asthma and chronic obstructive pulmonary disease (COPD), contributing to the symptoms and possibly exacerbating the inflammatory response. There is a close interrelationship between inflammation and neural responses in the airways, since inflammatory mediators may influence the release of neurotransmitters via activation of sensory nerves leading to reflex effects and via stimulation of pre-junctional receptors that influence the release of neurotransmitters [1]. In turn, neural mechanisms may influence the nature of the inflammatory response, either reducing inflammation or exaggerating the inflammatory response.

Afferent Nerves At least three types of afferent fiber have been identified in the lower airways (Fig. 41.1). Slowly adapting receptors. Myelinated fibers associated with smooth muscle of proximal airways are probably slowly adapting (pulmonary stretch) receptors that are involved in reflex control of breathing and in the cough reflex. Rapidly adapting receptors. Aδ myelinated fibers in the epithelium show rapid adaptation. Rapidly adapting


TABLE 41.1 Neurotransmitters in the Airways

Neurotransmitter Receptors

Airway Smooth Muscle

Mucus Secretion

Airway Vessels

Acetylcholine

M3

Constrict

Increase

Dilate

Norepinephrine

α1

No effect

No effect

Constrict

Nitric oxide

GC

Dilate

Increase

Dilate

VIP

VIP

Dilate

Increase

Dilate

CGRP

CGRP

Constrict?

No effect

Dilate

Substance P

NK1, NK2

Constrict

Increase

Dilate

Neurokinin A

NK2

Constrict

No effect

No effect

VIP, vasoactive intestinal polypeptide; GC, guanylyl cyclase; CGRP, calcitonin gene related peptide; NK, neurokinin.

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41. AuToNomIC CoNTRoL of THE LowER AIRwAys

Cholinergic Nerves

Muscarinic receptors. Three subtypes of muscarinic receptor are found in human airways [7]. M1-receptors are localized to parasympathetic ganglia and facilitate neurotransmission. M2-receptors serve as feedback inhibitory receptors on postganglionic nerves (and may be dysfunctional in asthma), whereas M3-receptors mediate the bronchoconstrictor and mucus secretory effect of ACh. Cholinergic reflexes. Reflex cholinergic bronchoconstriction may be activated by afferent receptors in the larynx or lower airways. Cholinergic reflexes are exaggerated in asthma and COPD because of increased responsiveness to ACh. Anticholinergics in airway disease. Muscarinic antagonists (anticholinergics) such as ipratropium and tiotropium, cause bronchodilatation in airway disease through the relief of intrinsic cholinergic tone. They are the bronchodilators of choice in COPD, but less effective than β2-adrenergic agonists in asthma in which several other bronchoconstrictor mechanisms are operative.

Cholinergic nerves are the major neural bronchoconstrictor mechanism in human airways, and are the major determinant of airway caliber [6].

Cholinergic Efferents Cholinergic nerve fibers arise in the nucleus ambiguus in the brainstem and travel down the vagus nerve and synapse in parasympathetic ganglia which are located within the airway wall. From these ganglia short post-ganglionic fibers travel to airway smooth muscle and submucosal glands and release acetylcholine (ACh) that acts on muscarinic receptors (Fig. 41.3).

mechanical stimuli capsaicin, bradykinin (bronchoconstriction) cigarette smoke, SO2 water, hyperosmolar solutions water, low Cl–

Airway epithelium

RAR Aδ-fiber

C-fiber

NP release SP, NKA, CGRP

Bronchial vessel

BRONCHODILATOR NERVES Cough Cholinergic reflex

Neural bronchodilator mechanisms exist in airways and there are considerable species differences.

Airway smooth muscle

Sympathetic nerves. Sympathetic innervation of human airways is sparse and there is no functional evidence for direct innervation of airway smooth muscle, although sympathetic nerves regulate bronchial blood flow and to a lesser extent mucus secretion (Fig. 41.4). Adrenergic tone in the airways is primarily regulated by circulating epinephrine.

SAR

FIGURE 41.1 Afferent nerves in airways. Slowly-adapting receptors (SAR) are found in airway smooth muscle, whereas rapidly adapting myelinated (RAR) and unmyelinated C-fibers are present in the airway mucosa. NP, neuropeptide; SP, substance P; NKA, neurokinin A; CGRP, calcitonin gene-related peptide.

Nodoseljugular ganglion

C.N.S.

Vagus nerve

Laryngeal Esophageal afferents

Parasympathetic nerve C-fiber Aδ-fiber

C-fiber receptors Irritant receptors

ACh

Parasympathetic ganglion Inflammatory cell ACh

ACh

Submucosal gland

Mediators

Airway epithelium Irritants (e.g. cigarette smoke)

FIGURE 41.2 Neurogenic inflammation. Possible neurogenic inflammation (axon reflex) in asthmatic airways via retrograde release of peptides from sensory nerves via an axon reflex. Substance P (SP) causes vasodilatation, plasma exudation and mucus secretion, whereas neurokinin A (NKA) causes bronchoconstriction and enhanced cholinergic reflexes and calcitonin gene-related peptide (CGRP) vasodilatation.


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NEuRAL CoNTRoL of AIRwAys IN DIsEAsE

CNS

M3-receptors ß2-receptors α-receptors

Sympathetic ganglion Vagus nerve

Sympathetic nerve Adrenal medulla

E

NE

Parasympathetic nerve

NE Parasympathetic ganglion NE

Bronchial vessel

ACh Airway smooth muscle

FIGURE 41.3 Cholinergic control of airway smooth muscle. Pre-ganglionic and post-ganglionic parasympathetic nerves release acetylcholine (ACh) and can be activated by airway and extra-pulmonary afferent nerves. NE, norepinephrine; E, epinephrine.

Epithelial shedding

Eosinophils Sensory nerve activation

Vasodilatation

Plasma exudation

Mucus secretion

Neuropeptide release SP, NKA, CGRP, …. Bronchoconstriction Cholinergic activation

Cholinergic facilitation

FIGURE 41.4 Adrenergic control of airway smooth muscle. Sympathetic nerves release norepinephrine (NE), which may modulate cholinergic nerves at the level of the parasympathetic ganglion or postganglionic nerves, rather than directly at smooth muscle in human airways. Circulating epinephrine (E) is more likely to be important in adrenergic control of airway smooth muscle. SP, substance P; NKA, neurokinin A; CGRP, calcitonin generelated peptide.

Inhibitory NANC nerves. The bronchodilator nerves in human airways are non-adrenergic non-cholinergic (NANC) and the major neurotransmitter is nitric oxide (NO). Neuronal NO synthase is expressed mainly in cholinergic neurons.

is a bronchoconstrictor and vasoconstrictor. The neuropeptides in sensory nerves (SP, NKA, CGRP) act as bronchoconstrictors and vasodilators, and also increase mucus secretion and inflammation in the airways [9].

NEURAL CONTROL OF AIRWAYS IN DISEASE

NEUROPEPTIDES Multiple neuropeptides have been localized to nerves in the respiratory tract and function as co-transmitters of classical autonomic nerves to fine-tune airway function [8]. Vasoactive intestinal peptide and related peptides act as bronchodilators and vasodilators, whereas neuropeptide Y

Autonomic control of airways may be abnormal and contribute to the pathophysiology in several airway diseases. Asthma. Neural mechanisms contribute to the pathophysiology of asthma is several ways [10]. Several triggers activate reflex cholinergic bronchoconstriction


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and inflammatory mediators, including neurotrophins, may sensitize the cough reflex. The role of neurogenic inflammation and neuropeptides is still uncertain, however. COPD. The structurally narrowing of the airways in COPD means that the normal vagal cholinergic tone has a relatively greater effect on caliber than in normal airways for geometric reasons. Cholinergic mechanisms are the only reversible component of COPD and may contribute to the mucus hypersecretion of chronic bronchitis.

References [1] Barnes PJ. Modulation of neurotransmission in airways. Physiol Rev 1992;72:699–729. [2] Undem BJ, Carr MJ. Targeting primary afferent nerves for novel antitussive therapy. Chest 2010;137:177–84.

[3] Freund-Michel V, Frossard N. The nerve growth factor and its receptors in airway inflammatory diseases. Pharmacol Ther 2008;117:152–76. [4] Geppetti P, Patacchini R, Nassini R, Materazzi S. Cough: the emerging role of the TRPA1 channel. Lung 2010;188(Suppl 1):S63–8. Epub;2009 Nov 30:S63–S68 [5] Barnes PJ. Cytokine modulators as novel therapies for airway disease. Eur Respir J Suppl 2001;34:67s–77s. [6] Racke K, Matthiesen S. The airway cholinergic system: physiology and pharmacology. Pulm Pharmacol Ther 2004;17:181–98. [7] Barnes PJ. Muscarinic receptor subtypes in airways. Life Sci 1993;52:521–8. [8] Barnes PJ, Baraniuk J, Belvisi MG. Neuropeptides in the respiratory tract. Am Rev Respir Dis 1991;144(1187-98):1391–9. [9] Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000;55:321–37. [10] Undem BJ, Carr MJ. The role of nerves in asthma. Curr Allergy Asthma Rep 2002;2:159–65.


C H A P T E R

42 Gastrointestinal Function Michael Camilleri Proper function of the gastrointestinal tract is essential for the orderly digestion, absorption, and transport of food and residue. Digestion requires secretion of endogenous fluids from the salivary glands, stomach, pancreas, and small bowel to facilitate intraluminal breakdown of foods. Fluids, electrolytes, and smaller building blocks of the macronutrients are then absorbed, leaving non-digestible residue to be excreted. The motor activity of the gut is one of the integrated functions that is essential for the normal assimilation of food. Gut motility facilitates the transport of nutrients, brings together digestive enzymes and their substrates, temporarily stores content for optimal absorption (particularly in the distal small bowel and right colon) and, finally, excretes non-digestible residue by defecation in a wellcoordinated function under voluntary control. The extrinsic autonomic nervous system is critically important for almost all secretory and motor functions in the digestive tract by modulating the intrinsic or enteric nervous system of the alimentary tract (Fig. 42.1).

Salivary Secretion Presentation of food to the mouth and olfactory stimulation trigger afferent nerves that stimulate secretory centers in the medulla. These reflexly stimulate efferent fibers along parasympathetic and sympathetic pathways: parasympathetic fibers course along the facial nerve to sublingual and submaxillary glands, and the glossopharyngeal nerve to the parotid gland. Synapse with postganglionic fibers occurs in or near the glands. Sympathetic fibers reach the salivary glands through the cervical sympathetic trunk, but the brainstem centers are unclear. Parasympathetic efferents stimulate secretion; sympathetic fibers serve to cause contraction of myoepithelial cells on the duct. The human salivary glands secrete 0.5–1.0 liter saliva per day at a maximal rate of 4 mL/min. Saliva facilitates speech, lubricates food for swallowing and contains the amylase ptyalin which begins the digestion of starch. Bicarbonate in saliva neutralizes noxious acidic ingesta.

Gastric Secretion Gastric secretion is stimulated by the act of eating (cephalic phase) and the arrival of food in the stomach


(gastric phase). Arrival of the food in the small intestine also controls gastric secretion (intestinal phase). The secreted fluid contains hydrochloric acid, pepsinogen, intrinsic factor, bicarbonate and mucus. Gastric secretion of acid and pepsinogen follows stimulation of oral and gastric vagal afferents. Efferent vagal pathways synapse with submucous plexus neurons which innervate secretory cells via several important bioactive molecules including gastrin, histamine, and somatostatin. In the stomach, there is some digestion of carbohydrate and protein, but very little absorption except for some fat-soluble substances. The mucus-bicarbonate layer protects the stomach lining from auto digestion by acid.

Pancreaticobiliary Secretion Pancreatic juice consists of alkaline (chiefly bicarbonate) fluid and enzymes; 200–800 mL is produced each day. The enzymes, trypsin, lipase, and amylase are essential for digestion of most of the protein, fat, and carbohydrate in the meal. The pancreas consists of exocrine and endocrine portions: bicarbonate and fluid are secreted by ductular cells, chiefly under the influence of secretin; enzymes are produced by acinar cells in response to vagal stimulation of intrapancreatic cholinergic neurons. Cholecystokinin (CCK), which is released from the duodenal mucosal enteroendocrine cells after chemical stimulation by food, activates pancreatic enzyme secretion by stimulating vagal afferents.

Bile Bile is continuously secreted by the liver as two fractions: the bile salt-independent fraction, controlled by secretin and CCK, is similar to the pancreatic ductular secretion; the bile salt-dependent fraction contains bile salts. Bile flow into the intestine is controlled by storage in the gallbladder and by the sphincter of Oddi. Postprandially, the gallbladder contracts under vagal and CCK stimulation, and the basal sphincter tone within the ampulla of Vater falls to allow bile to enter the duodenum. There is evidence that pancreaticobiliary secretion cycles during the interdigestive period, and the cycling is synchronous with the main phases of the gut’s cyclical migrating motor complex (vide infra).

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42. GAsTRoInTEsTInAl FunCTIon

Parasympathetic Cranial

Salivary secretion Appetite Swallowing

Gallbladder contraction

Hypothalamic and thalamic nuclei

Bile secretion

Sphincter of Oddi relaxation

Gastric secretion and motility

Pancreatic secretion Small bowel and colonic motility and absorption

Sympathetic Thoracolumbar

Visceral sensation, nociception Parasympathetic Sacral roots S2,3,4

Continence and defecation

FIGURE 42.1 Gastrointestinal physiology: functions under extrinsic autonomic control.

Intestinal Secretion and Absorption The small bowel produces about 5 liters of fluid per day during the equilibration of osmotic loads that arrive in the small intestine as ingested nutrients and are the result of intraluminal digestion. Yet, most of the 7 liters entering the digestive tract each day (5 liters from small intestine, 1 liter from stomach, 1 liter from liver and pancreas) is reabsorbed (about 80% in small bowel, 20% in colon) during the flow of chyme through the small bowel and colon; thus, stool weight is usually below 200 g/day in health. Water and electrolyte fluxes are generally independent of extrinsic neural control; on the other hand, the submucosal plexus is increasingly recognized as a key factor influencing mucosal blood flow and enterocyte function. Absorption of macro- and micronutrients is generally determined by concentration gradients or active carriermediated, energy-requiring transport processes. These are indirectly influenced by the autonomic nervous system through its effects on the secretion of salivary, gastric, and pancreaticobiliary juices and by the motor processes of mixing and delivery of substrate to sites of preferential absorption, e.g. B12 to the terminal ileum.

Control of Gut Motility The function of the gastrointestinal smooth muscle is intimately controlled by release of peptides and transmitters by the intrinsic (or enteric) nervous system; modulation of the latter input arises in the extrinsic autonomic nerves, the craniospinal (vagus, and S2, 3, 4 nerves) parasympathetic excitatory input, and the thoracolumbar

sympathetic outflow, which is predominantly inhibitory to the gut, but excitatory to the sphincters (Fig. 42.2). Gastrointestinal smooth muscle forms an electrical syncytium whereby the impulse that induces contraction of the first muscle cell results in efficient transmission to a sheet of sequentially linked cells in the transverse and longitudinal axes of the intestine. The pacemaker of the intestinal muscle syncytium is the network of interstitial cells of Cajal which serve to coordinate contraction circumferentially and longitudinally along the gut. In several species, including humans, the enteric nervous system is formed of a series of ganglionated plexuses, such as the submucosal (Meissner’s), myenteric (Auerbach), deep muscular (Cajal), mucosal and submucosal plexuses. Together these enteric nerves number almost 100 million neurons; this number is roughly equivalent to the number of neurons in the spinal cord. At the level of the diaphragm, the vagus nerve consists predominantly of afferent fibers. Thus, the classical concept that preganglionic vagal fibers synapse with a few motor neurons is not tenable, in view of the overwhelmingly larger number of effector cells that would need to be innervated by the smaller number of preganglionic nerves. The current concept (Fig. 42.3) is that each vagal command fiber supplies an integrated circuit that is hard-wired in the intestinal wall and results in a specific motor or secretory response. These hard-wired circuits in the enteric nervous system are also important in many of the automated responses of the gut, such as the peristaltic reflex. These hard-wired circuits persist and, therefore, they retain their functions even in a totally extrinsically denervated intestine. The enteric nerves also control pacemaker activity.


207

GAsTRoInTEsTInAl FunCTIon

Parasympathetic Cranial Hypothalamic and thalamic nuclei

cervical

Glossopharyngeal nucleus NA DMV Vagal nuclei NTS

}

thoracic LES

Vagus N.

pylorus CG (T6-9)

Sympathetic Thoracolumbar

SMG (T9-10)

IMG (T11-L1)

ICS IAS

Parasympathetic Sacral roots S2,3,4

Parasympathetic efferents to digestive tract

EAS

Sympathetic efferents to digestive tract Visceral afferents follow main nerve pathways

FIGURE 42.2 Neural pathways with sympathetic and parasympathetic nervous systems to the gastrointestinal tract. ICS, ileocolonic sphincter; IAS, internal anal sphincter; EAS, external anal sphincter; LES, lower esophageal sphincter; CG, celiac ganglion; SMG, superior mesenteric ganglion; IMG, inferior mesenteric ganglion; NA, nucleus ambiguous; DMV, dorsal motor nucleus of vagus; NTS, nucleus tractus solitarius; T, thoracic.

Vagal efferent fibers stimulate programmed patterns of neural activity; Sympathetic post-ganglionic efferent fibers inactivate neural circuits

Vagal command fiber

Intrinsic neuronal circuits

X

Parasympathetic Cranial

Vagal afferents: visceral reflexes, and CNS-mediated responses

S Sympathetic Thoracolumbar

Enteric effector neurons

Parasympathetic Sacral roots S2,3,4

Muscle cell

Epithelial cell

Visceral afferents: viscero-visceral reflexes, nociception Parasympathetic efferents to digestive tract Sympathetic efferents to digestive tract Visceral afferents follow main nerve pathways

FIGURE 42.3 Integration between extrinsic and intrinsic (or enteric) neural control. Hardwired programs controlling such functions of peristalsis are modulated by efferents in the vagus and sympathetic nerves, which also contain afferent fibers mediating visceral sensation, nociception, and reflex responses. CNS, central nervous system.


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42. GAsTRoInTEsTInAl FunCTIon

Pacemakers located on the greater curve of the gastric corpus and the duodenal bulb “drive” the maximum intrinsic frequency of contractions: three per minute in the stomach and 12 per minute in the small intestine. As in the heart, malfunction of the pacemaker with the highest frequency results in “take over” of pacemaker function by the region with the next highest intrinsic contractile frequency. Derangement of the extrinsic neural control of the gastrointestinal smooth muscle forms the basis for disorders of motility encountered in clinical practice, such as diabetic gastroparesis and fecal incontinence following obstetric trauma of the anal sphincters. Other diseases result from disorders of enteric neural function including achalasia or Hirschsprung's disease.

Normal Gastrointestinal Motor Function Swallowing involves chewing of food, transfer from the oral cavity to the hypopharynx, ejection of the bolus into the esophagus and esophageal peristalsis. The lower esophageal sphincter relaxes at the onset of the swallowing reflex and remains open for a period of about 8 seconds until the bolus passes through the entire esophagus. Then the sphincter contracts to prevent gastroesophageal reflux. Reflux is also prevented by the positive intra-abdominal pressure and contraction of the diaphragmatic crura that occlude the short intra-abdominal portion of the esophagus. Extrinsic neural control reaches the esophagus through efferent pathways in the glossopharyngeal (upper esophagus) and vagus nerves. The motor functions of the stomach and small bowel differ greatly between the fasting and postprandial periods.

(A)

During fasting, cyclical motor events sweep through the stomach and small bowel and are associated with similarly cyclical secretion from the biliary tract and pancreas. The cyclical motor activity is called the interdigestive migrating motor complex; this consists of a phase I of quiescence, phase II of intermittent pressure activity, and phase III or the activity front when contractions of the maximal frequency typical of each region (3 per minute in stomach, 12 per minute in the small bowel) sweep through the gut like a housekeeper, transporting non-digestible residue, products of digestion, epithelial debris and large numbers of commensal bacteria that constitute the normal microbionia and contribute to a large proportion of fecal mass towards the colon for subsequent excretion through the anal canal. The pacemaking functions, cyclical motor activity, and peristalsis are essentially controlled by intrinsic neural pathways, but they can be modulated by extrinsic autonomic nerves. Postprandially, this cyclical activity is abolished, and the different regions of the gut subserve specific functions. Tonic contractions in the gastric fundus result in the emptying of liquids; irregular but high amplitude antral contractions triturate solid food and propel particles that are less than 2 mm in size from the stomach. Particles that are digestible but larger than 2 mm in size are retained by a sieving function of the pylorus. Completion of gastric emptying of solids and liquids takes 3 to 4 hours, depending on the total caloric and fat content of the meal. Irregular, frequent contractions in the postprandial period serve to mix food with digestive juices in the duodenum and jejunum and to propel it aborally. The duration of small bowel transit is on average about 3 hours, and the ileum is a site of temporary storage of chyme,

(B)

At Rest

During Straining

Pubis

Puborectalis

External anal Sphincter Internal anal Sphincter

Coccyx Anorectal angle

Anorectal angle Descent of the pelvic floor

FIGURE 42.4 Dynamics of normal defecation: note the straightening of the rectoanal angle by relaxation of the puborectalis to facilitate evacuation.


GAsTRoInTEsTInAl FunCTIon

allowing salvage of nutrients, fluids and electrolytes, that were not absorbed upstream. Residues are finally discharged from the ileum to the colon in bolus transfers that probably result from prolonged propagated contractions or reestablished interdigestive cyclical motor activity (the “housekeeper”). The vagus nerve is critically important in efferent control of the fed phase; splanchnic and vagal afferents convey signals from the gut to the prevertebral ganglia, spinal cord and brain to evoke reflex responses and coordinate secretory and motor functions. In animal species such as the dog, the colon also demonstrates cyclical activity, but this is less understood than in the small bowel. This cyclical activity is not recognized in human colon. The proximal colon (ascending and transverse regions) stores solid residue. The ascending colon has variable patterns of emptying: relatively linear, or constant; intermittent; or sudden mass movements. The descending colon is mainly a conduit, and the rectosigmoid functions as a terminal reservoir allowing emptying under voluntary control when there is a call to defecate. Defecation results from a well-coordinated series of motor responses (Fig. 42.4). The rectoanal angle is maintained relatively acute by the puborectalis muscle sling or pelvic floor and this is also important to maintain continence. For defecation to occur, this sling relaxes, thereby

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opening the rectoanal angle to allow a straighter rectal conduit; the external anal sphincter is inhibited voluntarily by parasympathetic (S2, 3, 4) input, and the internal sphincter (under sympathetic control) is inhibited reflexly as a result of rectal distension. Propulsion is facilitated by intracolonic pressure increases, predominantly by a rise in intra-abdominal pressure associated with straining and by propagated contractions of the colon. In contrast, continence is maintained by contraction of the puborectalis (parasympathetic S2, 3, 4), contraction of the internal sphincter (sympathetic lumbar colonic nerves) and contraction of the external sphincter (pudendal nerve).

References [1] Davenport HW. Physiology of the digestive tract. 5th ed. 1982 [2] Camilleri M. Autonomic regulation of gastrointestinal motility. In: Low PA, editor. Clinical autonomic disorders: Evaluation and management (2nd ed.). Philadelphia: Lippincott-Raven; 1997. p. 135–45. [3] Camilleri M. Gastrointestinal motor mechanisms and motility: hormonal and neural regulation. In: Singer MV, Ziegler R, Rohr G, editors. Gastrointestinal tract and endocrine system. Dordrecht, The Netherlands: Kluwer Publishers; 1995. p. 237–53. [4] Cooke HJ. Role of “little brain” in the gut in water and electrolyte homeostasis. FASEB J 1989;3:127–38. [5] Lembo T, Camilleri M. Chronic constipation. N Engl J Med 2003;349:1360–8.


C H A P T E R

43 The Splanchnic Circulation Gregory D. Fink, John W. Osborn OVERVIEW The splanchnic circulation is composed of blood vessels serving the stomach, spleen, pancreas, small intestine, colon and liver. The urogenital organs, including the kidneys, are not part of the splanchnic system. The three major arteries serving the splanchnic organs are the celiac and superior and inferior mesenteric. These parallel circuits branch into numerous smaller arteries that in some instances may form anastomoses. Blood flow to the liver occurs through two distinct routes. Approximately one third enters via the hepatic artery (a branch of the celiac artery); another two-thirds arrives via the portal vein. The portal vein is formed by the confluence of smaller veins draining the spleen, pancreas, small intestine and colon. All venous blood leaving the splanchnic region passes through the three major hepatic veins.

Local Regulation The splanchnic organs receive ~25–30% of the cardiac output under normal conditions. Basal blood flow to the splanchnic region far exceeds that required by tissue oxygen utilization, thus total flow is not determined primarily by metabolic needs. Flow increases substantially, however, during feeding and digestion. Local (intrinsic) control mechanisms are responsible for most blood flow changes necessary to support specific functions of the splanchnic organs. For example, blood flow in the GI mucosa is regulated by intramural cholinergic vasodilator nerves activated in response to exposure of the mucosa to nutrients. Collectively the splanchnic vascular beds show some capacity for autoregulation of blood flow, but this is much weaker than in the renal circulation. Veins in the splanchnic region represent a major site for blood storage (capacitance). The splanchnic vessels contain 20–25% of total circulating blood volume. The high compliance and elasticity of veins allows passive forces to exert a major influence on their degree of filling. However, venous volume also is affected by the degree of contraction of smooth muscle in the wall of veins (venoconstriction). Myogenic tone and local humoral factors affect venous smooth muscle activity in response to hemodynamic and metabolic perturbations in the splanchnic organs.


Effect of Splanchnic Circulation on Overall Circulatory Function The splanchnic circulation powerfully influences systemic arterial pressure via two distinct mechanisms. Widespread contraction of arteries in the splanchnic bed reduces blood flow to the region. The low oxygen consumption of splanchnic organs allows for a very large reduction in blood flow without producing ischemia. Arterial constriction causes dramatic increases in systemic arterial pressure and total peripheral resistance. Cardiac output also may increase due to passive discharge of stored blood from downstream veins into the central circulation. Active constriction of veins in the splanchnic organs reduces regional blood volume. This has relatively little effect on total peripheral resistance but raises cardiac output and arterial pressure by increasing central blood volume and thus cardiac preload. Generalized arterial and venous constriction in the splanchnic circulation occur mainly in response to extrinsic neural and hormonal inputs. Catecholamines, angiotensin II and vasopressin are among the most powerful hormonal vasoconstrictors. They serve this function particularly in response to major challenges to overall circulatory homeostasis, for example acute hypovolemia caused by hemorrhage. Extrinsic neural regulation is achieved almost exclusively through the sympathetic branch of the autonomic nervous system. That topic is the focus of the remainder of this chapter.

SYMPATHETIC CONTROL OF THE SPLANCHNIC CIRCULATION – GENERAL FEATURES A large majority of the sympathetic nerves innervating the splanchnic organs originate from the T4–T9 segments of the spinal cord as cholinergic preganglionic neurons, pass through the greater splanchnic nerves, synapse in the celiac ganglion plexus, then distribute to various target sites as post-ganglionic adrenergic nerve fibers. Smaller numbers originate in the T10–T12 segments and pass through the lesser and least splanchnic nerves to the prevertebral ganglia. The major neurotransmitters released from sympathetic varicosities are norepinephrine (NE),

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43. THE SPlAnCHnIC CIRCulATIon

which binds to adrenoceptors, and ATP (or possibly a related purine), which binds to P2 purinoceptors and NPY, which binds to Y1 and Y2 receptors. Neurotransmission is regulated prejunctionally by feedback actions of the neurotransmitters, but also by a variety of other factors including angiotensin, nitric oxide, adenosine, and prostanoids. Removal of released transmitters is via neuronal and extraneuronal uptake, metabolism and simple diffusion. Both arteries and veins in the splanchnic region are densely innervated. The degree of innervation is not static: it can change in response to altered physiological demands [1]. In humans the density of sympathetic innervation is substantially greater in mesenteric veins than in arteries [2], presumably reflecting the importance of sympathetic control of splanchnic vascular capacitance during upright posture. The potential exists for distinct sympathetic control of splanchnic arteries versus veins, because they receive some separate postganglionic sympathetic inputs [3]. Furthermore, neurotransmission differs substantially in arteries and veins. For example, venoconstriction occurs at lower levels of nerve activity than does arterial constriction and is better maintained during sustained increases in activity. Alpha-2 adrenergic receptors play a larger role in constriction of mesenteric veins versus arteries. And the relative contribution of ATP and NPY to neurotransmission also varies in arteries versus veins. The functional significance of possible separate regulation of venous and arterial contraction in the splanchnic region remains a matter of dispute [ref 4, and related commentaries]. Splanchnic sympathetic nerve activity (splSNA) is controlled by arterial and cardiopulmonary baroreceptors, and by inputs from other somatic and visceral afferents. Some stimuli are able to alter splSNA in a selective fashion. For example, cholecystokinin and leptin decrease splSNA while generally increasing activity in other sympathetic nerves [5]. Selective changes in sympathetic activity appear to be enabled by the existence of neurons in the rostral ventrolateral medulla of the brainstem that specifically target the splanchnic region. It is unclear if central mechanisms can drive differential activation of arterial versus venous sympathetic nerves. As discussed earlier, sympathetic input to the splanchnic vasculature is not critical for normal functioning of the splanchnic organs, but instead mainly participates in overall regulation of the circulation. Systemic circulatory – and splanchnic hemodynamic – responses to splSNA have been well characterized using both nerve activation and inactivation (e.g. denervation or regional anesthesia). Maximal increases in splSNA can reduce splanchnic blood flow by ~80% and splanchnic blood volume by ~70%. Systemic arterial pressure, total peripheral resistance and cardiac output all increase; the relative proportion of resistance and output changes depends on circulating blood volume, with cardiac output changes predominant when blood volume is low. During sustained increases in splSNA, effects on cardiac output are

generally more persistent than on total peripheral resistance. Catecholamines from the adrenal medulla contribute to both regional and systemic circulatory responses to increased splSNA, especially at higher levels of nerve activity. In conscious, supine subjects inhibition of resting splSNA decreases arterial pressure and cardiac output, but not total peripheral resistance. Splanchnic vascular resistance is also generally unaffected; however, splanchnic blood volume is increased. Thus, basal levels of splSNA appear to regulate splanchnic vascular capacitance more strongly than resistance. This is consistent with evidence from animal studies that splSNA is low under basal conditions.

SYMPATHETIC CONTROL OF THE SPLANCHNIC CIRCULATION IN SPECIAL CIRCUMSTANCES Intact splanchnic sympathetic nerves are important for normal cardiovascular adjustments to upright posture in humans, and to exercise, heat stress and blood loss in humans and other animals. The relative contribution of changes in splanchnic blood flow versus blood volume in these conditions varies, but are driven both by increased splSNA to the vasculature and by release of adrenal catecholamines. Responsiveness of splSNA to these physiological perturbations is reduced with aging. Reduced sympathetically mediated splanchnic vasoconstriction also is an important cause of cardiovascular de-conditioning after bed rest or exposure to microgravity.

SYMPATHETIC CONTROL OF THE SPLANCHNIC CIRCULATION IN DISEASE Impaired sympathetic splanchnic vasoconstriction is an important cause of orthostatic hypotension and other orthostatic disorders such as postural tachycardia syndrome. In portal hypertension there is a very marked increase in sympathetic nervous system activity, including to the splanchnic organs. However, the density of sympathetic innervation in the mesenteric vascular bed is dramatically reduced [6]; this may contribute to the splanchnic vasodilation characteristic of portal hypertension. Autonomic nervous system neuropathy and dysfunction, including orthostatic hypotension, are a significant complication of diabetes. Sympathetic neurons innervating the splanchnic region are most likely to be affected, possibly due to their increased susceptibility to glucose-induced oxidative stress. Increased splSNA worsens intestinal ischemia in septic shock [7]. During the development of arterial hypertension in humans, increased vascular resistance is observed first in the splanchnic circulation. And the splanchnic sympathetic nerves clearly contribute to the pathophysiology of essential hypertension since early work in humans showed that


SymPATHETIC ConTRol of THE SPlAnCHnIC CIRCulATIon In DISEASE

splanchnicectomy or celiac ganglionectomy often lowered blood pressure. More recent studies in experimental animals also support the idea that sympathetically mediated splanchnic vasoconstriction can cause hypertension, at least in part by affecting vascular capacitance [8]. Interestingly, it appears that very selective increases in splSNA (with decreases or no change in sympathetic activity to muscle or kidney) account for this effect [9]. A relatively new area of interest is the interaction between sympathetic innervation to the intestine and inflammation. Major changes in sympathetic nerve density and neurotransmission have been reported, for example, in inflammatory bowel disease in humans and animal models [10]. Inflammatory factors may play a larger role than heretofore appreciated in dysregulated sympathetic neurotransmission in a variety of disease states.

References [1] Monos E, Lorant M, et al. Long-term adaptation mechanisms in extremity veins supporting orthostatic tolerance. News Physiol Sci 2003;18:210–4.

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[2] Birch DJ, Turmaine M, et al. Sympathetic innervation of human mesenteric artery and vein. J Vasc Res 2008;45(4):323–32. [3] Kreulen DL. Properties of the venous and arterial innervation in the mesentery. J Smooth Muscle Res 2003;39(6):269–79. [4] Rothe CF. Point: active venoconstriction is/is not important in maintaining or raising end-diastolic volume and stroke volume during exercise and orthostasis. J Appl Physiol 2006;101(4):1262–4. discussion 1265-6, 1270. [5] Sartor DM, Verberne AJ. Gastric leptin: a novel role in cardiovascular regulation. Am J Physiol Heart Circ Physiol 2010;298(2):H406–414. [6] Coll M, Martell M, et al. Atrophy of mesenteric sympathetic innervation may contribute to splanchnic vasodilation in rat portal hypertension. Liver Int 2010;30(4):593–602. [7] Daudel F, Freise H, et al. Continuous thoracic epidural anesthesia improves gut mucosal microcirculation in rats with sepsis. Shock 2007;28(5):610–4. [8] King AJ, Osborn JW, et al. Splanchnic circulation is a critical neural target in angiotensin II salt hypertension in rats. Hypertension 2007;50(3):547–56. [9] Osborn JW, Fink GD. Region-specific changes in sympathetic nerve activity in angiotensin II-salt hypertension in the rat. Exp Physiol 2010;95(1):61–8. [10] Lomax AE, Sharkey KA, et al. The participation of the sympathetic innervation of the gastrointestinal tract in disease states. Neurogastroenterol Motil 2010;22(1):7–18.


C H A P T E R

44 Autonomic Control of the Kidney Edwin K. Jackson INTRODUCTION The 1970s ushered in four decades of meticulous investigation of the renal autonomic nervous system. Several comprehensive reviews [1–4] describe the voluminous research underpinning our current state of knowledge of the renal autonomic nervous system and provide a detailed listing of citations in this regard.

INNERVATION OF THE KIDNEY Autonomic control of the kidney is predominantly sympathetic, and there is only scant evidence for parasympathetic innervation. The soma of neurons that project directly to renal sympathetic preganglionic neurons reside predominantly in the paraventricular nucleus of the hypothalamus (PVN), the A5 noradrenergic cell group in the caudal ventrolateral pons (A5), the caudal raphe nuclei (CRN) and the rostral ventrolateral medulla (RVLM) (Fig. 44.1). The RVLM is most critical because it provides the primary tonic excitatory input to preganglionic sympathetic neurons. In this regard, the nucleus tractus solitarius (NTS) receives direct inputs from the peripheral arterial and cardiopulmonary baroreceptors and communicates with the RVLM via the caudal ventrolateral medulla (CVLM). Moreover, the somatosensory and visceral systems gather information from chemoreceptors and mechanoreceptors and send direct inputs to the NTS, CVLM and RVLM. Finally, higher cortical centers may influence renal sympathetic tone via inputs to the aforementioned hypothalamic and brainstem nuclei. There is considerable inter-species and intra-species variation in the neuroanatomical arrangement of the renal sympathetic preganglionic and postganglionic neurons. The cell bodies of renal sympathetic preganglionic neurons reside primarily in the intermediolateral column of the thoracic spinal cord, and the axons of these neurons exit the spinal cord at the last few thoracic segments and first few lumbar segments. Sympathetic preganglionic neurons synapse with renal sympathetic postganglionic neurons both in paravertebral ganglia and in outlying prevertebral ganglia (including the celiac, superior mesenteric, aorticorenal and posterior renal ganglia, as well a variable number of smaller renal ganglia). The thoracic


and lumbar splanchnic nerves provide the dominant pathways for renal sympathetic innervation. Postganglionic sympathetic neurons densely innervate the kidneys and have varicosities on renal vascular smooth muscle cells in the interlobar, arcuate and interlobular arteries and afferent and efferent arterioles, on juxtaglomerular cells and on epithelial cells in proximal tubules, thick ascending limbs of Henle’s loops, distal convoluted tubules and collecting ducts. In this regard, the density of sympathetic varicosities is much greater on vascular structures compared with tubular elements.

AUTONOMIC RECEPTORS IN THE KIDNEY The basal discharge rate of renal sympathetic nerves is 0.5 to 2 Hz, and this causes the continuous basal release of the dominant neurotransmitter in the sympathetic varicosity, norepinephrine, along with lesser amounts of the co-transmitter, neuropeptide Y1–36 (Fig. 44.2). Both norepinephrine and neuropeptide Y1–36 cause autoinhibition (prejunctional negative feedback) of neurotransmitter release via prejunctional α2-adrenoceptors and Y2 receptors, respectively. In addition, several other humoral and paracrine factors also prejunctionally modulate renal noradrenergic neurotransmission, for example angiotensin II via AT1 receptors and epinephrine via β2-adrenoceptors, both of which facilitate neurotransmission. Studies involving direct electrical stimulation of renal nerves support the conclusion that different frequency threshold levels of efferent renal sympathetic nerve activity alter renin release (0.5 Hz), tubular transport (1 Hz) and renal hemodynamics (2.5 Hz). With regard to renin release, neuronally-released norepinephrine acts directly on granular juxtaglomerular cells, a type of modified vascular smooth muscle cell in afferent arterioles, to increase the rate of renin secretion. This effect of norepinephrine is mediated exclusively by β1-adrenoceptors and involves in part the following signal transduction process: (1) activation of Gs; (2) stimulation of adenylyl cyclase; (3) increased levels of cAMP; (4) stimulation of protein kinase A; (5) phosphorylation of proteins leading to increased H translocation into renin-containing granules; (6) increased KCl/H exchange; (7) osmotically-driven influx of H2O into the granule; (8) granule swelling; and (9) exocytosis of renin.

215


216

44. AuTonomIC ConTRol of THE KIdnEy

Arterial & Cardiopulmonary Baroreceptors

Somatosensory/VisceralSystems & Higher Cortical Centers

NTS CVLM

RVLM

Projecting Neuron

A5, CRN, PVN Projecting Neuron

Intermediolateral Column of Spinal Cord (Lower Thoracic & Upper Lumbar Segments)

Sympathetic Preganglionic Neuron

Sympathetic Preganglionic Neuron

Paravertebral Ganglia Prevertebral Ganglia (Celiac, Aorticorenal, Superior Mesenteric, Posterior Renal)

Sympathetic Postganglionic Neuron

Innervated Tubular Structures

Sympathetic Postganglionic Neuron

Innervated Vascular Structures

PT TALH DCT CD

Interlobar Arteries Arcuate Arteries Interlobular Arteries Afferent Arterioles Juxtaglomerular Cells Efferent Arterioles

FIGURE 44.1 Diagram of efferent innervation of the kidney. NTS, nucleus tractus solitarius; CVLM, caudal ventrolateral medulla; RVLM, rostral ventrolateral medulla; A5 area, A5 noradrenergic cell group in the caudal ventrolateral pons; CRN, caudal raphe nuclei; PVN, paraventricular nucleus of the hypothalamus; PT, proximal tubule; TALH, thick ascending limb of Henle’s loop; DCT, distal convoluted tubule; CD, collecting duct.

Low hydrostatic pressure in the afferent arteriole stimulates renin release via the “intrarenal baroreceptor mechanism.” Decreased influx of NaCl into columnar epithelial cells located at the end of the thick ascending limb of Henle’s loop augments renin secretion via the “macula densa mechanism.” Importantly, a powerful synergy exists between β1-adrenoceptor-induced and intrarenal baroreceptor- and macula densa-induced renin secretion. See Castrop et al. [5] for a recent, comprehensive review of mechanisms of renin release. Norepinephrine released from renal sympathetic varicosities also acts directly on renal epithelial cells to accelerate

the rate of solute and water reabsorption from the tubular lumen. This effect of noradrenergic neurotransmission occurs in all nephron segments, but is particularly pronounced in the proximal tubule and ascending limb of Henle’s loop. Although norepinephrine directly affects epithelial cell transport, norepinephrine-induced changes in renal blood flow and glomerular filtration rate may also contribute to sympathetically-induced decreases in renal excretion of electrolytes and water. The direct effects of norepinephrine to enhance tubular transport are mediated mostly by α1-adrenoceptors (predominantly α1Badrenoceptors), which appear to engage the following signal


217

REflEx REgulATIon of Blood VolumE

Juxtaglomerular Cell Renal Sympathetic Varicosity

Renin

(+)

β1

Renin Release

α2 α1 Basal Frequency = 0.5–2Hz

NE NPY

(–) (–)

Y1

Renal Vascular Smooth Muscle Cell

Other Prejunctional Modulators

(+)

Ang II (AT1) PGs Epi (β2) Histamine Ach ANP NO Bradykinin Adenosine (A1)

(+)

Vascular Contraction

(+)

Y2 (–)

(+) α2

NE NPY

α1 Renal Epithelial Cell (+)

Solute & H20

Sodium Reabsorption

FIGURE 44.2 Summary of autonomic receptors in the kidney. NE, norepinephrine; NPY, neuropeptide Y1–36; α1, α1-adrenoceptor; β1, β1-adrenoceptor; Y1, Y1 receptor for NPY; Y2, Y2 receptor for NPY; PGs, prostaglandins; Ach, acetylcholine; ANP, atrial natriuretic peptide; NO, nitric oxide; Ang II, angiotensin II; Epi, epinephrine; A1, A1 adenosine receptor; AT1, angiotensin type 1 receptor; β2, β2-adrenoceptor. “Threshold” refers to the frequency of renal nerve stimulation necessary to initiate the indicated response.

transduction process throughout the nephron: (1) activation of Gq; (2) stimulation of phospholipase C-β; (3) increased production of inositol trisphosphate; (4) release of intracellular calcium; (5) calcium-mediated activation of the phosphatase calcineurin; and (6) calcineurin-mediated dephosphorylation and activation of basolateral Na-K-ATPase. In addition, α1-adrenoceptors activate Na/H exchangers (NHE1 and NHE3) in the proximal tubule and the Na/K/2Cl cotransporter (NKCC2) in the thick ascending limb of Henle’s loop, and these actions also contribute to solute reabsorption. Stimulation of renal sympathetic nerves decreases renal blood flow and glomerular filtration rate. These effects are mediated mostly by norepinephrine-induced activation of α1-adrenoceptors (predominantly α1Aadrenoceptors) which mediate intense contraction of vascular smooth muscle cells leading to vasoconstriction of the renal microcirculation. The signal transduction pathway is in part: stimulation of Gq → activation of phospholipase C-β → inositol trisphosphate-induced calcium release  diacylglyerol production → activation of protein kinase C. In this regard, preglomerular microvessels appear to be more responsive to norepinphrine than are

postglomerular microvessels, an imbalance that contributes to norepinephrine-induced reductions in glomerular capillary hydrostatic pressure. Norepinephrine reduces single nephron glomerular filtration rate by decreasing both glomerular capillary hydrostatic pressure and single nephron blood flow. Although α1-adrenoceptors mediate most of the renal hemodynamic effects of renal sympathetic nerve stimulation, α2-adrenoceptors and Y1 receptors also participate, with considerable species variation. Both α2-adrenoceptors and Y1 receptors may contribute to renal vasoconstriction by inhibiting adenylyl cyclase via a Gi-mediated mechanism.

REFLEX REGULATION OF BLOOD VOLUME Autonomic control of the kidney enables an important renal reflex that buffers changes in blood volume and contributes to the rapid restoration of normal blood volume following a positive or negative perturbation in volume status due to for instance a large volume load or blood loss (Fig. 44.3). Increases in blood volume stimulate cardiopulmonary baroreceptors, particularly those


218


Positive Volume Perturbation (e.g., Large Volume Load)

(+) Blood Volume (–)

NaCl and H20 Excretion

Stimulation of Cardiopulmonary Baroreceptors

ADH Secretion

Integration of Signals in CNS

Negative Volume Perturbation (e.g., Large Blood Loss)

Renin Release

Renin Release

Tubular Transport

Tubular Transport

RBF & GFR

RBF & GFR

Postganglionic Sympathetic Activity

Postganglionic Sympathetic Activity

Preganglionic Sympathetic Activity

Preganglionic Sympathetic Activity

Premotor Neuron Activity

Premotor Neuron Activity

NaCl and H20 Excretion

(–) (+) Blood Volume

Inhibition of Cardiopulmonary Baroreceptors

ADH Secretion

Integration of Signals in CNS

FIGURE 44.3 Reflex regulation of blood volume by the renal autonomic system. ADH, antidiuretic hormone; RBF, renal blood flow; GFR, glomerular filtration rate; CNS, central nervous system.

in the left atrium. Stimulation of cardiopulmonary baroreceptors by an expanding blood volume increases afferent vagal signals to the NTS, which relays these incoming signals to cardiovascular centers in the brain for further integration. The net result is an inhibition of antidiuretic hormone secretion from the posterior pituitary and a reduction in efferent renal sympathetic nerve activity. The decrease in efferent renal sympathetic nerve activity decreases renin release and renal tubular epithelial transport and increases renal blood flow and glomerular filtration rate. These changes, in conjunction with decreases in antidiuretic hormone levels, markedly increase the excretion rate of NaCl and water, which accelerates the restoration of a normal blood volume. Conversely, a reduction in blood volume (for example by severe bleeding) increases antidiuretic hormone levels and efferent renal sympathetic nerve activity, and these changes reduce NaCl and water excretion to prevent further reductions in blood volume. As a general rule, evolution provides multiple homeostatic mechanisms controlling physiological parameters critical to life, and blood volume is no exception. Redundancy exists in the homeostasis of blood volume, and the extent to which autonomic control of the kidney contributes to blood volume regulation depends on multiple factors such as the magnitude of blood volume perturbation and the physiological status of other regulatory mechanisms.

THE RENORENAL REFLEX Since two kidneys regulate blood volume, pressure and composition, it is not surprising that a mechanism exists to balance these critical tasks between the two kidneys. This mechanism is the renorenal reflex (Fig. 44.4). Increased renal blood flow and glomerular filtration rate to one kidney results in ipsilateral increases in renal venous and pelvic pressures due to greater volumes of blood and urine, respectively, in those compartments. Pressure in the renal venous and pelvic structures activates renal mechanoreceptors residing in the major renal veins, the renal pelvis and the corticomedullary connective tissue. Release of substance P and calcitonin generelated peptide from afferent nerve endings, as well as local formation of prostaglandin E2, may augment the discharge of afferent renal sensory nerves. Renal afferent nerves have their cell bodies in the ipsilateral dorsal root ganglia of the lower thoracic and upper lumbar cord segments, and incoming signals pass via the spinal cord to cardiovascular/renal integration centers in the central nervous system. With increased incoming afferent traffic from the ipsilateral kidney, these centers of integration command a decrease in efferent renal sympathetic nerve activity to the contralateral kidney, which results in increases in renal blood flow to and glomerular filtration by the contralateral kidney, thereby increasing the workload on the contralateral kidney. Moreover, diuresis and


AuTonomIC ConTRol of THE KIdnEy In PATHoPHysIologICAl sTATEs

219

(–) Increased RBF & GFR to Ipsilateral Kidney

Increased Renal Pelvic Pressure

Increased Renal Venous Pressure Activation of Ipsilateral Renal Mechanoreceptors (Involves SP, CGRP, PGE2)

Activation of Ipsilateral Afferent Renal Nerves with Cell Bodies in DRG Spinal Pathways

Integration in CNS Spinal Pathways

Decreased Efferent Nerve Activity to Contralateral Kidney

Decreased Tubular Transport Increased RBF & GFR to Contralateral Kidney Diuresis and Natriuresis by Contralateral Kidney

FIGURE 44.4 How the renorenal reflex distributes workload evenly between the kidneys. RBF, renal blood flow; GFR, glomerular filtration rate; SP, substance P; CGRP, calitonin gene-related peptide; PGE2, prostaglandin E2; CNS, central nervous system; DRG, dorsal root ganglia.

natriuresis by the contralateral kidney gradually decreases arterial blood pressure, which ultimately reduces the renal blood flow and glomerular filtration rate of the ipsilateral kidney. The net result is a near equal renal blood flow and glomerular filtration rate between the kidneys and consequently an equal sharing of the workload of maintaining a constant blood volume, pressure and composition.

AUTONOMIC CONTROL OF THE KIDNEY IN PATHOPHYSIOLOGICAL STATES The relationship between mean arterial blood pressure and the renal excretion rate of sodium, i.e., the renal pressure-natriuresis curve, determines long-term levels

of arterial blood pressure [6]. Increased renal efferent sympathetic nerve activity impairs renal sodium excretion and shifts the renal pressure-natriuresis curve to the right such that higher long-term levels of blood pressure are required to maintain sodium excretion in balance with sodium intake. It is not surprising, therefore, that efferent renal sympathetic nerve activity contributes to the pathophysiology of hypertension. Evidence for this conclusion includes: (1) complete renal denervation delays and/or attenuates the development of hypertension in a wide spectrum of experimental animal models; (2) efferent renal sympathetic nerve activity is usually increased in hypertension; (3) chronic low-level renal nerve stimulation or chronic intrarenal infusions of norepinephrine induce hypertension; and (4) sympatholytic drugs lower blood


220


pressure. However, the pathophysiology of hypertension is multifactorial and efferent renal sympathetic nerves are only one among many participating mechanisms. Renal retention of NaCl and water is a prerequisite for edema formation in congestive heart failure, hepatic cirrhosis and nephrotic syndrome. In these pathophysiological states, blood pressure and intravascular extracellular fluid volume are often diminished, even though total extracellular fluid volume is usually expanded. These perturbations inappropriately engage an arterial baroreceptor-mediated and/or cardiopulmonary baroreceptormediated reflex increase in efferent renal sympathetic nerve activity which contributes importantly to the retention of NaCl and water and consequently to the edematous state. Accordingly, maneuvers that attenuate efferent renal sympathetic nerve activity, for example head-out water immersion, bilateral lumbar sympathetic anesthetic block and administration of sympatholytics, increase NaCl and water excretion in edema associated with heart, liver or kidney disease. Renal afferent nerves may also contribute to over-activation of the sympathetic nervous system in renal diseases. Renal ischemia, hypoxia and injury increases renal afferent signaling to the central nervous system, which activates sympathetic tone to the kidneys, heart and other innervated organs and may elevate arterial blood pressure. The important role of renal sympathetic efferent and

afferent nerves in cardiovascular regulation is underscored by recent advances in the use of catheter-based, radiofrequency ablation of renal efferent and afferent nerves for the treatment of drug-resistant hypertension [4, 7, 8, see Chapter 137].

References [1] DiBona GF, Kopp UC. Neural control of renal function. Physiol Rev 1997;77:75–197. [2] DiBona GF. Physiology in perspective: The Wisdom of the Body. Neural control of the kidney. Am J Physiol Regul Integr Comp Physiol 2005;289:R633–41. [3] Johns EJ, Kopp UC. Neural control of renal function. In: “Seldin and Giebisch’s the kidney: Physiology and pathophysiology. 4th ed. vol. 1, Chapter 33, Amsterdam: Elsevier; 2008. p. 925–46. [4] DiBona GF, Esler M. Translational medicine: the antihypertensive effect of renal denervation. Am J Physiol Regul Integr Comp Physiol 2010;298:R245–53. [5] Castrop H, Höcherl K, Kurtz A, Schweda F, Todorov V, Wagner C. Physiology of kidney renin. Physiol Rev 2010;90:607–73. [6] Johnson RJ, Feig DI, Nakagawa T, Sanchez-Lozada LG, RodriguezIturbe B. Pathogenesis of essential hypertension: historical paradigms and modern insights. J Hypertens 2008;26:381–91. [7] Schlaich MP, Sobotka PA, Krum H, Whitbourn R, Walton A, Esler MD. Renal denervation as a therapeutic approach for hypertension. Novel implications for an old concept. Hypertension 2009;54:1195–201. [8] Schlaich MP, Krum H, Sobotka PA. Renal sympathetic nerve ablation: The new frontier in the treatment of hypertension. Curr Hypertens Rep 2010;12:39–46.


C H A P T E R

45 Dopamine Mechanisms in the Kidney Robert M. Carey RENAL DOPAMINE (DA) FORMATION AND EXCRETION Circulating concentrations of DA (picomolar range) are generally insufficient to activate DA receptors, but concentrations within the kidney (high nanomolar to low micromolar range) are able to activate DA receptors. DA biosynthesis in the kidney occurs in proximal tubule cells (PTC) as a result of uptake of filtered L-dihydroxyphenylalanine (L-DOPA) by way of a sodium (Na) transporter in the apical (brush border) membrane. Once inside the PTC, L-DOPA is rapidly decarboxylated to DA via aromatic amino acid decarboxylase (AAAD), the activity of which is dependent on the Na load to the tubule. DA synthesized within the PTC is not stored but is secreted across the apical and basolateral membrane. The basolateral outward transporter also is dependent upon Na and pH. The supply of L-DOPA to the PTC is the major regulator of DA synthesis and secretion. In vivo, DA is preferentially APICAL MEMBRANE

BASOLATERAL MEMBRANE

L-dopa

L-dopa

Na+ Transporter Na+

DOPA DECARBOXYLASE Na+

DA

DA D1R

AC

secreted across the apical membrane into the tubule lumen, as the increase in urinary DA far exceeds its increment in the renal interstitial fluid [1]. In contrast to the usual pathway for DA biosynthesis in neurons, within the kidney DA is synthesized independently of nerve activity. Figure 45.1 depicts the scheme of DA formation and secretion in the PTC.

RENAL DA RECEPTOR EXPRESSION The D1-like and D2-like receptor families are expressed at post-junctional sites within the kidney. D1 receptors are localized in the smooth muscle cells of renal arterioles, juxtaglomerular (JG) cells, PTCs and cortical collecting ducts (CCD) both by immunohistochemistry and by in situ amplification of mRNA [2,3]. The D5 receptor is predominantly localized to the distal nephron. It is currently thought that D1 receptor function is preferentially exerted over that of the D5 receptor in the PTC, whereas the reverse is true in the distal nephron. The D3 receptor, the predominant D2-like receptor in the kidney, is localized in renal arterioles, glomeruli, PTC, medullary thick ascending limb cells and the CCD by immunohistochemistry [4]. The D4 receptor also is localized specifically in the cortical collecting duct. Renal DA receptor distribution is shown in Table 45.1 and the functions of renal DA receptors is shown in Table 45.2. TABLE 45.1 Renal Dopamine Receptor Expression

DA

ATP

PIP2

cAMP

DAG

PLC

Receptor Subtype

D 1R

IP3

PKC P

Na+ Protein

Protein

P

Na+K+ ATPase

NHE-3 H+

3Na+

Protein

2K+

FIGURE 45.1 Schematic depiction of dopamine formation and cell signaling mechanisms activating sodium transport across the proximal tubule cell. DA, dopamine; D1R, dopamine D1 receptor; PLC, phospholipase C; DAG, diacylglycerol; and AC, adenylyl clyclase.


221

Tissue Expression

D1

D2

D3

D4

D5

Arterioles











Glomerulus











Proximal tubule











mTal











Distal tubule











Cortical collecting duct











Renal nerves











Juxtaglomerular cells





?



?


222

45. DoPAMInE MECHAnIsMs In THE KIDnEy

TABLE 45.2 Effects of Renal Dopamine Receptor stimulation D1-LIKE RECEPTORS (D1 and D5) l l l l

Vasodilation of renal arterioles (requires circulating DA) Inhibition of proximal and distal tubule sodium reabsorption Stimulation of renin secretion Inhibition of angiotensin AT1 receptor expression

D2 RECEPTOR l

Inhibition of norepinephrine release from renal sympathetic neurons

D3 RECEPTOR l l l l l

Inhibition of renin secretion Inhibition of angiotensin AT1 receptor expression Possible increase in glomerular filtration Possible vasodilation of renal arterioles Possible inhibition of tubule sodium reabsorption (natriuresis)

D4 RECEPTOR l

Possible inhibition of sodium reabsorption in the cortical collecting duct

DOPAMINERGIC REGULATION OF RENAL Na EXCRETION D1-like Receptors The renal D1-like receptor family plays a major role in the regulation of tubule Na transport. The natriuretic action of DA is due to inhibition of both proximal and distal tubule Na reabsorption. The action of endogenous DA to inhibit tubule Na transport is distinct from the renal vasodilator action of exogenous DA, which depends upon increased plasma concentrations of DA [5]. A major cell signaling process whereby renal DA induces natriuresis is inhibition of Na, K-ATPase along the entire nephron (PTC, thick ascending limb of Henle’s loop, distal tubule and CCD). This action is thought to be mediated by the D1-like receptor family, although synergism between D1-like and D2-like receptors in the inhibition of Na, K-ATPase has been demonstrated. The signaling processes whereby DA inhibits Na/  K -ATPase activity are nephron-specific. Both protein kinase A (PKA) and protein kinase C (PKC) are involved in the PTC, while only PKA is required in the medullary thick ascending limb and CCD. PKA and PKC both phosphorylate the catalytic subunit of the enzyme inhibiting its activity. A primary component of the regulation of Na/  K -ATPase by DA is the action to inhibit the Na/H exchanger (NHE) and the Na/P1 co-transporter in the apical membrane of the tubule cell. This action of DA inhibits transport of Na into the cell, thus rendering intracellular Na too low to stimulate Na/ K-ATPase. DA’s ability to inhibit NHE activity is primarily due to activation of cyclic AMP and PKA, but can also occur via D1-like receptors directly without involving cAMP and

PKA. DA also can inhibit NHE activity by stimulation of P-450 eicosanoids such as 20-HETE. Endogenous intrarenal DA is a major physiological regulator of urinary Na excretion in vivo. About 60% of basal sodium excretion during normal Na balance is controlled by DA. There is clear evidence that renal DA acts as a paracrine substance (cell-to-cell mediator) locally modulating renal Na excretion by an action at the renal tubule independently of renal hemodynamic function [7]. This has been demonstrated both by pharmacologic blockade of the renal D1-like receptor family and also by antisense oligonucleotide-induced inhibition of D1A receptor protein expression [7,8].

D2-like Receptors Recent evidence suggests that the renal D3 receptor may increase glomerular filtration rate (GFR) via post-glomerular (efferent) arteriolar constriction. Also, a D2-like receptor, possibly D3 or D4, in the basolateral membrane of CCD cells is probably responsible for a natriuretic action of DA in this tubule segment. However, relatively little is known about the D2-like receptor family, as compared with D1like receptors, in the control of Na excretion.

PHYSIOLOGIC INTERACTIONS OF THE RENAL DOPAMINERGIC SYSTEM AND THE RENIN-ANGIOTENSIN SYSTEM (RAS) In addition to direct modulation of renal tubule Na transport, DA affects renin release from renal JG cells via the D1-like receptor family. The interaction of DA with the RAS (Fig. 45.2) is an area of continuing investigation. While D1 receptor activation stimulates renin secretion DOPAMINERGIC SYSTEM RENIN-ANGIOTENSIN SYSTEM D3RECEPTOR

ANGIOTENSINOGEN RENIN

DOPAMINE

ANG I ACE

D1-LIKE RECEPTOR

ANG II

AT1RECEPTOR ANTINATRIURESIS

AT2RECEPTOR NATRIURESIS

FIGURE 45.2 Schematic depiction of the interactions between the renal dopaminergic system and the renal renin-angiotensin system. ANG I, angiotensin I; ANG II, angiotensin II, ACE, angiotensin converting enzyme.


REnAl DA AnD HyPERTEnsIon

directly at JG cells, D1 receptors inhibit renin release by inhibiting macula densa cyclooxygenase activity in vivo [10]. DA also has been demonstrated to decrease PTC angiotensin AT1 receptor expression via the D1 receptor [9], so it appears that the net effect of DA is to increase intrarenal angiotensin II and down-regulate its actions. Current evidence suggests that DA may promote natriuresis both by direct action at the renal tubule and indirectly by decreasing the activity of the RAS and angiotensin II-stimulated Na reabsorption. The renal D3 receptor is also thought to inhibit renin secretion and additionally may inhibit AT1 receptor expression. Renal DA serves as one of several paracrine mediators of renal Na excretion. During low Na intake, Na is retained to meet the body’s requirement for Na homeostasis. Under these conditions, the RAS is stimulated and DA biosynthesis is markedly curtailed, both leading to antinatriuresis. During Na loading, on the other hand, the RAS is suppressed and DA biosynthesis is activated, both leading to natriuresis. When DA synthesis is increased, DA may inhibit renin secretion, reducing the activity of the RAS. Thus, the interaction between the RAS and renal dopaminergic systems leads to a series of overlapping counter-regulatory steps, which attempt to normalize each other to bring the control of Na excretion into equilibrium. In addition to the interactions of renal DA with AT1 receptors, recent evidence has demonstrated a major interaction of renal D1 and AT2 receptors. AT2 receptor activation induces vasodilation and natriuresis via a bradykinin - nitric oxide – cyclic GMP signaling cascade. DA-induced natriuresis is dependent on renal AT2 receptor activation in vivo [11].

RENAL DA AND HYPERTENSION Two fundamental defects in the renal DA system have been described in hypertension: [1] deficient DA production due to decreased PTC uptake and/or decarboxylation of DOPA and [2] defective D1-like receptor-G protein coupling so that DA is ineffective in transmitting a signal to inhibit Na excretion. The latter defect is confined to the PTC and current evidence suggests that the defect is due to hyperphosphorylation of the D1 receptor by a mutation in G-protein-coupled receptor kinase-4 (GRK-4). Under these circumstances, the hyperphosphorylated D1 receptor is internalized in the cytoplasm and desensitized. More work will have to be done to determine whether this defect is responsible for states of salt-sensitivity and sodium-dependent hypertension in humans.

223

Clearly, the role of DA receptors in hypertension has been substantiated by studies in animals in which a specific DA receptor has been disrupted. Knockout of the D1 receptor in mice leads to hypertension, but a mutation in the coding region of the receptor has not been found in human essential hypertension or in genetically hypertensive rats. Disruption of the D2 receptor also induces hypertension, but the increase in blood pressure is related to noradrenergic discharge at the whole body level, and there is no Na retention. Absence of the D3 receptor generates a renin-dependent form of hypertension with inability to excrete a Na load. While these studies show potentially interesting interactions, particularly between renin-angiotensin and dopaminergic systems, compensatory mechanisms may alter the resulting phenotype. Additional work needs to be done especially with renal- and nephron-specific knockout of the individual DA receptors and combinations of receptors of the DA and renin-angiotensin systems.

References [1] Wang Z-Q, Siragy HM, Felder RA, Carey RM. Intrarenal dopamine production and distribution in the rat: physiological control of sodium excretion. Hypertension 1997;29:228–34. [2] O’Connell DP, Botkin SJ, Ramos SP, Sibley DR, Ariano MA, Felder RA, et al. Localization of dopamine D1A receptor protein in the rat kidney. Am J Physiol 1995;268:F1185–F1197. [3] O’Connell DP, Aherne AM, Lane E, Felder RA, Carey RM. Detection of dopamine receptor D1A subtype-specific mRNA in rat kidney by in situ amplification. Am J Physiol 1998;43:232–41. [4] O’Connell DP, Vaughan CJ, Aherne AM, Botkin SJ, Wang Z-Q, Felder RA, et al. Expression of the dopamine D3 receptor protein in rat kidney. Hypertension 1998;32:886–95. [5] Hughes J, Ragsdale NV, Felder RA, Chevalier RL, King B, Carey RM. Diuresis and natriuresis during continuous dopamine-1 receptor stimulation. Hypertension 1988;11(Suppl 1):I-169–I-174. [6] Aperia AC. Intrarenal dopamine: a key signal in the interactive regulation of sodium metabolism. Ann Rev Physiol 2000;62:621–47. [7] Siragy HM, Felder RA, Howell NL, Chevalier RL, Peach MJ, Carey RM. Evidence that intrarenal dopamine acts as a paracrine substance at the renal tubule. Am J Physiol 1989;257:F469–77. [8] Wang Z-Q, Felder RA, Carey RM. Selective inhibition of renal dopamine subtype D1A receptor induces antinatriuresis in conscious rats. Hypertension 1999;33:504–10. [9] Cheng H-F, Becker BN, Harris RC. Dopamine decreases expression of type-1 angiotensin II receptors in renal proximal tubule. J Clin Invest 1996;97:2745–52. [10] Zhang MZ, Yao B, Fang X, Wang S, Smith JP, Harris RC. Intrarenal dopaminergic system regulates renin expression. Hypertension 2009;53:564–70. [11] Salomone LJ, Howell NL, McGrath HE, Kemp BA, Keller SR, Gildea JJ, et al. Intrarenal dopamine D1-like receptor stimulation induces natriuresis via an angiotnesin typr-2 receptor mechanism. Hypertension 2007;49:155–61. [12] Felder RA, Sanada H, Xu J, Yu P-Y, Wang Z, Wang W, et al. G protein-coupled receptor kinase 4 gene variants in human essential hypertension. Proc Nat Acad Sci USA 2002;97:3872–7.


C H A P T E R

46 Autonomic Control of the Lower Urinary Tract Lori Birder, William C. de Groat INTRODUCTION The storage and periodic elimination of urine is controlled by neural circuitry in the brain and spinal cord that regulates the activity of two functional units in the lower urinary tract: (1) a reservoir (the urinary bladder); and (2) an outlet (consisting of bladder neck, urethra, and striated sphincter muscles). Under normal conditions, the urinary bladder and urethral outlet exhibit reciprocal activity. During storage, the bladder neck and proximal urethra are closed and the detrusor muscle is quiescent, allowing intravesical pressure to remain low over a wide range of bladder volumes. During voluntary micturition the initial event is a reduction of intraurethral pressure, which reflects a relaxation of the pelvic floor and the urethral striated muscles followed in a few seconds by a detrusor contraction and a rise in intravesical pressure that is maintained until the bladder empties. These effects are mediated by three sets of peripheral nerves: sacral parasympathetic (pelvic nerves), thoracolumbar sympathetic nerves (hypogastric nerves and sympathetic chain), and sacral somatic nerves (primarily the pudendal nerves) (Fig. 46.1).

INNERVATION Parasympathetic Pathways The sacral parasympathetic outflow, which in humans originates from S2 to S4 segments of the spinal cord, provides the major excitatory input the bladder. Cholinergic preganglionic neurons located in the intermediolateral region of the sacral spinal cord send axons to cholinergic ganglion cells in the pelvic plexus and in the bladder wall. Transmission in bladder ganglia is mediated by a nicotinic cholinergic mechanism, which is sensitive to modulation by various transmitter systems, including muscarinic, adrenergic, purinergic, and peptidergic (Table 46.1). The ganglion cells in turn excite the bladder smooth muscle. A large proportion of the ganglia and nerves supplying the human lower urinary tract contain acetylcholinesterase


(AChE) as well as the vesicular ACh transporter (VAChT), and therefore must be cholinergic. AChE- and VAChTpositive nerves are abundant in all parts of the bladder but are less extensive in the urethra. Parasympathetic neuroeffector transmission in the bladder is mediated by ACh acting on postjunctional muscarinic (M) receptors. Both M2 and M3 muscarinic receptor subtypes are expressed in bladder smooth muscle, however, studies with subtype-selective muscarinic receptor antagonists and muscarinic receptor knockout mice have revealed that the M3 subtype is the principal receptor involved in excitatory transmission. In bladders of various animals, stimulation of parasympathetic nerves also produces a noncholinergic contraction that is resistant to atropine and other muscarinic receptorblocking agents. ATP (Table 46.1) has been identified as the excitatory transmitter mediating the noncholinergic contraction. ATP excites the bladder smooth muscle by acting on P2X receptors that are ligand-gated ion channels. Among the seven types of P2X receptors that have been identified in the bladder, the P2X1 subtype is the major subtype expressed in the rat and human bladder smooth muscle. Although purinergic excitatory transmission is not important in the normal human bladder, it has been identified in bladders from patients with pathological conditions such as chronic urethral outlet obstruction or interstitial cystitis. Smooth muscle contractions are initiated by an increase in intracellular Ca2 concentration that can occur by intracellular release of Ca2 from the sarcoplasmic reticulum or by influx of Ca2 from the extracellular fluid. The former mechanism is an essential step in the cholinergic activation of the detrusor muscle. It has been shown that stimulation of M3 receptors triggers the formation of inositol triphosphate (IP3) and this in turn activates IP3 receptors on the sarcoplasmic reticulum, which then causes the release of Ca2. On the other hand, activation of P2X purinergic receptors causes the influx of extracellular Ca2 as well as depolarization of the cells, leading to an opening of voltage-gated Ca2 channels. This triggers intracellular Ca2-induced Ca2 release by activation of ryanodine-sensitive receptors in the sarcoplasmic reticulum.

225


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46. AUTonomIC ConTRoL of THE LowER URInARy TRACT

FIGURE 46.1 Nerve pathways controlling urine storage and voiding. (A) Urine storage. During the storage of urine, distention of the bladder produces a low level of firing in vesical afferent axons, which in turn stimulates (1) the sympathetic outflow to the bladder outlet (base and urethra) and (2) pudendal outflow to the external urethral sphincter (EUS). These responses occur by spinal reflex pathways and represent guarding reflexes, which promote continence. Sympathetic firing also inhibits the detrusor muscle and modulates transmission in bladder ganglia. A region in the rostral pons (the pontine urine storage center) increases activity of the EUS. (B) Voiding reflexes. During elimination of urine, intense firing of bladder afferents activates spinobulbospinal reflex pathways passing through the pontine micturition center, resulting in stimulation of the parasympathetic outflow to the bladder and urethral smooth muscle and inhibition of the sympathetic and pudendal outflows to the urethral outlet. Ascending afferent input from the spinal cord may pass through relay neurons in the periaqueductal gray (PAG) before reaching the pontine micturition center. () or , excitatory mechanisms or synapses; () or , inhibitory mechanisms or synapses. ON, motor neurons in Onuf’s nucleus; PPN, parasympathetic preganglionic neurons; SPN, sympathetic preganglionic neurons.

Intracellular Ca2 combines with calmodulin to activate the contractile proteins. Activation of M2 muscarinic receptors also appears to enhance contractions by suppressing β-adrenergic inhibitory mechanisms by blocking adenylyl cyclase or K channels. Parasympathetic pathways to the urethra induce relaxation during voiding. In various species, the relaxation is not affected by muscarinic receptor antagonists and therefore is not mediated by ACh. However, inhibitors of NOS block the relaxation in vivo during reflex voiding or block the relaxation of urethral smooth muscle strips induced in vitro by electrical stimulation of intramural nerves, indicating that nitric oxide is the inhibitory transmitter involved in relaxation.

Sympathetic Pathways Sympathetic preganglionic pathways that arise from the T11 to L2 spinal segments pass to the sympathetic chain ganglia and then to prevertebral ganglia in the superior hypogastric and pelvic plexuses, and also to short adrenergic neurons in the bladder and urethra. Sympathetic postganglionic nerves that release norepinephrine provide and

excitatory input to smooth muscle of the urethra and bladder base, an inhibitory input to smooth muscle in the body of the bladder, as well as inhibitory and facilitatory inputs to vesical parasympathetic ganglia. Histofluorescence microscopy in animals and humans has shown that adrenergic terminals richly innervate the smooth muscle of the bladder base, but the bladder body has a considerably weaker adrenergic innervation. Radioligand receptor binding studies show that α-adrenergic receptors are concentrated in the bladder base and proximal urethra, whereas β-adrenergic receptors are most prominent in the bladder body. These observations are consistent with pharmacological studies showing that sympathetic nerve stimulation or exogenous catecholamines produce β-adrenergic receptor-mediated inhibition of the bladder body and strong α-adrenergic receptor-mediated contractions of the base and urethra and weak contractions of the bladder body. Molecular and contractility studies have shown that β3-adrenergic receptors elicit inhibition and α1-adrenergic receptors elicit contractions. The α1A-adrenergic receptor subtype is most prominent in normal bladders, but the α1D subtype is upregulated in bladder from patients with outlet obstruction due to benign prostatic hyperplasia. This


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THERAPy

TABLE 46.1 Receptors for Putative Transmitters in the Lower Urinary Tract Tissue

Cholinergic

Adrenergic

Other

Bladder body

 (M2)  (M3)

– (β2) – (β3)

Bladder base

 (M2)  (M3)

(α1)

Urothelium

 (M2)  (M3)

α β

Urethra

 (M)

 (α1)  (α2) – (β)

 Purinergic (P2X1) – VIP  Substance P (NK2) – VIP  Substance P (NK2)  Purinergic (P2X)  TRPV1  TRPM8  P2X  P2Y  Substance P  Bradykinin (B2)  Purinergic (P2X) – VIP – Nitric oxide

(α1)

– NPY

(α1)

– NPY

Sphincter striated  (N) muscle Adrenergic nerve – (M2/4) terminals  (M1) Cholinergic nerve – (M2/4) terminals  (M1) Afferent nerve terminals Ganglia

 (N)  (M1)

 Purinergic (P2X2/3) (α1) –(α2) (β)

 TRPV1 – Enkephalinergic (δ) – Purinergic (P1)  Purinergic (P2X)  Substance P

VIP, vasoactive intestinal polypeptide; NPY, neuropeptide Y; TRP, transient receptor potential. Letters in parentheses indicate receptor type (M, muscarinic; N, nicotinic; NK2, neurokinin-2 receptor). Plus and minus signs indicate excitatory and inhibitory effects.

finding raises the possibility that enhanced α1-adrenergic receptor excitatory mechanisms in the bladder body might contribute to irritative lower urinary tract symptoms in patients with prostatic disease. Activation of β-adrenergic receptors in bladder smooth muscle stimulates adenylyl cyclase and increases cyclic adenosine monophosphate (cAMP), which in turn activates protein kinase A. Protein kinase A is thought to act in part by inducing a hyperpolarization of the cells, either by opening of K channels or by stimulating an electrogenic ion pump. Excitatory responses in the urethra and bladder neck mediated by α1-adrenergic receptors are attributed to an increased release of Ca2 from intracellular stores.

CENTRAL NEURAL CONTROL Urine storage is facilitated by sympathetic and pudendal nerve reflex mechanisms organized in the lumbosacral spinal cord; while voiding is dependent on neural circuitry in the brain that inhibits spinal storage reflexes and activates the parasympathetic outflow to the bladder and urethra. Pathways in the forebrain that are responsible for voluntary control of voiding modulate reflex mechanisms in the pontine micturition center in the brain stem that mediate the coordination between the bladder and urethral sphincter.

NEUROPATHOLOGY Injuries to the neuraxis that interrupt connections between pontine micturition center and the lumbosacral spinal cord lead to complete loss of bladder function and in turn urinary retention. In most spinal cord injured patients, bladder reflexes slowly recover as a result of a reorganization of synaptic connections in the spinal cord and the emergence of sacral reflex mechanisms that initiate involuntary bladder contractions. However, micturition in these patients is usually compromised due to a lack of coordination between bladder and sphincter activity (a condition termed detrusor-sphincter dyssynergia). This condition is characterized by simultaneous contractions of the bladder and the striated urethral sphincter causing incomplete emptying and urinary retention. Experimental studies in animals and humans indicate that the emergence of involuntary voiding reflexes following spinal cord injury is due in part to plasticity in bladder afferent pathways and the unmasking of reflexes triggered by capsaicin-sensitive, C-fiber bladder afferent neurons. C-fiber afferents have also been implicated in the bladder hyperactivity associated with other neurological disorders such as multiple sclerosis. Damage to peripheral neural pathways to the lower urinary tract or to the lumbosacral spinal cord (i.e. a lower motoneuron lesion) causes a loss of bladder sensations as well as loss of voluntary and reflex voiding. On the other hand more selective injury to the motor nerves of the urethral sphincter or the pelvic floor or injury to these striated muscles can often occur during pregnancy and/or childbirth resulting in decreased urethral closure mechanisms and involuntary loss of urine (stress urinary incontinence) during straining, sneezing or coughing that increase intraabdominal pressure.

Somatic Motor Pathways to the Urethral Sphincter The innervation of the striated sphincter muscle of the urethra arises from cholinergic motoneurons in the sacral spinal cord and travels in the pudendal nerves. Transmission in the sphincter muscle is mediated by acetylcholine and activation of nicotinic receptors.

THERAPY Neurogenic involuntary voiding occurring as a result of central nervous system lesions or idiopathic urinary frequency, urgency and urgency incontinence (the overactive bladder syndrome) is usually treated with antimuscarinic


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agents that reduce involuntary bladder contractions. These drugs increase bladder capacity and reduce urgency sensations as well as incontinent episodes. Patients resistant to antimuscarinic therapy can be treated with botulinum toxin injected into the bladder wall. This toxin is also injected into the urethral sphincter to suppress detrusorsphincter-dyssynergia in patients with spinal cord injury.

Further Reading Andersson KE. Detrusor myocyte activity and afferent signaling. Neurourol. Urodyn 2010;29:97–106. Andersson KE, Hedlund P. Pharmacologic perspective on the physiology of the lower urinary tract. Urology 2002;60:13–20.

Birder L, de Groat W, Mills I, Morrison J, Thor K, Drake M. Neural control of the lower urinary tract: peripheral and spinal mechanisms. Neurourol Urodyn 2010;29:128–39. de Groat WC. Integrative control of the lower urinary tract: preclinical perspective. Br J Pharmacol 2006;147:S25–40. Fowler CJ, Griffiths D, de Groat WC. The neural control of micturition. Nat Rev Neurosci 2008;9:453–66. Klausner AP, Steers WD. The neurogenic bladder: an update with management strategies for primary care physicians. Med Clin N Amer 2011;95:111–20. Tai C, Roppolo JR, de Groat WC. Spinal reflex control of micturition after spinal cord injury. Restor Neurol Neurosci 2006;24:69–78. Yoshimura N, Kaiho Y, Miyazato M, Yunoki T, Tai C, Chancellor MB, et al. Therapeutic receptor targets for lower urinary tract dysfunction. Naunyn Schmied Arch Pharmcol 2008;377:437–48.


C H A P T E R

47 Bladder Function in Health and Disease Marcus J. Drake, Brian A. Parsons The lower urinary tract (LUT) stores and intermittently expels urine; the alternation between these functions is termed the micturition cycle. The control of the micturition cycle depends on several levels of the neuraxis (see Chapter 46), which enables low pressure urine storage, and volitional initiation of voiding. The complexity of control renders it vulnerable to problems, and LUT symptoms are prevalent clinically.

STRUCTURE OF THE LOWER URINARY TRACT The bladder and its outlet comprise a reservoir and a conduit respectively through which urine is periodically expelled. The bladder is a smooth muscle organ which in the human can vary in volume between 0 and 500 mL. The smooth muscle is termed the detrusor, and is primarily controlled by the parasympathetic innervation. Additional functional regulation is derived from interstitial cells within the detrusor and subjacent to the urothelial lining [1]. The urothelium itself releases active substances which can influence the subjacent afferent nerves and maybe also the detrusor [2]. The conduit of the outlet is the urethra; the urethral smooth muscle also contains interstitial cells – the urothelium here is not as active pharmacologically as in the bladder, but is important in maintaining urethral closure by coaptation. The outlet is supported by the pelvic floor; the skeletal muscle external urethral sphincter contributes to maintaining outlet closure by compression and kinking. The bladder neck in men is relatively welldeveloped and receives a sympathetic nervous system supply mediated by α-1-adrenergic receptors; this structure is important in maintaining bladder neck closure during ejaculation. In health, the bladder alternates between storage and voiding functions, the transition between the two phases being mediated by the periaqueductal grey (PAG) and pontine micturition center (PMC), with extensive input from other CNS centers [3]. The PMC is fundamental to healthy bladder function in ensuring that the bladder and its outlet behave synergistically – which is to say that at any one time only one structure is actively contracting; the bladder during voiding and the outlet during storage.


PROPERTIES OF THE HEALTHY LOWER URINARY TRACT Properties of the LUT are summarized in Table 47.1. Sensory information from the bladder is derived from the suburothelial afferent complex and from in-series stretch receptors in the detrusor [4]. Once the sensory information reaches the cerebral cortex, it gives rise to perceived sensations, comprising awareness of filling, normal desire to void, and strong desire to void [5]. The sensations are perceived intermittently and can be suppressed, except for a strong desire to void. As the bladder fills, adaptive relaxation means that the pressure change between full and empty is very small (“compliance”). The human bladder is capable of holding around about 500 mL, but typical voided volumes are often less, as people tend to make decisions about the timing of voiding based on practical influences (e.g., anticipated activities) rather than leaving it until their bladder reaches capacity. Volitional control is mediated by higher centers of the central nervous system meaning that people can make a conscious decision regarding initiation of voiding. TABLE 47.1 Sensory and Motor Properties of the Bladder and LUT Outlet During the Phases of the Micturition Cycle Storage

Voiding

BLADDER Motor

Sensory

Low amplitude micromotions [6] Generalized contraction Non-micturition contractions of detrusor by No widespread propagation parasympathetic of motor activity innervation In-series stretch receptors and Off-loading of stretch suburothelial afferents [4] receptors Intermittent conscious awareness [5]

OUTLET Motor

Sensory

229

Tonic contraction of circular smooth muscle Tonic contraction of skeletal muscle sphincter, with exertional or voluntary enhancement (“guarding”) Minimal afferent activity

Active relaxation of circular smooth muscle and sphincters Shortening of longitudinal muscle of urethra Reporting of urethral flow through pudendal afferents


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Voiding should result in complete emptying of the bladder. Clinically, urine flow is characterised by key parameters of pattern, maximum flow rate (Qmax) and voided volumes. The latter is important to assessing individual patients as poor urinary flow can result if the bladder is suboptimally filled at the time of testing (Fig. 47.1). Animals have additional voiding characteristics; territorial marking is a rapid release of small volumes of urine not resulting in complete emptying, and voiding in some rodents is associated with high frequency oscillations in sphincter activity. The LUT has to coordinate with the genital tract. The bladder neck remains closed at the time of ejaculation in males, whilst the main urethral sphincter is open to allow expulsion.

CLINICAL EVALUATION LUT symptoms [7] comprise those affecting urine storage (incontinence, urgency, frequency and nocturia), voiding (retention, poor stream, hesitancy, dribbling) or post-voiding (dribbling and sensation of incomplete emptying). Other symptoms include: pain, hematuria (blood in the urine), reduced awareness of filling, and dysuria (burning sensation with urine flow). Physical examination includes a general overview of the patient’s health, checks for any neurological impairment, bladder palpation after voiding, and pelvic examination to check for voluntary pelvic floor squeeze and for prostate enlargement in men. Symptom assessment tools such as the International Consultation on Incontinence Questionnaires [8] and the International Prostate Symptom Score are used to quantify symptoms and measure the associated quality of life

impairment. A frequency volume chart (FVC) is used to measure voided volumes over periods of a few days to evaluate maximum and typical voided volumes, day- and night-time urinary frequency, and overall output [9] (Fig. 47.2). Symptom tools and FVC are assessed before and after treatment to gauge response. Flow rate testing (Fig. 47.1) is used to assess outlet function and bladder contractility. A normal flow rate signifies good bladder contraction, satisfactory outlet channel, and good neurological coordination of the components. Ultrasound bladder scanning after flow rate testing can assess completeness of emptying. Filling and voiding cystometry, “urodynamics”, is an invasive test to measure bladder pressures during storage and voiding. A filling line is placed in the bladder so that fluid can be introduced. A separate line is used to measure pressure in the bladder. Since the bladder is an intra-abdominal organ, additional recording to correct for abdominal pressure changes is done, using a rectal or vaginal line. Detrusor contraction can be assumed if bladder pressure goes up in the absence of a rise in rectal pressure, when both lines are measuring accurately (Fig. 47.3). Additional tests may include cystoscopy which is a visual examination of the inside of the bladder and outlet. Radiological imaging can assess whether adjacent organs are influencing the bladder or whether there is any neurological abnormality.

CLINICAL CONDITIONS Incontinence Incontinence is the involuntary loss of urine. It arises when bladder pressure exceeds outlet resistance, which can occur if bladder pressure is abnormally high, outlet

FIGURE 47.1 Flow rate testing. Three successive flow rate tests on a woman with stress urinary incontinence (left) and a man with multiple sclerosis (right); both patients were aged 34. The left hand traces are basically normal, with a rapid rise, a high maximum flow rate and short duration; the importance of voided volumes is illustrated, as maximum flow rate is clearly lower for the small volume void at the top. On the right, maximum flow rate is clearly lower, and the flow is more protracted. The fluctuations in flow are because the patient used abdominal straining to try to improve flow, suggesting impaired bladder contractility – a well-recognized problem in multiple sclerosis.


CLInICAL COnDITIOnS

resistance is abnormally low, or from a combination of both. Detrusor overactivity (DO) results when spontaneous or provoked bladder contractions arise inappropriately during the storage phase (Fig. 47.3); if DO contractions are very strong, they can overcome outlet resistance, causing DO incontinence. DO (with or without

231

incontinence) is a subgroup of the overactive bladder syndrome (OAB), in which patients complain of excessive urinary urgency – the sudden compelling desire to pass urine which is difficult to defer [7]. OAB is defined by the presence of the urgency symptom, but DO can only be diagnosed by performing urodynamic testing.

FIGURE 47.2 Frequency volume charts. Two three-day frequency volume charts in the same patient, recorded before starting treatment and after one month starting treatment, which included an antimuscarinic drug. Time and volume (if measured) of each void is recorded, allowing the physician to evaluate maximum voided volume (in this case “functional bladder capacity” is 450 ml), typical voided volume and total voided volume per 24 hours. Additional parameters recorded in this case are an urgency score (1–5, with 5 being severe) and number of incontinence pads used. After treatment, daytime frequency, urgency score and number of pads used is lower. DNM, did not measure.

FIGURE 47.3 Urodynamic test. A filling and voiding cystometry trace. The patient was a woman with symptoms of urgency urinary incontinence. Illustrated are traces showing the bladder pressure (Pves; top trace) and abdominal pressure recorded from the rectum (Pabd; second trace). The third trace is the detrusor pressure (Pdet), which the urodynamic computer calculates by subtracting Pves from Pabd. The bottom trace shows urine flow, recorded by a flow rate meter placed under the commode on which the woman was seated. Time in minutes and seconds is shown at the very top. Between time 0 and 10:00, the bladder is being filled with saline at a rate of 30 ml/min (not illustrated); this is the filling phase. The first filling phase event is when the patient was asked to cough, at about 10 seconds; this caused a sharp spike in Pves and Pabd – coughs are repeated throughout, and since each event is associated with minimal deflection of Pdet, it serves as a useful check that pressures are being recorded successfully. Other fluctuations simultaneously present in Pabd and Pves (such as at 1:50) may be due to the patient speaking. At 5:00, the Pves rises, but Pabd does not, so Pdet also rises. This is detrusor overactivity (DO); since it also leads to urine flow, the diagnosis is DO incontinence. At 10:00, the patient reported that she had a strong desire to void. Filling was stopped and the patient was given permission to void, which is illustrated at 10:50. Another cough after the void is included to confirm that the process of voiding did not displace the bladder line, which would lead to erroneous pressure recording.


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Stress urinary incontinence (SUI) results from weakening of the bladder outlet. Physical stresses and straining increase abdominal pressure; this is transmitted to the bladder, and will cause leakage if outlet resistance is impaired. The most common etiology is following childbirth in women, as the pelvic floor is no longer able to support the sphincter as effectively. SUI can occur in men who have suffered loss of sphincter function as a complication of prostatectomy. Mixed urinary incontinence means that both DO incontinence and SUI are present. OAB is managed with fluid advice, bladder training and antimuscarinic medication [10]. Stress incontinence is managed by pelvic floor exercises; surgical measures may be needed to support the bladder outlet, such as placement of a tape at the midpoint of the urethra. Artificial urinary sphincter devices have been developed for SUI in men and neurological SUI.

Bladder Outlet Obstruction The prostate encircles the proximal urethra in men. It enlarges with age and encroaches on the urethral lumen, reducing the calibre of the outlet conduit. This gives rise to both voiding and post micturition symptoms, and is diagnosed according to the fact that detrusor pressures are high, and maximum flow rates during voiding reduced compared with normal [11]. Conservative, medical and surgical interventions are available according to degree of quality of life impairment and confounding risk factors for this older patient population.

Neurourology LUT dysfunction is highly prevalent in neurological disease because of the fundamental importance of several CNS centers in controlling LUT activity. As a consequence, various combinations of the following clinical LUT dysfunctions are possible: 1. DO incontinence: DO is common, as the CNS should inhibit generalised detrusor activity during the storage phase. In low spinal lesions, the adaptive detrusor relaxation underlying filling compliance can be impaired, so pressure rises at higher bladder volumes. 2. SUI: the CNS regulates outlet closure, so CNS disease can weaken sphincter and pelvic floor function. 3. Voiding dysfunction is common, including difficulty initiating voiding (hesitancy) and failure to maintain detrusor contraction until the bladder is emptied. An important subgroup is outlet obstruction caused by detrusor sphincter dyssynergia (DSD), which is simultaneous sphincter and detrusor contraction. 4. Reduced awareness of bladder filling or urine flow, as afferent traffic and conscious perception can be impaired at multiple points in the neuraxis. 5. Ejaculatory dysfunction results from impaired coordination of the sympathetic and parasympathetic spinal nuclei.

6. Autonomic hyperreflexia affects some high spinal cord injury patients. It is a life-threatening hypertension resulting from uninhibited activity of the spinal sympathetic nucleus, elicited by noxious stimuli in patients [12]. Neurourological management [13] primarily consists of ensuring renal function is protected and that the patient remains safe in the longer term. Factors that can impair renal function are poor bladder compliance and DSD, as increased bladder pressure hinders emptying of the ureters, raising pressure within the renal collecting systems. In the longer term this leads to renal failure and was the major cause of death in spinal cord injury up until recent years. In controlling symptoms, measures to improve urine storage include antimuscarinic medication and botulinum injections into the bladder to counteract DO, autologous sling placement or artificial urinary sphincter placement where the outlet function is impaired. To manage voiding dysfunction, intermittent self-catheterization or indwelling catheter drainage may be used.

Painful Bladder Syndrome A rare syndrome which can have a very severe impact on quality of life [14]. Bladder pain is reported during filling and can be slightly eased by voiding. As a consequence, affected patients report continuous pain and frequent trips to the toilet to pass urine, day and night. Bladder capacity is markedly impaired and in extreme cases the bladder may hold only 50 mL.

CONCLUSIONS Normal LUT function requires co-ordination of several structures to achieve the two contrasting roles of storage and voiding. Synergic function of the bladder and its outlet, the latter further complicated in males by the genital outflow, is achieved by complex regulatory mechanisms throughout the neuraxis. Consequently, clinical LUT problems are highly prevalent.

References [1] McCloskey KD. Interstitial cells in the urinary bladder – localization and function. Neurourol Urodyn 2010;29(1):82–7. [2] Birder LA. Urothelial signaling. Auton Neurosci 2010;153(1–2):33–40. [3] Drake MJ, Fowler CJ, Griffiths D, Mayer E, Paton JF, Birder L. Neural control of the lower urinary and gastrointestinal tracts: supraspinal CNS mechanisms. Neurourol Urodyn 2010;29(1):119–27. [4] Kanai A, Andersson KE. Bladder afferent signaling: recent findings. J Urol 2010;183(4):1288–95. [5] Wyndaele JJ, De Wachter S. Cystometrical sensory data from a normal population: comparison of two groups of young healthy volunteers examined with 5 years interval. Eur Urol 2002;42(1):34–8. [6] Drake MJ, Harvey IJ, Gillespie JI, Van Duyl WA. Localized contractions in the normal human bladder and in urinary urgency. BJU Int 2005;95(7):1002–5.


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COnCLUSIOnS

[7] Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U, et al. The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn 2002;21(2):167–78. [8] Abrams P, Avery K, Gardener N, Donovan J. The International Consultation on Incontinence Modular Questionnaire: www.iciq. net. J Urol 2006;175(3 Pt 1):1063–6. [discussion 66] [9] Bright E., Drake M.J., Abrams P. Urinary diaries: evidence for the development and validation of diary content, format, and duration. Neurourol Urodyn 2011; In press. [10] Abrams P, Andersson KE, Birder L, Brubaker L, Cardozo L, Chapple C, et al. Fourth International Consultation on Incontinence Recommendations of the International Scientific Committee:

[11]

[12]

[13]

[14]

Evaluation and treatment of urinary incontinence, pelvic organ prolapse, and fecal incontinence. Neurourol Urodyn 2010;29(1):213–40. Rosario DJ, Woo HH, Chapple CR. Definition of normality of pressure-flow parameters based on observations in asymptomatic men. Neurourol Urodyn 2008;27(5):388–94. Khastgir J, Drake MJ, Abrams P. Recognition and effective management of autonomic dysreflexia in spinal cord injuries. Expert Opin Pharmacother 2007;8(7):945–56. Stohrer M, Blok B, Castro-Diaz D, Chartier-Kastler E, Del Popolo G, Kramer G, et al. EAU guidelines on neurogenic lower urinary tract dysfunction. Eur Urol 2009;56(1):81–8. Hanno P, Nordling J, Fall M. Bladder pain syndrome. Med Clin North Am 2011;95(1):55–73.


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48 Physiology and Pathophysiology of Female Sexual Function Max J. Hilz In a 1999 survey, female sexual dysfunction (FSD) was prevalent in 43% of 1749 American women younger than 60 years [1]. In a British study sexual dysfunction was prevalent in 34.8% of men and 53.8% of women for prob lems lasting for at least one month, and in 6.2% of men and 15.6% of women for dysfunction lasting for at least 6 months during the previous year [2]. Common classifications are the International Classifi cation of Diseases (ICD)10 [3] and the Diagnostic and Statistical Manual of Mental Disorders (DSMIVTR) [4]. Both include four major FSD categories: disorders of desire, arousal, orgasm and pain. The Second Consensus of Sexual Medicine [5] revised FSD definitions as follows: (A) Hypoactive sexual desire/interest disorder: Absent or diminished feelings of sexual interest or desire, sexual thoughts or fantasies, and a lack of responsive desire; scarce or absent motivations for attempting to become sexually aroused, lack of interest is beyond normal decrease experienced with lifecycle and relationship duration. (B) Arousal disorders: Distress is caused by absence of arousal. Subtypes are subjective (diminished feelings of sexual arousal), genital (complaints of absent/ impaired genital sexual arousal, e.g. minimal vulvar swelling, reduced sexual sensations), combined genital and subjective arousal disorder, and persistent genital arousal disorder (with spontaneous, unwanted genital arousal without sexual interest). (C) Women’s orgasmic disorder: Despite sexual arousal no or delayed low intensity orgasm. (D) Dyspareunia: Pain with attempted or complete vaginal entry. (E) Vaginismus: Persistent or recurrent difficulties to allow vaginal entry despite expressed desire. (F) Sexual aversion disorder: Extreme anxiety/disgust at anticipation or attempt of sexual activity. Masters and Johnson described the sexual response cycle as a linear sequence of excitement, plateau, orgasm and resolution phases. Whipple and BrashMcGreer introduced a circular female sexual response pattern that implements positive or negative reinforcement of sexual


function, with pleasant and satisfying experiences during one sexual experience promoting pleasant further sexual experiences, or vice versa with negative experiences [6].

PHYSIOLOGY OF THE FEMALE SEXUAL RESPONSE CYCLE Sexual Arousal Sexual arousal causes increased pelvic blood flow, geni tal vasocongestion, vaginal dilatation and lengthening, engorgement of the labia minora and tissue surround ing the urethral lumen, clitoris and vestibular bulbs. Sites of genital sensory activation include the clitoris, clitoral sheath, anterior vaginal wall, labial and introital area, ure thra, Halban’s fascia between the anterior vaginal wall and bladder, and the controversially discussed Gspot, an anterior vaginal wall area along the urethra [6,7]. Afferent and Central Pathways Afferent arousal pathways include several sensory sys tems, e.g., somatosensory, auditory, vestibular and gusta tory stimuli reaching the nucleus tractus solitarii, as well as visual and olfactory stimuli. Stimuli from the pudendal, pelvic hypogastric, and genitofemoral nerves contribute to arousal. There is evidence of a direct vagus nerve innerva tion of the upper vagina and cervix [8] that bypasses the spinal cord, and thus allows women to experience arousal and orgasm, even after spinal cord injury above the spinal entry level of the genitospinal nerves [8]. Impulses reach ing the spinal cord ascend to the thalamus the somato sensory cortex [6,9]. Among the central areas involved in arousal and responses are the ventral medullary reticular formation that facilitates sexual excitation, the nucleus paragiganto cellularis in the ventral medulla that mediates descending inhibitory impulses suppressing the so called urethro genital reflex [9]. The periaqueductal grey, mesencephalic ventral tegmental area, neurons in the central tegmental mesencephalic region, and the medial amygdala acti vate the medial preoptic area (MPOA), that is involved

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in arousal [6]. The basomedial hypothalamus seems more important for female than male sexual behavior [6,9]. Neurotransmitters Among central neurotransmitters mediating arousal are dopamine (enhances sexual desire, arousal and willingness towards sexual engagement), norepineph rine (gates sensory input from genitalia and maintain ing sexual arousal), acetylcholine (mediates peripheral lubrication and vaginal engorgement), histamine, and serotonin (decreases hippocampal norepinephrine release and diminishes excitatory effects of norepinephrine and dopamine, [7] promotes arousal and orgasm in periph eral tissue, but also interferes with arousal and orgasm, as evidenced by anorgasmia often associated with intake of selective serotononin reuptake inhibitors [9]). Prolactin causes sexual satiety, reduces libido and gonadal func tion, offsets central dopamine effects after orgasm and correlates with postorgasmic sexual satiety and relief. Oxytocin increases during arousal and orgasm, promotes sexual receptivity, social bonding, care for offspring (“cud dling hormone”) [6,7].

Innervation of Female Sexual Function Descending motor impulses innervate rhabdosphinc ters and striated pelvic floor muscles and thus contrib ute to male and female sexual responses. Motor neurons

originate from the Onuf’s nucleus (S2–S4) that receives impulses from the primary motor cortex, ipsilateral para ventricular hypothalamus, ipsilateral caudal pontine lateral reticular formation, contralateral caudal nucleus retroambiguus, and other areas. Impulses, e.g., from the ventromedial nucleus of the hypothalamus (VMN) via the mesencephalon to pelvic floor motor neurons modulate motor sexual responses and, e.g., mediate female lordosis [9]. During orgasm, involuntary rhythmic pelvic floor con tractions assure male ejaculation while they squeeze the outer third of the vagina and the anal sphincter [6,7]. Central autonomic structures and peripheral autonomic innervation contribute essentially to female sexual func tion [6,9]. Sympathetic and parasympathetic pathways are similar in men and women and reach sexual organs via the inferior hypogastric, ovaric and uterovaginal plexus. The inferior hypogastric plexus receives sympathetic fibers from T10–L2, from the sympathetic trunk via sacral splanchnic nerves (from sacral sympathetic ganglion S2– S5), and parasympathetic fibers from sacral spinal nerves via the pelvic splanchnic nerves [6]. Pelvic splanchnic nerve fibers carry parasympathetic fibers from S2–S5 [6]. The inferior hypogastric plexus innervates bladder, rectum, cervix, vagina, urethra, vestibular bulbs and clitoris [6]. The pudendal nerve (S2–S4) innervates the genita lia and perineum with somatic sensory and motor fibers, parasympathetic fibers from the sacral spinal cord and sympathetic fibers from the sacral sympathetic trunk [6].

FIGURE 48.1 Autonomic and somatic innervation of female genitalia. (adapted from Rees et al., Lancet, 2007 with permission from the authors and Elsevier). Note: There is evidence that the pudendal nerve also contains sympathetic and parasympathetic fibers supplying the external genitalia and vagina [6].


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237

There is controversy regarding the functional contribu tion of parasympathetic and sympathetic innervation to female sexual responses [6]. There might be a dual auto nomic mediation of sexual arousal with cerebral sym pathetic activation during arousal being accompanied by spinal parasympathetic activation. Arousal induced tumescence of clitoris and vaginal bulbs is mediated by trabecular smooth muscle relaxation, similar to male erec tion [6]. Vaginal smooth muscle contraction very likely occurs only during late excitation, before orgasm, and is mediated via alphaadrenoreceptors [6]. Sympathetic activation dominates during late arousal and orgasm [6]. Knowledge about peripheral neu rotransmitters of female sexual response is still limited. Adrenergic, cholinergic, nonadrenergicnoncholinergic (NANC) neurotransmitters, vasoactive intestinal polypep tide (VIP), nitric oxide synthase, neuropeptide Y, calcito nin generelated peptide, substance P, pituitary adenylate cyclase activating polypeptide, helospectine and peptide histidine methionine have been identified in the female genital tract [6,7].

Common Etiologies of Female Sexual Dysfunction

Hormones Influencing Female Sexual Function

Neurogenic Etiologies

Progesterone seems to further receptivity to part ner approach. Estrogens are vasoprotective and support female sexual response by enhancing desire, arousal, nerve transmission, sensory thresholds, vaginal, clitoral and urethral arterial blood flow. Menopausal estrogen decrease leads to vaginal atrophy, inadequate vasoconges tion and lubrication, often causing painful intercourse and other FSDs with reduced genital sensation, sexual desire, activity and responsiveness, or anorgasmia. Testosterone is also relevant for desire and initiation of sexual activ ity. Decreased levels may also cause reduced wellbeing, libido, sexual receptivity, and pleasure, and induce fatigue and dysphoria, loss of pubic hair, and thinning of the vagi nal mucosa [6,7]. Correlations between patient complaints, FSD and age related androgen decrease are difficult to assess. Multiple factors may decrease sexual arousability and desire. Female testosterone replacement has gained interest but diagnosis of female androgen insufficiency requires care ful differential exclusion of psychosocial issues, psycho logical/psychiatric disorders, multiple medical conditions, effects of pharmacologic and recreational drugs, and is not proven by a single – difficult – assessment of low andro gen levels [6,7,10]. Transdermally applied testosterone improves sexual desire, responsiveness and activity, and bone mineral den sity in postmenopausal women. However, there are sig nificant concerns regarding increased cardiovascular risk and effects on breast cancer with testosterone supplementa tion. In postmenopausal women, it is nonphysiological to prescribe testosterone only, without concomitant estrogen therapy. Breast or uterine cancer, cardiovascular or liver dis eases are contraindications against testosterone therapy [10].

These include stroke, epilepsy, Parkinson’s disease, movement disorders, spinal cord injuries, multiple sclero sis, cauda equina syndrome, peripheral neuropathies, and surgical disruption of the genital autonomic nerve supply. Impairment of pelvic neurovascular and autonomic struc tures may compromise sexual function [6,7].

These include psychogenic and psychiatric disorders (e.g., emotional and relational problems, low selfesteem, body image, depression etc.), cardiovascular, atheroscle rotic, neurological or muscular problems (e.g., pelvic floor spasticity causing dyspareunia or vaginism), trauma (or chronic perineal pressure), endocrine problems includ ing menopause, hypothalamicpituitaryadrenal axis dys function, premature ovarian failure, medical or surgical castration, metabolic syndrome, diabetes, androgen defi ciency, hyperprolactinemia, hypo and hyperthyroidism, adrenal insufficiency, female hyperandrogenic disorders and estrogen deficiency. Pelvic, genital or general infec tious diseases may cause FSDs. Medications causing FSDs include antihypertensives, antidepressants, antacids, anti psychotics, anticholinergic substances, and oral contracep tives. Selective serotonin reuptake inhibitors (SSRIs) often inhibit libido and orgasm. SSRIrelated FSDs may improve with dosage reduction or switching, e.g., to bupropion or mirtazapine [6,7].

Assessment of Female Sexual Dysfunction Assessment includes a detailed history, physical, geni tal examination, neurological and neurophysiological examination. Pressure and touch sensitivity of the genita lia should be quantified using Semmes–Weinstein mono filaments. Mechanical pain thresholds can be measured by weighted pinprick stimulators. Quantitative sensory testing (QST) of vibratory thresholds evaluates afferent, thickly myelinated A betafibers, Paccinian corpuscles and Meissner bodies. QST assessment of cold, warm or heat– pain perception evaluates function of thinlymyelinated A deltafibers and unmyelinated Cfibers, i.e., small nerve fibers that mediate much of the sensation from erogenous zones [6]. Bulbocavernosus reflex testing or pudendal nerve somatosensory evoked potentials and tests such as puden dal nerve distal motor latencies or central motor conduc tion time to pelvic floor muscles are useful in suspected central, spinal or peripheral motor pathway lesions. Vaginal photoplethysmography assesses vaginal perfu sion. Transcutaneous partial oxygen pressure correlates with sexual arousal and orgasm and may be of diagnos tic use. Doppler ultrasound measures clitoral, labial, urethral, vaginal and uterine systolic peak blood flow velocity and enddiastolic velocity at rest and during


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sexual stimulation. Vaginal pH measurement determines lubrication as transsudate during arousal increases vaginal pH [6]. l

Laboratory Investigation Laboratory investigation may include microscopic and cultural examination of vaginal discharges and cervical Papanicolaou smear testing, as well as blood cell count, glu cose, thyroid and thyroidstimulating hormone levels [6].

COMMON THERAPEUTIC APPROACHES IN FEMALE SEXUAL DYSFUNCTION [6,7] l

l

l

l

l

Psychoeducation and open discussion are a first step towards therapy. Estrogen replacement may help in postmenopausal women with FSD complaints such as dyspareunia due to sore or atrophic vaginal tissue. Treatment of female sexual arousal disorder is difficult. Multiple formulations, mostly without documented safety or efficacy, have been suggested. Phosphodiesterase type 5 inhibitors seem to be ineffective in female arousal disorders. The FDA approved “EROSClitoral Therapy Device”, a battery operated device producing a gentle vacuum and lowlevel vibratory genital sensation seems to increase blood flow to clitoris, vagina and pelvis [11]. Treatment of orgasmic disorders is also difficult and requires sex therapy including cognitive behavioral therapy, sensate focus therapy, encouraging self stimulation, pelvic muscle exercises or biofeedback. Benefits may occur with the “EROSClitoral Therapy Device” [11]. Sexual pain treatment should address the underlying cause, e.g., vaginal entry pain due to vestibulitis, vaginal dryness or atrophy, or deep pain due to endometriosis or levator spasm. Physiotherapy, vaginal dilators, biofeedback have been helpful. Hypoactive sexual desire disorder is the most common FSD. Often androgen replacement therapy is initiated, yet the approach is controversial. So far, non pharmacologic treatment, psychoeducation, lifestyle

changes or stress management and psychological therapies remain the mainstay of managing female sexual interest and desire disorders. A novel treatment option was expected with the development of flibanserin, a 5HT1a receptor agonist, 5HT2A receptor antagonist and partial dopamin D4receptor agonist that was supposed to increase dopamine and norepinephrine and decrease serotonin in selective brain regions and thus facilitate sexual excitation [12]. However, the substance was not approved by the Food and Drug Administration.

References [1] Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the United States: Prevalence and predictors. Jama 1999;281:537–44. [2] Mercer CH, Fenton KA, Johnson AM, Copas AJ, Macdowall W, Erens B, et al. Who reports sexual function problems? Empirical evi dence from Britain’s 2000 national survey of sexual attitudes and lifestyles. Sex Transm Infect 2005;81:394–9. [3] World Health Organization (WHO). International statistical clas sification of diseases and related health problems 10th revision. Geneva: World Health Organization (WHO); 1994. [4] American Psychiatric Association. Diagnostic and statistical man ual of mental disorders. Washington, DC: American Psychiatric Association Press; 2000. [5] Basson R, Althof S, Davis S, FuglMeyer K, Goldstein I, Leiblum S, et al. Summary of the recommendations on sexual dysfunctions in women. J Sex Med 2004;1:24–34. [6] Hilz MJ. Female and male sexual dysfunction. In: Low PA, Benarroch EE, editors. Clinical autonomic disorders. Philadelphia: Lippincott Williams & Wilkins; 2008. p. 657–711. [7] Clayton AH, Hamilton DV. Female sexual dysfunction. Psychiatr Clin North Am 2010;33:323–38. [8] Komisaruk BR, Whipple B, Crawford A, Liu WC, Kalnin A, Mosier K. Brain activation during vaginocervical selfstimulation and orgasm in women with complete spinal cord injury: FMRI evi dence of mediation by the vagus nerves. Brain Res 2004;1024:77–88. [9] Schober JM, Pfaff D. The neurophysiology of sexual arousal. Best Pract Res Clin Endocrinol Metab 2007;21:445–61. [10] North American Menopause Society. The role of testosterone ther apy in postmenopausal women: Position statement of the North American Menopause Society. Menopause 2005;12:496–511. [11] Wilson SK, Delk 2nd JR, Billups KL. Treating symptoms of female sexual arousal disorder with the Erosclitoral therapy device. J Gend Specif Med 2001;4:54–8. [12] Clayton AH, Dennerstein L, Pyke R, Sand M. Flibanserin: A poten tial treatment for hypoactive sexual desire disorder in premeno pausal women. Women’s Health (Lond Engl) 2010;6:639–53.


C H A P T E R

49 Control of the Pupil Karen M. Joos, Mark R. Melson The iris is surrounded by aqueous humor. The actions of the sphincter and dilator muscles on the size of the pupil are not impeded by bulky tissue, are visible, and measureable. It is not surprising that 80 to 100 years ago, at the very beginning of autonomic pharmacology, the pupil was frequently used as an indicator of drug action. In those years it was shown that parasympathetic and sympathetic neural impulses to the iris muscles could be modified by drugs at the synapses and at the effector sites because it was at these locations that the transmission of the impulse depended on chemical mediators. In the following paragraphs these well-known, autonomically active drugs are grouped according to the site and mechanism of their action. Precaution should be taken about the interpretation of pupillary responses to topically instilled drugs. There are large interindividual differences in the responsiveness of the iris to typical drugs, and this becomes most evident when weak concentrations are used. For example, 0.25% pilocarpine will produce a minimal constriction in some patients and an intense miosis in others. This means that the most secure clinical judgments stem from comparisons with the action of the drug on the other, normal eye. The general status of the patient will also influence the size of the pupils. If the patient becomes uncomfortable or anxious while waiting for the drug to act, both pupils may dilate. If the patient becomes drowsy, both pupils will constrict. Thus, if a judgment is to be made about the dilation or contraction of the pupil in response to a drug placed in the conjunctival sac, one pupil should be used as a control whenever possible [1,2].

PARASYMPATHOLYTIC (ANTICHOLINERGIC) DRUGS The belladonna alkaloids occur naturally. They can be found in various proportions in deadly nightshade (Atropa belladonna), henbane (Hyoscyamus niger), and jimsonweed (Datura stramonium). Potions made from these plants were the tools of professional poisoners in ancient times. The word “belladonna” (“beautiful lady”) was derived from the cosmetic use of these substances as mydriatics in sixteenth-century Venice. The mischief caused by the ubiquitous jimsonweed is typical of this group of plants.


Jimsonweed has been used as a poison, has been taken as a hallucinogen, and has caused accidental illness and death, and it can cause an alarming accidental mydriasis. These solanaceous plants, which are related to the tomato, potato, and eggplant, are still cultivated for medical purposes. Atropine and scopolamine block parasympathetic activity by competing with acetylcholine at the effector cells of the iris sphincter and ciliary muscle, thus preventing depolarization. After conjunctival instillation of atropine (1%), mydriasis begins within about 10 minutes and is fully developed at 35 to 45 minutes; cycloplegia is complete within 1 hour. The pupil may stay dilated for several days, but accommodation usually returns in 48 hours. Scopolamine (0.2%) causes mydriasis that lasts, in an uninflamed eye, for about 2 days; it is less effective cycloplegic than atropine. Tropicamide and cyclopentolate (are synthetic parasympatholytics with a relatively short duration of action. Tropicamide (1%) is an effective, short-acting mydriatic (3 to 6 hours), which results in only a very transient paresis of accommodation. Compared with tropicamide, cyclopentolate (1%) seems to be a more effective cycloplegic and a slightly less effective mydriatic, especially in dark eyes; accommodation takes about half a day to return and the pupil still may not be working perfectly after more than 24 hours. Botulinum toxin blocks the release of acetylcholine, and hemicholinium interferes with the synthesis of acetylcholine both at the preganglionic and at the postganglionic nerve endings, thus interrupting the parasympathetic pathway in two places. The outflow of sympathetic impulses is also interrupted by systemic doses of these drugs, since the chemical mediator in sympathetic ganglia is also acetylcholine.

PARASYMPATHOMIMETIC (CHOLINERGIC) DRUGS Pilocarpine and methacholine are structurally similar to acetylcholine and are capable of depolarizing the effector cell, thus causing miosis and spasm of accommodation. Methacholine is still sometimes used in a weak (2.5%) solution to test for cholinergic supersensitivity of the

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sphincter muscle in autonomic failure. It has been generally replaced by weak pilocarpine (0.1%). Arecoline is a naturally occurring substance with an action similar to that of pilocarpine and methacholine; its chief advantage is that it acts quickly; a 1% solution produces a full miosis in 10 to 15 minutes (compared to 20 to 30 minutes for 1% pilocarpine) [3]. Carbachol acts chiefly at the postganglionic cholinergic nerve ending to release the stores of acetylcholine. There is also some direct action of carbachol on the effector cell. A 1.5% solution causes intense miosis, but the drug does not penetrate the cornea easily and is therefore usually mixed with a wetting agent (1:3500 benzalkonium chloride). Acetylcholine is liberated at the cholinergic nerve endings by the neural action potential and is promptly hydrolyzed and inactivated by cholinesterase. Cholinesterase, in turn, can be inactivated by any one of the many anticholinesterase drugs. These drugs either block the action of cholinesterase or deplete the stores of the enzyme in the tissue. They do not act on the effector cell directly, they just potentiate the action of the chemical mediator by preventing its destruction by cholinesterase. It follows from their mode of action that these drugs will lose their cholinergic activity once the innervation has been completely destroyed. Physostigmine (eserine) is the classic anticholinesterase. Along the Calabar coast of West Africa the native tribes once conducted trials “by ordeal” using a poison prepared from the bean of the plant Physostigma venenosum. The local name for this big bean was the “esere nut”. The organic phosphate esters (echothiophate [phospholine], isoflurophate [diisopropyl fluorophosphates – DFP], tetraethyl pyrophosphate, hexaethyltetraphosphate, parathion), many of which are in widespread use as insecticides, cause a much longer lasting miosis than the other anticholinesterases, but even this potent effect, thought to be due to interference with cholinesterase synthesis, can be reversed by pralidoxime chloride (P-2-AM).

SYMPATHOMIMETIC (ADRENERGIC) DRUGS Epinephrine stimulates the receptor sites of the dilator muscle cells directly. When applied to the conjunctiva, the 1:1000 solution does not penetrate into the normal eye in sufficient quantity to have an obvious mydriatic effect. If, however, the receptors have been made supersensitive by previous denervation, or if the corneal epithelium has been damaged, allowing more of the drug to get into the eye, then this concentration of epinephrine will dilate the pupil. Phenylephrine in the 10% solution has a powerful mydriatic effect. Its action is almost exclusively a direct alpha stimulation of the effector cell. The pupil recovers in 8 hours and shows a “rebound miosis” lasting several days. A 2.5% solution is now commonly used for mydriasis. Ephedrine acts chiefly by releasing endogenous

norepinephrine from the nerve ending, but it also has a definite direct stimulation effect on the dilator cells. Tyramine (5%) and hydroxyamphetamine (1%) have an indirect adrenergic action; they release norepinephrine from the stores in the postganglionic nerve endings; as far as is known this is their only effective mechanism of action. Cocaine (5% to 10%) is applied to the conjunctiva as a topical anesthetic, a mydriatic, and a test for Horner syndrome. Its mydriatic effect is the result of an accumulation of norepinephrine at the receptor sites of the dilator cells. The transmitter substance builds up at the neuroeffector junction because cocaine prevents the reuptake of the norepinephrine back into the cytoplasm of the nerve ending. Cocaine itself has no direct action on the effector cell nor does it serve to release norepinephrine from the nerve ending, and it does not retard the physiologic release of norepinephrine form the stores in the nerve ending. Its action is indirect, it interferes with the mechanism for prompt disposition of the chemical mediator, and in this respect its action is analogous to that of the anticholinesterases at the cholinergic junction. If the nerve action potentials along the sympathetic pathway are interrupted, as in Horner syndrome, the transmitter substance will not accumulate and the pupil will not dilate. The duration of cocaine mydriasis is quite variable; it may last more than 4 hours. It does not show “rebound miosis.” Apraclonidine (0.5%) is a relatively selective alpha2adrenergic agonist which is used topically to lower intraocular pressure. It will also dilate the pupil in Horner syndrome [4] including pupillary sympathetic denervation in diabetes mellitus [5]. This drug is easily obtainable and is a positive test for sympathetic denervation. Less than 0.5 mm dilation occurs in normal eyes.

SYMPATHOLYTIC DRUGS (ADRENERGIC BLOCKERS) Thymoxamine HCl (0.5%) and dapiprazole are alphaadrenergic blockers that will reverse phenylephrine mydriasis by taking over the alpha-receptor sites on the iris dilator muscle.

OTHER AGENTS l

l

l

l

Substance P affects the sphincter fibers directly, and will constrict the pupil of a completely atropinized eye. The chief pupillary action of morphine is to cut off cortical inhibition of the iris sphincter nucleus in the midbrain, with resultant miosis. Topical morphine, however, even in strong solutions (5%), has a minimal miotic effect on the pupil. Nalorphine and levallorphan are antinarcotic drugs that, given parenterally, reverse the miotic action of morphine. Intravenous heroin seems to produce miosis in proportion to its euphoric effect. In a habituated


PuPIl SymPATHETIC DEfECTS

l

l

heroin user, the same dose of the drug seems to produce less pupillary constriction than in a naïve subject. Thus, given the plasma drug concentration and the size of the pupil in darkness it should be possible to come up with a measure of the degree of physical dependence in a given individual. During the induction of anesthesia the patient may be in an excited state and the pupils are often dilated. As the anesthesia deepens, supranuclear inhibition of the sphincter nuclei is cut off and the pupils become small. If the anesthesia becomes dangerously deep and begins to shut down the midbrain, the pupils become dilated and fail to react to light. The concentration of calcium and magnesium ions in the blood may affect the pupil. Calcium facilitates the release of acetylcholine, and when calcium levels are abnormally low, the amount of acetylcholine liberated by each nerve impulse drops below the level needed to produce a postsynaptic potential, thus effectively blocking synaptic transmission and causing dilation. Magnesium has an opposite effect: a high concentration of magnesium can block transmission and this may dilate the pupil.

241

IRIS PIGMENT AND PUPILLARY RESPONSE TO DRUGS In general, the more pigment in the iris, the more slowly the drug takes effect and the longer its action lingers. This is probably due to the drug being bound to iris melanin and then slowly released. It should be noted that there are wide individual differences in pupillary responses to topical drugs. There is probably a greater range of responses among blue eyes than there is between the average response of blue eyes and the average response of dark brown eyes. Some of these individual differences are due to corneal penetration of the drug [6]. Intense exercise will significantly increase pupil diameter [7]. Melanopsincontaining intrinsically photosensitive retinal ganglion cells also influence pupil diameter besides circadian clock synchronization, sleep, and pineal melatonin production [8].

PUPIL SYMPATHETIC DEFECTS A defect in the sympathetic pathway (Fig. 49.1) affects the pupillary dilator muscle and results in a Horner

FIGURE 49.1 The innervation of the iris muscles, showing the pathways and the terminology in general use. Note that an alerting stimulus dilates the pupil in two ways – both of them with a noradrenergic step in the pathway. The alerting stimulus inhibits the iris sphincter nucleus and, at the same time sends a message down to the cervical cord and the out along the cervical sympathetic pathway. This arrives at the iris about half a second after the sphincter-relaxing message and causes the radial dilator muscle to tighten, thus widening the pupil. III. AUTONOMIC PHYSIOLOGY

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syndrome. If unilateral, asymmetry of pupil diameter is more obvious in darkness than in light. Disorders causing a Horner syndrome include: (1) a distal third-order neuron lesion such as internal carotid dissection, surgery around the carotid artery, or tumor extension into the cavernous sinus; (2) a second-order neuron lesion such as apical lung tumor, chest surgery, thoracic aortic aneurysms, pediatric neuroblastoma, or brachial plexus injury; (3) a central firstorder neuron lesion such as vascular occlusion near the lateral medulla, or tumors or disc disease near the upper cervical spinal cord. Bilateral defects have been reported in diabetes mellitus [5], pure autonomic failure [9], and dopamine beta-hydroxylase deficiency [10].

PUPIL PARASYMPATHETIC DEFECTS A defect in the parasympathetic pathway (Fig. 49.1) affects the pupillary sphincter muscle and results in a larger pupil. If unilateral, asymmetry of pupil diameter is more obvious in the light than in darkness. Disorders of postganglionic parasympathetic defects include lesions of the ciliary ganglion or short posterior ciliary nerve causing a tonic Adie pupil [11]. Bilateral defects have been reported in Miller Fisher syndrome [9], acute pandysautonomia [9], paraneoplastic autonomic neuropathy [9], Sjögren syndrome [9,10], and rarely systemic lupus erythematosus [9]. Pupil involvement accompanied by a third cranial nerve palsy requires investigation for an aneurysm at the junction of the internal carotid and posterior communicating arteries.

PUPIL COMBINED SYMPATHETIC AND PARASYMPATHETIC DEFECTS

in Guillain–Barre syndrome [9], Lambert–Eaton myasthenic syndrome [9], diabetes mellitus [9], acute and subacute dysautonomia [10], and amyloidosis [10].

Acknowledgements This chapter was updated with permission by the author in previous editions: H. Stanley Thompson, M.D., Oxford, Iowa. Support: NIH Core Grant 2P30EY008126-22 and an unrestricted departmental grant from Research to Prevent Blindness, Inc., NY.

References [1] Loewenfeld IE. The pupil: anatomy, physiology and clinical applications. Ames, IA: Iowa State University Press; 1993. pp 797-826 and 1255-1558. (Reprinted by Butterworth-Heinemann in 1997) [2] Thompson HS. The Pupil. In: Hart WmM, editor. Alder’s physiology of the eye (9th ed.). St. Louis: Mosby-Year Book; 1992. p. 429. [3] Babikian PV, Thompson HS. Arecoline miosis [Letter]. Am J Ophthalmol 1984;98:514–5. [4] Brown SM, Aouchiche R, Freedman KA. The utility of 0.5% apraclonidine in the diagnosis of Horner syndrome. Arch Ophthalmol 2003;121:1201–3. [5] Koc F, Kansu T, Kavuncu S, Firat E. Topical apraclonidine testing discloses papillary sympathetic denervation in diabetic patients. J Neuro-Ophthalmol 2006;26:25–9. [6] Kardon R. Drop the Alzheimer’s drop test. [Editorial]. Neurology 1998;50:588–91. [7] Hayashi N, Someya N, Fukuba Y. Effect of intensity of dynamic exercise on pupil diameter in humans. J Physiol Anthropol 2010;29:119–22. [8] Bailes HJ, Lucas RJ. Melanopsin and inner retinal photoreception. Cell Mol Life Sci 2010;67:99–111. [9] Toth C, Fletcher WA. Autonomic disorders and the eye. [Editorial]. J Neuro-Ophthalmol 2005;25:1–4. [10] Bremner FD, Smith SE. Pupil abnormalities in selected autonomic neuropathies. J Neuro-Ophthalmol 2006;26:209–19. [11] Bremmer F. Pupil evaluation as a test for autonomic disorders. Clin Auton Res 2009;19:88–101.

Defects in both pathways (Fig. 49.1) affect the pupillary dilator and sphincter muscles. Disorders of the sympathetic and parasympathetic pathways have been reported


C H A P T E R

50 Central Thermoregulation Shaun F. Morrison Central neural circuits orchestrate a homeostatic repertoire to maintain body temperature during environmental temperature challenges (Fig. 50.1) and to alter body temperature during the inflammatory response. Body temperature regulation is effected primarily through dedicated pathways in the brain which function to produce an optimal operating temperature for neurons and for the many tissues on which the brain depends for survival. The principal non-behavioral effector mechanisms for cold defense, recruited in order of increasing energy costs, include heat conservation resulting from cutaneous vasoconstriction (CVC) and piloerection and heat production from thermogenesis, a byproduct of the inefficiency of mitochondrial ATP production and of ATP utilization, in brown adipose tissue (BAT), the heart and skeletal muscle (shivering). Heat loss mechanisms for heat defense include cutaneous vasodilation and evaporative cooling. The activation of these effectors is regulated by parallel but distinct, effector-specific, core efferent pathways within the central nervous system that share a common peripheral thermal sensory input. A wide variety of non-thermal physiological parameters, disease processes, neurochemicals and drugs can influence the central regulation of body temperature and their effects are hypothesized to result from an alteration of the activity within the core neural circuit for thermoregulation. The core central thermoregulatory network (Fig. 50.2) comprises the fundamental pathways through which cutaneous cold and warm sensation and/or reductions or elevations in brain temperature elicit changes in thermoregulatory effector tissues to counter or protect against deviations from a homeostatic temperature of the brain and other critical organ tissues.

CUTANEOUS THERMAL RECEPTOR AFFERENT PATHWAY The central thermoregulatory system receives signals related to changes in environmental temperature through thermoreceptors in primary sensory nerve endings distributed in the skin. Members of the transient receptor potential (TRP) family of cation channels comprise the molecular mechanisms of cutaneous cool and warm thermoreception [1]. Primary thermal somatosensory afferents


synapse on thermoreceptive-specific, lamina I spinal (or trigeminal) dorsal horn cells that respond linearly to graded, innocuous cooling or warming stimuli, but are not activated further in the noxious temperature range. In turn, spinal and trigeminal lamina I neurons collateralize and innervate the thalamus and the pontine lateral parabrachial nucleus (LPB) (Fig. 50.2). Densely clustered neurons that project principally to the midline subregion of the POA, including the median preoptic nucleus (MnPO), from the dorsal (LPBd) and external lateral (LPBel) subnuclei of the LPB are activated following warm or cold exposure, respectively, in parallel with respective skin warming-evoked inhibitions and skin cooling-evoked activations of BAT sympathetic nerve activity (SNA) and BAT thermogenesis [2,3]. Activation of LPBd or LPBel neurons evokes respective decreases or increases in BAT thermogenesis, metabolism and heart rate (HR) that mimic skin warming-evoked or skin cooling-evoked physiological response. Either inhibition of LPBel neurons or blockade of their glutamate receptors eliminates skin cooling-evoked cold-defense responses, including the activation of BAT and shivering thermogenesis and increases in metabolism and in HR [2]. Similar inhibition of LPBd neurons eliminates skin warming-evoked heat-defense responses, including the inhibition of cutaneous vasoconstrictor SNA (mediating cutaneous vasodilation) [3]. Thus, activations of LPBd and LPBel neurons, likely by glutamatergic inputs from lamina I neurons, driven respectively by cutaneous warming and cooling signals, transmit the respective warm and cold cutaneous thermal afferent stimuli that initiate heat defense and cold defense responses to defend body temperature during environmental thermal challenges (Figs 50.1, 50.2). The spinoparabrachiopreoptic thermal afferent pathway that triggers involuntary thermoregulatory responses is distinct from the spinothalamocortical pathway, in which lamina I neurons synapse on neurons in the thalamus that project to the primary somatosensory cortex, which leads to perception and discrimination of cutaneous temperature [2] (Fig. 50.2). The relative contributions of the spinothalamic vs. spinoparabrachial pathways in initiating thermoregulatory behaviors, the stereotypical somatic motor acts directed toward minimizing or optimizing heat transfer from the body to the environment, remain to be elucidated.

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50. CEnTRAl THERmoREgulATIon

THERMOREGULATORY SENSORIMOTOR INTEGRATION IN THE PREOPTIC AREA

FIGURE 50.1 Modulation of thermal effectors by activation of cutaneous thermal receptors. (A) Reductions in skin temperature (Tskin) elicit increases in brown adipose tissue (BAT) sympathetic nerve activity (SNA), in BAT thermogenesis and BAT temperature (Tbat), in expired CO2, an indicator of increased metabolism, in heart rate (HR), but little change in arterial pressure (AP). (B) With lowered core and brain temperatures (Tbrain), reductions in skin temperature elicit shivering, indicated by marked increases in neck muscle EMG. Note the simultaneous increase in BAT temperature and thermogenesis. (C) Increases in skin temperature inhibit the cutaneous vasoconstrictor (CVC) sympathetic outflow monitored as the action potentials of a single postganglionic fiber (unit) in the sural nerve. Note also the increase in tail skin temperature (Ttail), an indicator of cutaneous vasodilation promoting heat loss.

Glutamatergic stimulation of MnPO neurons, rather than those in medial (MPO) or lateral preoptic areas, evokes thermogenic, metabolic and tachycardic responses similar to those evoked during cold-defense [4]. Colddefense responses triggered either by LPBel stimulation or by skin cooling are blocked by antagonizing glutamate receptors in the MnPO. Similarly, skin warming signaling, mediated via LPBd neurons, is transmitted preferentially to neurons in MnPO and in the rostral dorsomedial portions of MPO and blockade of glutamate receptors in this region of the POA interrupts skin warming-evoked responses [3]. Thus, activation of MnPO neurons mediates cold- or warm-defensive responses to environmental cooling or warming challenges (Figs 50.1, 50.2). Transection of the neural pathways immediately caudal to the POA or reducing the activity of neurons in the MPO produces hyperthermia by stimulating BAT thermogenesis, metabolism, shivering and CVC [1]. Local warming of the POA inhibits CVC and eliminates shivering, whereas cooling of the local environment of POA neurons evokes BAT and shivering thermogenesis. These findings are indicative of a tonically-active, local-warming-mediated mechanism in the POA capable of driving a potent inhibition of cold-defense effectors responsible for heat conservation and thermogenesis (Fig. 50.2). The neuronal substrate for these effects likely resides in GABAergic, warm-responsive neurons [5,6] in the POA whose tonic discharge is reduced by skin cooling. Whether warm-sensitive POA neurons project axons outside of the POA remains to be demonstrated. BAT and shivering thermogenesis, as well as increases in metabolism and HR that are evoked by skin cooling are blocked by antagonizing GABAA receptors in the MPO [7], suggesting that cutaneous cool signals received by MnPO neurons drive a GABAergic inhibition of inhibitory warmsensitive, MPO projection neurons [4] (Fig. 50.2). Thus, warm-sensitive, GABAergic POA projection neurons integrate cutaneous and local thermal information and are tonically active at thermoneutral temperatures to suppress, to varying degrees, shivering and non-shivering thermogenesis and cutaneous vasoconstriction (Fig. 50.2). Different populations of warm-sensitive, GABAergic POA projection neurons, whose firing rates contribute significantly to the balance of thermoregulatory effector activation that determines core body temperature, are expected to control the activation of different thermal effectors, thereby providing the substrate for the graded thermal thresholds for the cold defense activation of different thermal effectors. Further, the binding of prostaglandin E2 (PGE2), an intermediary in the fever cascade, to inhibitory EP3 receptors on POA inhibitory neurons that project to the dorsomedial hypothalamus (DMH) or to the rostral raphe pallidus (rRPa) region (Fig. 50.2) provides a substrate for the disinhibitory activation of cold defense effectors during fever [1].


THERmoREgulAToRy EffECToR DRIvE fRom THE DoRsomEDIAl HyPoTHAlAmus

245

FIGURE 50.2 Functional neuroanatomical and neurotransmitter model for the core pathways providing the thermoregulatory control and pyrogenic activation of cutaneous vasoconstriction (CVC), brown adipose tissue (BAT) and shivering thermogenesis. Cool cutaneous thermal signals or prostaglandin (PG) E2 stimulate CVC-mediated heat retention and BAT and shivering thermogenesis. Warm cutaneous thermal signals produce inhibitions of BAT and shivering thermogenesis and of CVC sympathetic outflow, the latter allowing cutaneous vasodilation and thereby promoting heat loss. DRG, dorsal root ganglia; DH, dorsal horn; GLU, glutamate; LPBel, external lateral subnucleus of the lateral parabrachial nucleus; LPBd, dorsal subnucleus of the LPB; POA, preoptic area; MnPO, median preoptic; W-S, warm-sensitive; MPO, medial preoptic; rRPa, rostral raphe pallidus; IML, intermediolateral nucleus; 5-HT, serotonin; DMH, dorsomedial hypothalamus; α, alpha motoneuron; γ, gamma motoneuron.

THERMOREGULATORY EFFECTOR DRIVE FROM THE DORSOMEDIAL HYPOTHALAMUS The dorsal portion of the rostral DMH and the dorsal hypothalamic area contain neurons mediating the BAT thermogenic and HR responses to skin cooling and to injection of PGE2 into the POA [1,7,8]. Activation or

disinhibition of neurons in this region of the DMH elicits potent increases in BAT thermogenesis, HR, and metabolism [1]. In contrast, although activation of DMH neurons can increase CVC sympathetic outflow, the activity of DMH neurons is not required for the cutaneous vasoconstriction stimulated by cooling or by injection of PGE2 into the MPO [8]. Thus, skin cooling- and febrile-evoked BAT and cardiac sympathoexcitatory and somatic shivering


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50. CEnTRAl THERmoREgulATIon

excitatory signals are respectively transmitted to BAT and cardiac sympathetic and somatic shivering premotor neurons in the rRPa from those DMH neurons that are disinhibited following cold cutaneous or pyrogenic stimuli in the POA (Fig. 50.2). In contrast, the parallel activations of CVC sympathetic outflow are mediated by POA projection neurons that bypass the DMH. Although many POA neurons branch to innervate both the DMH and the rRPa regions, almost none of these express the EP3 receptor, suggesting that at least the febrile activations of BAT and shivering thermogenesis and of CVC are activated by relief of a tonic inhibition from separate populations of POA neurons (Fig. 50.2).

ROSTRAL RAPHE PALLIDUS AREA CONTAINS PREMOTOR NEURONS FOR THERMOREGULATORY EFFECTORS The rRPa is a prominent site of neurons that multi-synaptically innervate BAT, the heart, skeletal muscle fibers and cutaneous blood vessels. Activation or disinhibition of neurons in the rRPa elicits pronounced increases in BAT SNA, BAT thermogenesis, shivering, HR and CVC [9]. Blockade of neuronal activity in the rRPa inhibits and prevents skin-cooling and febrile stimulations of BAT and shivering thermogenesis, of HR and of CVC heat retention [7]. Thus, neurons in the rRPa and the immediately surrounding rostral ventromedial medulla play a key role as sympathetic and somatic premotor neurons controlling BAT and shivering thermogenesis and CVC, providing essential excitatory drives to activate spinal motor networks during cold defense and fever (Fig. 50.2).

SPINAL SYMPATHETIC MECHANISMS CONTROLLING THERMAL EFFECTORS The discharges of BAT, cardiac and CVC sympathetic preganglionic neurons and those of alpha and gamma motoneurons that determine the levels and the rhythmic bursting characteristics of BAT, cardiac and CVC SNAs and of skeletal muscle shivering and, in turn, BAT, cardiac and shivering thermogenesis and cutaneous heat loss, are governed primarily by their supraspinal inputs, but also by the excitability of the networks of spinal interneurons (Fig. 50.2) that influence the discharge of the spinal motor neurons for these thermal effectors. A significant fraction of the BAT, cardiac and CVC sympathetic and the somatic shivering premotor neurons in the rRPa are glutamatergic and/or serotonergic neurons, giving rise to at least a portion of the 5-hydroxytryptamine (5-HT)-containing and VGLUT3-containing terminals in the IML. Spinal glutamate and serotonin receptors in the intermediolateral nucleus (IML) play significant roles in mediating the activation of BAT thermogenesis [10] and CVC, including the cold-evoked and rRPa stimulus-evoked increases in BAT

thermogenesis and CVC. Different synaptic mechanisms and postsynaptic targets are engaged in the spinal 5-HT regulation of different thermal effectors (Fig. 50.2).

SUMMARY The activity of thermal effectors is strongly influenced by shared cutaneous thermal afferent signals in a pathway that includes synapses in the spinal dorsal horn leading to glutamatergic activation of neurons in the LPB, where cool and warm afferent signals are processed by anatomically distinct, POA projecting neurons that influence, in turn, the discharge of different, effector-specific populations of warm-sensitive, GABAergic POA projection neurons, that provide for the integration of local temperature and of peripheral thermal sensory signals. The core efferent pathway for thermoregulatory activation of BAT and shivering thermogenesis and HR involves a tonicallyactive inhibitory input from the POA to sympathoexcitatory neurons in the DMH, which project to sympathetic premotor neurons in the rRPa, which, in turn, provide the excitatory drive to spinal sympathetic preganglionic and somatic motor neurons that is transmitted to brown adipocytes, cardiac pacemaker cells and somatic muscle cells. The core efferent pathway for CVC also involves a tonically-active inhibition emanating from the POA. However, these POA projection neurons send axons to the rRPa where they influence the discharge of CVC sympathetic premotor neurons and, consequently, the level of excitation to CVC sympathetic preganglionic neurons to elicit cutaneous vasoconstriction. Identifying key structures and neurochemical mechanisms within each of these core thermoregulatory pathways provides a framework for understanding how body temperature regulation is influenced by a wide variety of neurotransmitters, peptides, cytokines, and genetic, nutritional and perinatal manipulations, as well as how thermoregulation is integrated with other homeostatic systems regulating oxygen and fuel substrate availability, body water, salt appetite and energy balance.

References [1] Morrison SF, Nakamura K, Madden CJ. Central control of thermogenesis in mammals. Exp Physiol 2008;93:773–97. [2] Nakamura K, Morrison SF. A thermosensory pathway that controls body temperature. Nat Neurosci 2008;11:62–71. [3] Nakamura K, Morrison SF. A thermosensory pathway mediating heat-defense responses. Proc Natl Acad Sci USA 2010;107:8848–53. [4] Nakamura K, Morrison SF. Preoptic mechanism for cold-defensive responses to skin cooling. J Physiol 2008;586:2611–20. [5] Lundius EG, Sanchez-Alavez M, Ghochani Y, Klaus J, Tabarean IV. Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons. J Neurosci 2010;30:4369–81. [6] Griffin JD, Kaple ML, Chow AR, Boulant JA. Cellular mechanisms for neuronal thermosensitivity in the rat hypothalamus. J Physiol 1996;492(Pt 1):231–42.


summARy

[7] Nakamura K, Morrison SF. Central efferent pathways mediating skin cooling-evoked sympathetic thermogenesis in brown adipose tissue. Am J Physiol 2007;292:R127–36. [8] Rathner JA, Madden CJ, Morrison SF. Central pathway for spontaneous and prostaglandin E2-evoked cutaneous vasoconstriction. Am J Physiol 2008;295:R343–354.

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[9] Morrison SF, Sved AF, Passerin AM. GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue. Am J Physiol 1999;276:R290–7. [10] Madden CJ, Morrison SF. Serotonin potentiates sympathetic responses evoked by spinal NMDA. J Physiol 2006;577:525–37.


C H A P T E R

51 Sweating Phillip A. Low INTRODUCTION

Density and Distribution

There are two types of human sweat glands, apocrine and eccrine. The full complement of eccrine sweat glands is present at birth and undergoes a gradual reduction in numbers with increasing age. It is primarily innervated by sympathetic cholinergic nerve fibers. There is much complexity of the sweat gland. It is adrenergic in utero and undergoes an adrenergic to cholinergic innervational switch during development. Apart from cholinergic and adrenergic innervation of M3 receptors on sweat glands, human eccrine sweat glands are also innervated by VIP, CGRP, and SP fibers. The density of human sweat glands varies greatly, being most dense around the palms. The primary role of sweat glands is thermoregulation. With repeated stimulation, sweat glands undergo hypertrophy. Males have the same number of glands as women, but the volume of each gland is several-fold larger. With denervation, preganglionic or postganglionic, sweat gland size and function become greatly reduced and undergo significant atrophy. Tran-synaptic degeneration has also been suggested. Regulation of sweating in humans serves an important role in thermoregulation (Chapter 50) and loss of the ability to sweat can result in thermoregulatory failure, including heat stroke. The focus of this chapter will be on the anatomy, function, and innervation of the sweat gland.

ANATOMY AND FUNCTION OF THE SWEAT GLAND

Eccrine sweat glands are of greater neuroscience interest and the rest of the description will focus on eccrine sweat glands. They each weigh between 30–40 μg [1]. They first appear in the 3½-month-old fetus in the volar surface of the hands and feet. Eccrine glands show area differences with the greatest density in the palms and soles. They vary in density from 400/cm2 on the palm to about 80/cm2 on the thigh and upper arm. The total numbers are approximately 2 to 5 million. Males and females have the same number of sweat glands. However, the size and volume secreted by each gland is about five times greater in males [2]. Surrounding the secretory cells are myoepithelial cells whose contraction is thought to aid the expulsion of sweat. These glands receive a rich supply of blood vessels and sympathetic nerve fibers but are unusual in that sympathetic innervation is largely cholinergic. The full complement of eccrine glands develops in the embryonic state [3]. No new glands develop after birth.

Physiology of Sweat Glands The physiology of human sweat response is known from the detailed in vitro studies of Sato [4]. Acetylcholine secretion results in the production of an ultrafiltrate (isotonic) by the secretory coil. Directly collected sweat

TABLE 51.1 Comparison of Eccrine with Apocrine Sweat Gland

Type

Parameter

Eccrine

Apocrine

There are two types of sweat glands, eccrine and apocrine. The eccrine sweat glands are simple tubular glands that extend down from the epidermis to the lower dermis. The lower portion is a tightly coiled secretory apparatus consisting of two types of cells. The apocrine gland is a dark basophilic cell that secretes mucous material, and the eccrine sweat gland is a light acidophilic cell that is responsible for the passage of water and electrolytes. Differences between the two types of glands are described in Table 51.1. Apocrine sweat glands are found in the axilla, the anogenital zone, the areola of the nipple, and the external auditory meatus.

Size Duct Ductal opening

Relatively small Long and thin Skin surface (near hair)

Secretory coil

Small ext diameter very narrow lumen Secretory (clear); dark myoepithelial Present

Large Short and thick Directly into upper follicular canal Large ext diameter wide lumen Columnar secretory; myoepithelial Absent

Present at birth Chol  β-adr  α-adr Continuous high Serous

Present at birth Chol  β-adr Intermittent variable Milky protein-rich


Cell type Intercellular cannaliculi Development Pharmacology Sweat secret rate Secretory product

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51. SwEATInG

samples yield Na and K values identical with plasma. Reabsorption of sodium ions by the eccrine sweat duct results in hypotonic sweat, confirmed in directly collected sweat samples from proximal duct (Na 20–80 mM; K 5–25 mM; [1]). Extracellular Ca2 is important since removal of periglandular Ca2 with EGTA completely inhibits sweat secretion, while Ca ionophore A23187 strongly and persistently stimulates sweating [1]. Magnesium ions appear to be unimportant.

INNERVATION OF SWEAT GLAND

Parameter

Comments

Mental stress

Greatest effect on palmar, sole, axillary sites

Exercise

precedes rise in core temperature

Rehydration

Lowers core temperature

Male sex

Greater sweat gland volume

Race

Blacks  whites but difference quite small

Acclimatization

Increased gain (sweating/temperature change); reduced sweat sodium content

Circadian rhythm

Higher in PM

Seasonal variation

Greater response in winter

Innervation is mainly by sympathetic postganglionic cholinergic fibers. In isolated human eccrine sweat gland regulation, the regulation of sweating is cholinergic and muscarinic, being completely inhibited by atropine [1]. Sudomotor function is metabolically active. It is inhibited by cold, involves active transport, and inhibited by metabolic inhibitors. Microtubules may be important since vinblastine strongly but reversibly inhibits sweating. Endogenous cyclic AMP appears to be the second messenger, since theophylline by phosphodiesterase inhibition markedly increases the sweat response. The muscarinic receptor subtype is M3 [6]. The prostaglandin PGE1 has a sudorific effect in vitro comparable to ACh and was thought to act via cAMP. Histochemical labeling studies show prominent innervation with vasoactive intestinal polypeptide and CGRP fibers and presence of sP and tyrosine hydroxylase fibers [7]. There is, however, dual innervation with a loose network of catecholamine-containing nerves around sweat glands. Innervation of human sweat glands show similarities to rat and mouse sweat glands. In rodents, innervation is initially completely adrenergic followed by an adrenergic to cholinergic switch during development [8]. It is assumed that such a switch occurs in humans during development, so that human sweating is predominantly cholinergic. There is some plasticity and there is some indication that a switch-back to adrenergic sweating can occur with neuropathic states [9]. There are a number of observations that suggest adrenergic innervation increases with certain diseases. Human sweat gland responds to intradermal and intra-arterial adrenaline (10% of cholinergic) [10]. There is evidence of both α-adrenergic (blocked by dibenaline) and β-adrenergic mechanism (blocked by propranolol but not phentolamine). Prominent hyperhidrosis and adrenergic sensitivity occurs in certain neuropathies and Chronic Regional Pain Syndrome I (CRPS I) suggesting increased sympathetic innervation. Indeed the postganglionic sympathetic neuron has recently been shown to enhance adrenergic sweating in CRPS I [9]. In vitro studies suggest the following rank order of sudorific effect ACh  epinephrine (α  β)isoproterenol (β)  phenylephrine (α).

Alcohol and drugs

Cutaneous vasodilatation; reduced hypothalamic set-point

Denervation

Function The major function of the sweat gland in humans is thermoregulatory (see Chapter 50). There are a number of factors that affect the sweat response (Table 51.2). With repeated episodes of profuse sweating, the salt content of the sweat progressively declines. In individuals that have acclimatized to a hot climate, the salt content is reduced, probably reflecting an increase of mineralocorticoids in response to thermal stress [3]. Sweat glands are very prone to atrophy and hypertrophy. Repeated stimulation can result in a several-fold increase in the size and function of the gland. Diffuse loss or absence of sweat can occur due to absence of sweat glands or widespread denervation. Heat intolerance can be a major problem, especially in young patients with widespread anhidrosis, as in the condition chronic idiopathic anhidrosis [5]. TABLE 51.2 Factors that Affect the Sweat Responses FACTORS THAT INCREASE THE SWEAT RESPONSE

FACTORS THAT REDUCE THE SWEAT RESPONSE Parameter

Comments

Skin pressure

Mechanoreceptor stimulation; inhibition of local sympathetic efferents

Hydromeiosis

Water on skin surface reduces sweating rate

Dehydration

Reduced skin blood flow

Hyperosmolarity

Reduced skin blood flow

Cold stimulus

Inhibition of sudomotor activity

Denervation of sweat glands occurs in preganglionic lesions (such as spinal cord injury or multiple system atrophy) or postganglionic lesions (as in the autonomic neuropathies). The size and function of the sweat gland under these circumstances undergoes dramatic atrophy (Fig. 51.1 [4]). Another mechanism of injury is that of transynaptic degeneration of postganglionic axons that could occur with chronic preganglionic lesions. There is also evidence that in early or mild neuropathy affecting the feet, there is excessive forearm response suggesting hypertrophy. The


InnERvATIon oF SwEAT GlAnd

251

References

FIGURE 51.1 Human eccrine sweat gland from a normal subject (A) and a patient with chronic idiopathic anhidrosis (B). There is marked sweat gland atrophy secondary to disuse. D, duct; SC, secretory coil. Reprinted with permission from. Sato K. (1997). Normal and abnormal sweat gland function. In: Clinical Autonomic Disorders: Evaluation and Management. P.A. Low, ed. Philadelphia: Lippincott-Raven, pp. 97–108.

postganglionic sweat response progressively fails with increasing age [11]. The sweat loss is associated with a loss of cholinergic unmyelinated fiber stained with the panaxonal marker PGP9.5 and AChE [12]. In summary, the eccrine sweat gland is an important appendage, subserving thermoregulation. Its absence results in heat intolerance. Alterations in its function provide important clues to the status of the autonomic nervous system.

[1] Sato K, Sato F. Individual variations in structure and function of human eccrine sweat gland. Am J Physiol 1983;245:R203–8. [2] Ogawa T, Low PA. Autonomic regulation of temperature and sweating. In: Low PA, editor. Clinical autonomic disorders: evaluation and management. Boston: Little, Brown and Company; 1993. p. 79–91. [3] Kuno Y. Human perspiration. Springfield, IL: Charles C. Thomas; 1956. [4] Sato K. Normal and abnormal sweat gland function. In: Low PA, editor. Clinical autonomic disorders: evaluation and management. Philadelphia: Lippincott-Raven; 1997. p. 97–108. [5] Low PA, McLeod JG. Autonomic neuropathies. In: Low PA, editor. Clinical autonomic disorders: evaluation and management. Philadelphia: Lippincott-Raven; 1997. p. 463–86. [6] Torres NE, Zollman PJ, Low PA. Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography. Brain Res 1991;550:129–32. [7] Low PA, Kennedy WR. Cutaneous effectors as indicators of abnormal sympathetic function. In: Morris JL, Gibbins IL, editors. Autonomic innervation of the skin. Amsterdam: Harwood Academic Publishers; 1997. p. 165–212. [8] Landis SC. 1988 Neurotransmitter plasticity in sympathetic neurons. In: Handbook of chemical neuroanatomy: the peripheral nervous system. Amsterdam: Elsevier; p. 65–115. [9] Chemali KR, Gorodeski R, Chelimsky TC. Alpha-adrenergic supersensitivity of the sudomotor nerve in complex regional pain syndrome. Ann Neurol 2001;49:453–9. [10] Sato K. Sweat induction from an isolated eccrine sweat gland. Am J Physiol 1973;225:1147–52. [11] Low PA. The effect of aging on the autonomic nervous system. In: Low PA, editor. Clinical autonomic disorders: evaluation and management. Philadelphia: Lippincott-Raven; 1997. p. 161–75. [12] Abdel-Rahman TA, Collins KJ, Cowen T, Rustin M. Immunohistochemical, morphological and functional changes in the peripheral sudomotor neuro-effector system in elderly people. J Auton Nerv Syst 1992;37:187–97.


C H A P T E R

52 Regulation of Metabolism Christopher Bell

CONTRIBUTION OF THE SYMPATHOADRENAL SYSTEM TO COMPONENTS OF TOTAL DAILY ENERGY EXPENDITURE

CONTRIBUTION OF SYMPATHETICALLY STIMULATED ORGANS/TISSUES TO RESTING METABOLIC RATE

The sympathoadrenal system, predominantly via stimulation of β-adrenergic receptors (β-ARs), is a significant biological determinant of total daily energy expenditure [1]. The most compelling evidence comes from animal data: compared with wild-type mice, mice genetically modified such that they do not express any of the three β-AR subtypes demonstrate accelerated weight gain, despite similar energy intake [2]. In humans, the largest component of total daily energy expenditure is resting metabolic rate (RMR). RMR accounts for up to 75% of 24-hour caloric utilization (Fig. 52.1 and Reference [3]). During intra-venous administration of the non-selective β-AR antagonist, propranolol, RMR is decreased [4]. Similarly, during transdermal administration of the centrally-acting, pre-junctional alpha-2-adrenergic receptor (α2-AR) agonist, clonidine, sympathoadrenal activity is inhibited and RMR is decreased [5]. The thermogenic effect of feeding (TEF) is the increase in energy expenditure above RMR following consumption of food. TEF accounts for approximately 10% of total daily energy expenditure and, similarly to RMR, also receives support from the sympathoadrenal system. During sympathoadrenal inhibition or β-AR blockade, TEF is decreased. Further, TEF is positively associated with the thermogenic response to intravenous administration of the non-selective β-AR agonist, isoproterenol [6]. Given the overall contribution of RMR and TEF to total daily energy expenditure, and the important contribution of β-AR stimulation to each of these components, then observations of weight gain in patients prescribed β-AR blockers are perhaps not surprising. From a strictly bioenergetic perspective, the remaining component of total daily energy expenditure, physical activity, is generally unaffected by sympathoadrenal inhibition. During β-AR blockade, submaximal oxygen consumption at the same absolute work rate is not altered, although perceived rates of exertion are increased.


In order to appreciate the metabolic demands, and hence energy requirements, of adult humans, the quantification of the metabolic rate of specific organs and tissues is of obvious significance. This quantification has been attempted using a variety of techniques, including the combination of indirect calorimetry, magnetic resonance imaging and dual-energy X-ray absorptiometry [7]. These techniques have revealed that, per unit mass of tissue, the most thermogenic organs/tissues (in descending order) are the heart, kidneys, brain, liver, skeletal muscle and adipose tissue, all of which receive considerable input from the sympathoadrenal system (Fig. 52.2).

MOBILIZATION/UTILIZATION OF SPECIFIC MACRONUTRIENTS: CARBOHYDRATE The liver and pancreas are the tissues most important for mobilization of carbohydrate (glucose). Stimulation of α-ARs on the beta cells of the pancreas inhibits the secretion of insulin, while stimulation of β-ARs on the alpha cells promotes the release of glucagon. In the liver, in addition to the effects of glucagon, stimulation of β-ARs will promote gluconeogenesis and glycogenolysis. With respect to utilization, epinephrine impairs insulin mediated glucose uptake, possibly by inhibition of insulin receptor substrate-1 (IRS-1)-associated activation of phosphatidylinositol 3-kinase (PI3-kinase), decreased glucose transporter protein type-4 (GLUT4) translocation, and/or inhibition of hexokinase and glucose phosphorylation [8].

MOBILIZATION/UTILIZATION OF SPECIFIC MACRONUTRIENTS: LIPID Stimulation of β-ARs in adipose tissue activates hormone sensitive lipase, initiating lipolysis, the breaking down of triacyglycerols into free fatty acids and glycerol.

253


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52. REgulATIon of METAbolIsM

Once mobilized, these free fatty acids bind to albumin in the blood, and are delivered to organs/tissues. Free fatty acids gain entry into skeletal muscle through various active transport proteins, and subsequent entry into the mitochondria by the use of carnitine pathways. In the mitochondria they are oxidized via beta-oxidation. During β1-AR stimulation, energy expenditure and fatty acid oxidation are increased, however these are both attenuated when

lipolysis is inhibited [9]. This suggests that β1-ARs are more important for mobilization than utilization of lipid. A recent and exciting development pertaining to lipid regulation and thermogenesis is the confirmation of functional brown adipose tissue in humans [10]. Using positron emission tomography (PET), in combination with computed tomography (CT) and administration of [18F] fluoro-deoxyglucose (18F-FDG), metabolically active brown adipose tissue has been detected. Brown adipose tissue appears to be predominantly located in the supraclavicular and anterior neck regions, but also in the anterior thorax. Uptake of 18F-FDG by brown adipose tissue is attenuated during β-AR blockade, suggesting that the brown fat is under sympathoadrenal control.

MOBILIZATION/UTILIZATION OF SPECIFIC MACRONUTRIENTS: PROTEIN

FIGURE 52.1 Contribution of the sympathoadrenal system to components of total daily energy expenditure. % Values represent estimated contribution of each component to total daily energy expenditure. Shaded area represents the contribution of the sympathoadrenal system to each component. TEF: Thermic effect of feeding. Data based on references [3,5,14].

In contrast to the sympathetic regulation of the mobilization/utilization of carbohydrate and lipid, relatively little is known about the sympathetic contribution to protein oxidation. Instead, more is understood about the potential role of the sympathoadrenal system in protein turnover; that is, the balance between protein synthesis and breakdown, and not necessarily the use of protein as a substrate for adenosine tri-phosphate (ATP) production. In skeletal muscle, β2-AR stimulation appears to be important for protein synthesis (anabolic effect), possibly via activation of PI-3 kinase and Akt signaling, and inhibition of calpain

FIGURE 52.2 Contribution of sympathetically stimulated organs/tissues to resting metabolic rate. % Values refer to estimated contribution of specific organs/tissues to resting metabolic rate. Unaccounted % attributed to contribution of residual mass. Listed beneath are the major (metabolic) adrenergic receptors specific to that organ/tissue and their influence on substrate mobilization/utilization. ↑ Stimulation. ↓ Inhibition. Data based on reference [7].


RolE of THE syMPATHoAdREnAl sysTEM In THE dysREgulATIon of METAbolIsM

activity [11]. While this has been clearly demonstrated in animals (mostly rodents), it has been more difficult to document in humans.

ROLE OF THE SYMPATHOADRENAL SYSTEM IN THE DYSREGULATION OF METABOLISM Studying physiological systems during sickness and disease can often provide valuable regulatory information. There are many examples of metabolic dysregulation that lead to a variety of common disease states, including obesity, diabetes, hypercholesterolemia and hypertriglyceridemia. Arguably, the most prevalent of these disease states is obesity. Sympathoadrenal activation is typically high in obesity, with a few notable population exceptions that include the Pima Indians. Visceral obesity is thought to be a particularly important determinant of high sympathoadrenal activation [1], although the reasons for this increased activation remain poorly understood. Several theories exist that are not necessarily mutually exclusive: increased adipose tissue leads to greater secretion of various adipocyte-derived and/ or adiposity-associated hormones including leptin, insulin and angiotensin II; all of which are able to stimulate direct central sympathetic outflow. Further, insulin has vasodilatory properties and thus may also increase sympathoadrenal activation via a baroreflex mediated response. Given the role of the sympathoadrenal system in lipid mobilization/utilization and adipocyte proliferation [12], elevated sympathoadrenal activity might intuitively seem like a favorable response to obesity. Unfortunately this persistently high degree of β-AR stimulation eventually leads to β-AR desensitization and down-regulation. Recent evidence [13] suggests obesity-associated sympathoadrenal activation evokes the cyclic-adenosine monophosphate (cAMP) response element binding protein (CREB) coactivator, CRTC3. This, in turn, leads to upregulation of the GTP-ase activating protein, Rgs2, thereby inhibiting adenyl cyclase activity. Consequently, greater than usual sympathoadrenal stimulation is required in order to elicit a given β-AR mediated response, leading to a vicious cycle of further desensitization and increased tonic activation. New

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data suggest that the thermogenic response to β-AR stimulation is augmented following short-term sympathoadrenal inhibition [5]. Clearly the sympathoadrenal system remains an important therapeutic target for obesity-intervention.

References [1] Davy KP, Orr JS. Sympathetic nervous system behavior in human obesity. Neurosci Biobehav Rev 2009;33:116–24. [2] Bachman ES, Dhillon H, Zhang CY, Cinti S, Bianco AC, Kobilka BK, et al. betaAR signaling required for diet-induced thermogenesis and obesity resistance. Science 2002;297:843–5. [3] Ravussin E, Lillioja S, Knowler WC, Christin L, Freymond D, Abbott WG, et al. Reduced rate of energy expenditure as a risk factor for body-weight gain. N Engl J Med 1988;318:467–72. [4] Monroe MB, Seals DR, Shapiro LF, Bell C, Johnson D, Jones PP. Direct evidence for tonic sympathetic support of resting metabolic rate in healthy adult humans. Am J Physiol Endocrinol Metab 2001;280:E740–4. [5] Newsom SA, Richards JC, Johnson TK, Kuzma JN, Lonac MC, Paxton RJ, et al. Short-term sympathoadrenal inhibition augments the thermogenic response to beta-adrenergic receptor stimulation. J Endocrinol 2010;206:307–15. [6] Stob NR, Bell C, van Baak MA, Seals DR. Thermic effect of food and beta-adrenergic thermogenic responsiveness in habitually exercising and sedentary healthy adult humans. J Appl Physiol 2007;103:616–22. [7] Wang Z, Ying Z, Bosy-Westphal A, Zhang J, Schautz B, Later W, et al. Specific metabolic rates of major organs and tissues across adulthood: evaluation by mechanistic model of resting energy expenditure. Am J Clin Nutr 2010;92:1369–77. [8] Hunt DG, Ivy JL. Epinephrine inhibits insulin-stimulated muscle glucose transport. J Appl Physiol 2002;93:1638–43. [9] Schiffelers SL, Brouwer EM, Saris WH, van Baak MA. Inhibition of lipolysis reduces beta1-adrenoceptor-mediated thermogenesis in man. Metabolism 1998;47:1462–7. [10] Lee P, Greenfield JR, Ho KK, Fulham MJ. A critical appraisal of the prevalence and metabolic significance of brown adipose tissue in adult humans. Am J Physiol Endocrinol Metab 2010;299:E601–6. [11] Koopman R, Gehrig SM, Leger B, Trieu J, Walrand S, Murphy KT, et al. Cellular mechanisms underlying temporal changes in skeletal muscle protein synthesis and breakdown during chronic {beta}adrenoceptor stimulation in mice. J Physiol 2010;588:4811–23. [12] Foster MT, Bartness TJ. Sympathetic but not sensory denervation stimulates white adipocyte proliferation. Am J Physiol Regul Integr Comp Physiol 2006;291:R1630–R1637. [13] Song Y, Altarejos J, Goodarzi MO, Inoue H, Guo X, Berdeaux R, et al. CRTC3 links catecholamine signalling to energy balance. Nature 2010;458:933–41. [14] Tappy L. Thermic effect of food and sympathetic nervous system activity in humans. Reprod Nutr Dev 1996;36:391–7.


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53 Autonomic Innervation of the Skeleton Florent Elefteriou, J. Preston Campbell BONE SYMPATHETIC INNERVATION: ONTOGENY AND ANATOMY Autonomic innervation of the skeleton is documented at the gross anatomical and histological levels with both long and flat bones being innervated with myelinated sensory and unmyelinated sympathetic nerve fibers [1–3]. Retrograde tracing and axotomy experiments have demonstrated that these intraosseus nerve fibers are structurally and functionally connected to primary afferent neurons in dorsal root ganglia and paravertebral sympathetic ganglia [4]. Although the terminal nerve branches feeding the bones have been largely unstudied, they are likely derived from the overlying muscle innervations. Postganglionic fibers from cervical sympathetic ganglion and glossopharyngeal nerve innervate external and internal bones of the skull. The long bones of the upper extremities receive nerve supply from the brachial plexus which then branches to the median nerve to innervate the humerus and the ulnar and radian nerves which supply the forearm bones. Osseus innervation of the flat rib bones is achieved via the anterior branches of the 12 pairs of intercostals nerves. Sympathetic innervation of the lower limbs originates in the lumbar plexus which supplies the femoral and deep saphenous nerves to the femur, and the tibial, medial, and popliteal nerves to the tibia and fibula. Basivertebral nerves in the spine supply intraosseus autonomic innervations of the vertebral bodies [5]. Due to the inherent technical difficulties of working with osseous tissue, little research exists concerning the development of bone innervation. In rats, nerves penetrate uncalcified osseous tissues relatively late, after gestational day 17, and continue to develop more fully after birth [4]. These nerve fibers follow alongside blood vessels and grow into the bone presumably in response to bone marrow expression of neurotrophic factors [6] though this has not been demonstrated in vivo. Despite their presence in developing embryonic tissues, it is still unclear whether sympathetic nerves have a functional role in early bone developmental processes. The lack of obvious bone developmental or morphogenesis abnormalities in mutant mouse models lacking Dopamine β-hydroxylase, the β1, 2 and 3-adrenergic receptor, NPY or the Y1, 2 or 4 receptors for instance argues against a major role of sympathetic nerves during development, despite the


demonstrated need for NE for embryonic cardiovascular development [7]. The first detailed description and histology of bone innervation was given by de Castro (1930) who developed a silver staining technique that demonstrated ramification of neurons with mesenchymal cells in bone. Subsequent research has shown that intraosseous nerves express tyrosine hydroxylase [8], indicating catecholamine synthesis, PGP 9.5, vasoactive intestinal peptide, calcitonin related gene product, and substance P, indicating both sensory and autonomic innervation (see [9] for review). The location of these neural markers show that neurosteal fibers can be divided into two major groups: marrow innervation and periosteal innervation (Fig. 53.1A). Most nerve fibers in the periosteum are sensory, but autonomic TH-positive fibers are also present. These sympathetic nerves branch around the periosteum and enter the cortical bone where they are organized parallel to the long axis of the bone in the Haversian canals or perpendicular, alongside Sharpey’s fibers, which anchor the periosteum to the cortical bone (Fig. 53.1B). In the marrow, nerves accompany blood vessels through a nutrient foramen into the bone’s interior where they can be seen to branch and make vascular and cellular contacts (Fig. 53.1C). The majority of these nerves are perivascular, forming spiral patterns around blood vessels, and can be categorized as vasomotor, controlling the vascular hemodynamics in the bone marrow. Schwann cells are also present in the marrow though they are believed to accompany sensory nerves rather than the sympathetic axons. Electronic microscopy studies have shown nerve fibers at the proximity of osteoblasts but the existence of synapses has not been demonstrated (Fig. 53.1D) [10].

EFFECT OF SYMPATHETIC NERVES ON BONE REMODELING The cells forming bone and allowing its constant remodeling during adult life include osteoblasts, the bone-forming cells of mesenchymal origin, osteoclasts, the bone resorbing cells of monocytic origin, and osteocytes, which are fully mature and bone matrix-embedded osteoblasts. Both osteoblasts and osteoclasts express the

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recognized, and the effect of sympathetic nerves on both lineages, hematopoietic and mesenchymal, is likely to be interrelated. The recently identified effect of sympathetic nerves on hematopoietic stem cell egress from the bone marrow, mediated by osteoblasts, supports this hypothesis [15]. NPY is co-released with NE from sympathetic nerves and a role of this neuropeptide in the regulation of bone remodeling is also well established. The Y2 receptor expressed by hypothalamic neurons exerts an inhibitory influence of bone formation, as demonstrated by the high bone mass phenotype of mice lacking the Y2 receptor specifically in these neurons [16]. On the other hand, the Y1 receptors expressed by bone stromal cells appear to regulate the number of mesenchymal stem cells in bone, downstream of NPY signals from bone sympathetic nerves [17]. Surprisingly, NPY is also expressed by osteocytes, which are fully differentiated osteoblasts embedded within bone matrix [18].

RELEVANCE TO BONE PHYSIOLOGY AND DISEASES

FIGURE 53.1 Intraosseal innervation. Branches of the tibial nerve (yellow) supply the bones of the lower limb (A) alongside the post. tibial and peroneal branches of the popliteal artery (red). Blood vessels and nerves enter tibia (arrow) through the nutrient foramen. Neurosteal fibers in the periosteum run parallel with Sharpey’s fibers and throughout the canaliculi of the cortex (B). Most nerve fibers in the central canal are perivascular vasomotor nerves (C) though some branch into the bone. Some sympathetic fibers ramify near mesenchymal cells such as osteoblasts (D), but whether these derive from the periosteal or perivascular nerves is unknown.

β2AR and are thus responsive to catecholamines [10]. Catecholamines and adrenergic receptor agonists indeed potently increase intracellular cAMP levels in osteoblasts and trigger changes in gene expression for osteoclastogenesis (RANKL) and proliferation (G1 cyclin and Ap1) related genes [11,12]. In agreement with these cellular effects, isoproterenol administration in rodents causes severe bone loss caused by increased bone resorption and decreased bone formation [10,13]. On the other hand, pharmacological blockade of the βAR by propranolol increases bone mass in mice and rats by inhibiting bone resorption and promoting bone formation [10,14]. The use of mutant mouse models corroborated these findings, with the demonstration that genetic lack of Dopamine β-hydroxylase (DBH) or of the β2AR in mice causes an increase in bone mass [10,11]. In addition to their structural function, bones are also hematopoietic tissues and host a wide variety of immune cells at different stages of differentiation, most of which express βARs. The fact that immune cells are involved in the complex regulation of bone remodeling is increasingly

Despite the fairly well established nature of these findings in murine models, it is still unclear to what extent sympathetic signaling regulates bone mass or contributes to bone pathologies in humans. Most studies in humans are restricted to the effect of β-blockers on bone mineral density and tend to support a protective effect of this class of drugs on bone mineral density [19]. They did not yet investigate drugs or conditions influencing sympathetic outflow or responsiveness. Sympathetic signaling in bone may be regulated by several physiological and/or pathological signals. Activation of the cannabinoid receptor 1 (CB1) by 2-arachidonoylglycerol (2AG) may represent such a signal since it regulates sympathetic signaling at the presynaptic level in bone nerve endings by reducing NE release and the antiosteogenic effect of the sympathetic nervous system [20]. Glucocorticoids on the other hand may promote sympathetic responsiveness by osteoblasts to induce bone loss, by their action on β2AR expression and signaling [21]. The effect of thyroid hormones on bone may involve a similar mechanism, although this has not been demonstrated yet. Sympathetic outflow is also regulated centrally and particularly by brainstem and hypothalamic centers controlling major bodily homeostatic functions, including body weight and reproduction. The observation that β-blockade by propranolol or β2AR deficiency prevented the bone catabolic effect of leptin hypothalamic administration in mice clearly linked hypothalamic neurons to the regulation of bone remodeling, and functionally positioned the skeleton as a target organ of central neurons, via the sympathetic nervous system [11] (Fig. 53.2). Severe stress and depression are two CNS pathological conditions that may stimulate sympathetic outflow and have effects on bone via this mechanism, as suggested by a study in mice [22] and by


RElEvAnCE To BonE PHySIology And dISEASES

FIGURE 53.2 The sympathetic nervous system links central neurons to bone cells and regulates the endocrine function of the skeleton. HSC, hematopoietic stem cell; NE, norepinephrine.

the association between clinically diagnosed depression and low BMD [23]. Lastly, the skeleton is increasingly recognized as a true endocrine organ, whose secretion is controlled by sympathetic nerves. Osteocalcin secretion by osteoblasts is indeed controlled by catecholamines and regulates glycemia and testosterone production [24,25].

References [1] Hurrell DJ. The Nerve Supply of Bone. J Anat 1937;72(Pt 1):54–61. PMCID: PMC1252438. [2] Calvo W. The innervation of the bone marrow in laboratory animals. Am J Anat 1968;123(2):315–28. [3] Duncan CP, Shim SS. J. Edouard Samson Address: the autonomic nerve supply of bone. An experimental study of the intraosseous adrenergic nervi vasorum in the rabbit. J Bone Joint Surg Br 1977;59(3):323–30. [4] Gajda M, Litwin JA, Tabarowski Z, Zagolski O, Cichocki T, Timmermans JP, et al. Development of rat tibia innervation: colocalization of autonomic nerve fiber markers with growth-associated protein 43. Cells Tissues Organs 2010;191(6):489–99. [5] Antonacci MD, Mody DR, Heggeness MH. Innervation of the human vertebral body: a histologic study. J Spinal Disord 1998;11(6):526–31. [6] Wang J, Ding F, Gu Y, Liu J, Gu X. Bone marrow mesenchymal stem cells promote cell proliferation and neurotrophic function of Schwann cells in vitro and in vivo. Brain Res 2009;1262:7–15. [7] Thomas SA, Matsumoto AM, Palmiter RD. Noradrenaline is essential for mouse fetal development. Nature 1995;374(6523):643–6. [8] Bjurholm A, Kreicbergs A, Terenius L, Goldstein M, Schultzberg M. Neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-immunoreactive nerves in bone and surrounding tissues. J Auton Nerv Syst 1988;25(2–3):119–25.

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[9] Elefteriou F. Neuronal signaling and the regulation of bone remodeling. Cell Mol Life Sci 2005;62(19–20):2339–49. [10] Takeda S, Elefteriou F, Levasseur R, Liu X, Zhao L, Parker KL, et al. Leptin regulates bone formation via the sympathetic nervous system. Cell 2002;111(3):305–17. [11] Elefteriou F, Ahn JD, Takeda S, Starbuck M, Yang X, Liu X, et al. Leptin regulation of bone resorption by the sympathetic nervous system and CART. Nature 2005;434(7032):514–20. [12] Fu L, Patel MS, Bradley A, Wagner EF, Karsenty G. The molecular clock mediates leptin-regulated bone formation. Cell 2005;122(5):803–15. [13] Bonnet N, Benhamou CL, Brunet-Imbault B, Arlettaz A, Horcajada MN, Richard O, et al. Severe bone alterations under beta2 agonist treatments: bone mass, microarchitecture and strength analyses in female rats. Bone 2005;37(5):622–33. [14] Bonnet N, Benhamou CL, Malaval L, Goncalves C, Vico L, Eder V, et al. Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions. J Cell Physiol 2008;217(3):819–27. [15] Katayama Y, Battista M, Kao WM, Hidalgo A, Peired AJ, Thomas SA, et al. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 2006;124(2):407–21. [16] Baldock PA, Allison S, McDonald MM, Sainsbury A, Enriquez RF, Little DG, et al. Hypothalamic regulation of cortical bone mass: opposing activity of y2 receptor and leptin pathways. J Bone Miner Res 2006;21(10):1600–7. [17] Lundberg P, Allison SJ, Lee NJ, Baldock PA, Brouard N, Rost S, et al. Greater bone formation of Y2 knockout mice is associated with increased osteoprogenitor numbers and altered Y1 receptor expression. J Biol Chem 2007;282(26):19082–091. [18] Igwe JC, Jiang X, Paic F, Ma L, Adams DJ, Baldock PA, et al. Neuropeptide Y is expressed by osteocytes and can inhibit osteoblastic activity. J Cell Biochem 2009;108(3):621–30. PMCID: 2754602 [19] de Vries F, Souverein PC, Cooper C, Leufkens HG, van Staa TP. Use of beta-blockers and the risk of hip/femur fracture in the United Kingdom and The Netherlands. Calcif Tissue Int 2007;80(2):69–75. [20] Tam J, Ofek O, Fride E, Ledent C, Gabet Y, Muller R, et al. Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling. Mol Pharmacol 2006;70(3):786–92. [21] Ma Y, Nyman JS, Tao H, Moss HH, Yang X, Elefteriou F. β2-Adrenergic receptor signaling in osteoblasts contributes to the catabolic effect of glucocorticoids on bone. Endocrinology 2011; 152(4): 1412-22. [22] Yirmiya R, Goshen I, Bajayo A, Kreisel T, Feldman S, Tam J, et al. Depression induces bone loss through stimulation of the sympathetic nervous system. Proc Natl Acad Sci U S A 2006;103(45):16876–881. [23] Cizza G, Ravn P, Chrousos GP, Gold PW. Depression: a major, unrecognized risk factor for osteoporosis? 2001;12(5):198–203. Trends Endocrinol Metab 2001;12(5):198–203. [24] Hinoi E, Gao N, Jung DY, Yadav V, Yoshizawa T, Myers Jr. MG, et al. The sympathetic tone mediates leptin's inhibition of insulin secretion by modulating osteocalcin bioactivity. J Cell Biol 2008;183(7):1235–42. [25] Oury F, Sumara G, Sumara O, Ferron M, Chang H, Smith CE, et al. Endocrine Regulation of Male Fertility by the Skeleton. Cell 2011.


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54 Sex Differences in Autonomic Function Emma C. Hart, Nisha Charkoudian, Michael J. Joyner INTRODUCTION In humans, both normal and pathophysiological mechanisms of autonomic control of blood pressure are modified by sex. The incidence of hypertension is lower in young women than in young men, and the incidence of “hypotensive” disorders such as orthostatic intolerance is much greater in young women. However, even in healthy normotensive humans, resting autonomic tone and autonomic support of blood pressure tend to be different between men and women. Interestingly, aging increases the risk of developing hypertension and other cardiovascular diseases in both sexes, but this effect is even more marked in women after the menopause than it is in men of a similar age range. In the context of this discussion, there has been some debate regarding whether the term “gender” or “sex” should be used when referring to differences between men and women, particularly with regard to biomedical findings. The word “gender” originates from the old French word “gendre”, referring to a grammatical category which indicates whether a word is masculine or feminine. More recently (over the last century) the word gender is typically used to refer to the general sociological roles of females and males, and a person who is biologically of one sex or the other can identify with either gender. In contrast, sex differences refer to biological differences based on reproductive organs and function. In the present chapter, the word “sex” is used to refer to biological differences between men and women. The cultural and sociological influences of gender (i.e., differences based on how a person defines him or herself by gender) are outside the scope of the present discussion.

SEX DIFFERENCES IN NORMAL AUTONOMIC FUNCTION Tonic sympathetic nerve activity can be measured using microneurography to measure muscle sympathetic nerve activity (MSNA) or norepinephrine spill-over techniques [1,2]. Studies utilizing either approach typically indicate that resting sympathetic nerve activity is lower in young women compared to men [3]. In addition, baseline MSNA


is altered during different phases of the menstrual cycle, where MSNA is higher during the luteal (high hormone) compared to the early follicular (low hormone) phase in women not taking oral contraceptives [4]. These differences in MSNA appear to be partially mediated via the effects of the female sex hormones on central autonomic nuclei [5]. Central estrogen administration also increases resting vagal tone in female mice, suggesting the sex hormones might also influence parasympathetic outflow to the heart [5]. However, whether resting cardiac vagal tone is different in men and women is unclear, with some studies (using heart rate variability analysis) reporting higher vagal tone in women vs. men [6] and others indicating that there are no differences between men and women [7]. Despite the fact that MSNA is tightly linked to arterial pressure via the baroreflex, tonic levels of MSNA do not determine baseline arterial pressure in young normotensive people. In young men and women, there is no relationship between tonic MSNA and resting arterial pressure [1,8,9] (Fig. 54.1). Investigations into inter-individual differences in arterial blood pressure regulation have been essential in explaining this apparent paradox. In young men, MSNA is positively related to TPR and inversely related to cardiac output [8] (Fig. 54.1). Therefore, lower cardiac output values balance high levels of MSNA, (which contribute to high TPR) in young men, thus explaining the lack of direct relationship between MSNA and arterial pressure in young men. Interestingly, in young women, MSNA is not related to TPR or cardiac output (Fig. 54.1). Consequently, MSNA does not appear to determine the overall level of peripheral vasoconstrictor tone in young women. Therefore, in young women other factors must offset the vasoconstrictor influence of sympathetic vasoconstrictor nerves. Other mechanisms may relate to the potent vasodilator effect of estrogen (for review see ref [10]) and differences in β-adrenergic receptor sensitivity between men and women [11]. Because norepinephrine released from adrenergic nerves can stimulate both the α- and β-adrenergic receptors, α-adrenergic vasoconstriction may be offset by increased β-adrenergic receptor mediated vasodilation in young women. In this context, recent data suggest that when the β-adrenergic receptors are blocked, the relationship between MSNA and TPR becomes positive in young women [12].

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FIGURE 54.1 Relationship of muscle sympathetic nerve activity (MSNA) to mean arterial pressure (MAP) cardiac output (CO) and total peripheral resistance among young men and women. There is no relationship between MSNA and MAP among men and women. However, in young men MSNA is positively related to TPR and inversely related to CO. In young women, there is no such relationship. Modified from Hart et al. [9].

ORTHOSTATIC INTOLERANCE AND HYPOTENSION Both orthostatic intolerance and orthostatic hypotension are more common in young women compared to

young men. Normotensive, otherwise healthy women, are less tolerant to changes in central blood volume compared to men [13]. That is, young women tend to develop symptoms (dizziness, lightheadedness, or fainting) earlier during orthostatic stress such as lower body negative


ovERAll SummARy AnD ConCluSIonS

pressure. This tendency is exacerbated after spaceflight or prolonged bed rest [13]. Moreover, individuals suffering from postural orthostatic tachycardia syndrome (POTS) are most often young women under the age of 35 years. Individuals with POTS tend to have similar MSNA responses to orthostatic stress as age matched controls, suggesting that lower MSNA is not a major contributor to POTS symptoms. Patients with POTS have a smaller stroke volume and blood volume compared to healthy controls [14], which might explain why more women suffer from POTS; i.e., women have smaller stroke volumes and blood volumes vs. men (due to smaller body size). Consequently, a chronic decrease in stroke volume (due to various reasons including physical de-conditioning) may be less well tolerated in women and thus more women express symptoms of POTS. A recent study suggests that exercise training improves left ventricular mass and blood volume in women with POTS and can improve or even cure the symptoms of POTS [14]. It is well accepted that even healthy young women who do not have symptoms of POTS are predisposed to orthostatic intolerance or hypotension at a greater rate compared to men. The exact mechanisms underlying this predisposition are unclear. First, differences in baroreflex function in men and women could explain why more women experience syncope during head-up-tilt or lower body negative pressure. However, this is unlikely since the sympathetic responses and increases in total peripheral resistance to postural stress in women tend to be similar to that in men [15]. Furthermore, studies indicate that sympathetic baroreflex sensitivity is not different between men and women. In women, however, the heart rate response to orthostatic stress appears to be greater than that in men [13]. This may be due to differences in the cardiac physiology between men and women. Along these lines, women have a lower stroke volume and stroke index preceding syncope vs. men [13]. Furthermore, reduced orthostatic tolerance in women appears to be associated with a steeper Frank–Starling curve; that is, for a given decrease in pulmonary capillary wedge pressure, there is a larger decrease in stroke volume in women vs. men [13]. Finally, the withdrawal of sympathetic nerve activity precipitates, and invariably results in, syncope. It is possible that women reach the threshold for sympathetic withdrawal earlier than men due to larger decreases in cardiac filling during orthostatic stress [13,15].

SEX, AGING AND HYPERTENSION The sympathetic nerve system is involved in the pathogenesis of hypertension, with many studies demonstrating that sympathetic nerve activity is elevated among individuals with hypertension. In general young men are more at risk of developing hypertension than women of the same age, which may be related to differential effects that the sympathetic nerves appear to have on the vasculature of

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men and women. Aging is associated with an increased risk of developing hypertension. In this context, aging is associated with a gradual augmentation in sympathetic nerve activity and arterial pressure [1]. Although SNA is not related to blood pressure in young men or women, this relationship becomes significantly positive in older men and women, so that higher sympathetic nerve activity is related to increased arterial pressure [3]. This might explain why arterial pressure is typically higher in older men and women vs. young men and women. The slope of this relationship between arterial pressure and MSNA is steeper in older women, so that for a given increase in MSNA there is a greater increase in arterial pressure in women vs. men of the same age. Consequently, sympathetic nerve activity becomes more important in determining resting arterial pressure in older women. This might be explained by the loss of the protective influence of estrogen after menopause. Along these lines, the risk of developing hypertension is greater in postmenopausal women than in men of the same age.

OVERALL SUMMARY AND CONCLUSIONS In summary, sex and sex hormones have substantial influences on the interaction between the autonomic and cardiovascular systems, such that resting arterial pressure is controlled differently in men and women across the lifespan. In young women, the sex hormones modulate the transduction of sympathetic nerve activity into peripheral vasoconstriction. “Hypotensive” disorders such as orthostatic intolerance and orthostatic hypotension occur more frequently in young women than in young men. Although women tend to have lower resting sympathetic vasoconstrictor nerve activity, this may not be a major contributor to orthostatic intolerance, as recent data suggest that, orthostatic intolerance/hypotension in women appears to be primarily due to less cardiac filling and/or a smaller cardiac size. Interestingly, as humans age, the overall level of arterial pressure becomes related to sympathetic nerve activity; this relationship is especially prominent in women. This might explain why the increase in the rate of hypertension is greater in older women after the menopause vs. men. Overall, understanding sex-based differences in autonomic function is becoming fundamental for the treatment of autonomic dysfunction and related disorders such as hypertension.

References [1] Sundlof G, Wallin BG. Human muscle nerve sympathetic activity at rest. Relationship to blood pressure and age. J Physiol 1978;274:621–37. [2] Wallin BG, Thompson JM, Jennings GL, Esler MD. Renal noradrenaline spillover correlates with muscle sympathetic activity in humans. J Physiol 1996;491(Pt 3):881–7. [3] Narkiewicz K, Phillips BG, Kato M, Hering D, Bieniaszewski L, Somers VK. Gender-selective interaction between aging,


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blood pressure, and sympathetic nerve activity. Hypertension 2005;45:522–5. Minson CT, Halliwill JR, Young TM, Joyner MJ. Influence of the menstrual cycle on sympathetic activity, baroreflex sensitivity, and vascular transduction in young women. Circulation 2000;101:862–8. Saleh TM, Connell BJ. 17 beta-estradiol modulates baroreflex sensitivity and autonomic tone of female rats. J Auton Nerv Syst 2000;80:148–61. Liao D, Barnes RW, Chambless LE, Simpson Jr. RJ, Sorlie P, Heiss G. Age, race, and sex differences in autonomic cardiac function measured by spectral analysis of heart rate variability – the ARIC study. Atherosclerosis Risk in Communities. Am J Cardiol 1995;76:906–12. Evans JM, Ziegler MG, Patwardhan AR, Ott JB, Kim CS, Leonelli FM, et al. Gender differences in autonomic cardiovascular regulation: spectral, hormonal, and hemodynamic indexes. J Appl Physiol 2001;91:2611–8. Charkoudian N, Joyner MJ, Johnson CP, Eisenach JH, Dietz NM, Wallin BG. Balance between cardiac output and sympathetic nerve activity in resting humans: role in arterial pressure regulation. J Physiol 2005;568:315–21. Hart EC, Charkoudian N, Wallin BG, Curry TB, Eisenach JH, Joyner MJ. Sex differences in sympathetic neural-hemodynamic balance: implications for human blood pressure regulation. Hypertension 2009;53:571–6.

[10] Miller VM, Duckles SP. Vascular actions of estrogens: functional implications. Pharmacol Rev 2008;60:210–41. [11] Kneale BJ, Chowienczyk PJ, Brett SE, Coltart DJ, Ritter JM. Gender differences in sensitivity to adrenergic agonists of forearm resistance vasculature. J Am Coll Cardiol 2000;36:1233–8. [12] Hart EC, Charkoudian N, Wallin BG, Roberts SK, Johnson CP, Joyner MJ. Sex differences in the sympathetic balance of blood pressure: the role of the alpha-adrenergic receptors. FASEB J 2010;24:594–9. [13] Fu Q, Arbab-Zadeh A, Perhonen MA, Zhang R, Zuckerman JH, Levine BD. Hemodynamics of orthostatic intolerance: implications for gender differences. Am J Physiol Heart Circ Physiol 2004;286:H449–57. [14] Fu Q, Vangundy TB, Galbreath MM, Shibata S, Jain M, Hastings JL, et al. Cardiac origins of the postural orthostatic tachycardia syndrome. J Am Coll Cardiol 2010;55:2858–68. [15] Fu Q, Witkowski S, Okazaki K, Levine BD. Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress. Am J Physiol Regul Integr Comp Physiol 2005;289:R109–16.


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55 Autonomic Control During Pregnancy Virginia L. Brooks, Belinda H. McCully, Priscila A. Cassaglia The physiological adaptations to pregnancy are profoundly complex yet exquisitely tuned to ensure adequate development of the fetus. These changes are largely orchestrated directly or indirectly by the placenta, which is a source of a broad array of hormones, including neuropeptides, steroid hormones, pituitary-like hormones, growth factors, vasoactive hormones, and metabolic hormones. The cardiovascular system and its autonomic innervation are particularly impacted. An early event in gestation is systemic vasodilation (Fig. 55.1), which occurs even before uteroplacental vascular growth and blood flow are significantly increased [1,2]. As a result, arterial pressure falls. However, the decrease is modest because of parallel increases in cardiac output (Fig. 55.1), due in part to cardiac remodeling and enlargement, but also to simultaneous activation of fluid-retaining factors, such as the renin-angiotensin-aldosterone system, and increases in blood volume [2].

PREGNANCY ACTIVATES THE SYMPATHETIC NERVOUS SYSTEM In parallel to increases in fluid-retaining hormones, alterations in the autonomic nervous system accompany normal pregnancy. Using microneurography in humans, several studies have documented that muscle sympathetic nerve activity is increased [3,4]. In experimental animals, elevations of basal renal sympathetic nerve activity have also been observed [3]. In line with these direct measurements of sympathetic nerve firing rates, indirect assessments, including quantification of heart rate and arterial pressure variability in both the frequency and time domains [3,5], also suggest that pregnancy increases basal sympathetic activity. Activation of the sympathetic nervous system would be expected to cause vasoconstriction, which could nullify the primary decreases in systemic vascular resistance. However, this effect is minimized during pregnancy, because the vasculature becomes resistant to the actions of vasoconstrictors, including norepinephrine [3]. While pregnancy induces sympathoexcitation, simultaneously, basal parasympathetic tone decreases [5,6]. One consequence of increased cardiac sympathetic activity, and decreased parasympathetic activity, is increased heart rate, which is also evident early in gestation (Fig. 55.1) [1].


The mechanisms by which pregnancy modifies basal autonomic tone have received limited attention. Central actions of increases in circulating angiotensin II appear to contribute to the sympathoexcitation [3]. In contrast, decreased parasympathetic control of the heart is due in part to reductions in the responsiveness of the heart to acetylcholine [6].

PREGNANCY IMPAIRS THE BARORECEPTOR REFLEX While most physiological changes induced by pregnancy are relatively benign, one deleterious consequence is a marked suppression of the function of the baroreceptor reflex [3–5]. As a result, pregnant women are prone to orthostatic hypotension, and pregnant animals are less able to maintain arterial pressure during hemorrhage [3]. Given that hemorrhage accompanies every delivery and that the baroreflex is largely responsible for arterial pressure maintenance during hemorrhage, it is understandable why peripartum hemorrhage is a major cause of maternal mortality [3]. Baroreflex dysfunction has been documented in several species besides humans, including rabbits, rats, goats, sheep, and dogs [3]. Pregnancy attenuates baroreflex control of multiple efferents, such as renal and muscle sympathetic nerve activity, heart rate, as well as hormones like vasopressin and ACTH [3]. The decrease in baroreflex control of the autonomic nervous system has been detected using several methodological approaches, including noninvasive techniques in women employing the analysis of heart rate and arterial pressure variability, assessments of spontaneous baroreflex sensitivity, and infusion of vasoactive drugs to construct complete sigmoidal baroreflex relationships between arterial pressure and heart rate or sympathetic nerve activity (Fig. 55.2) [3,5]. Three features of these curves are commonly attenuated: the maximum gain or slope of the most linear segment of the curves, the maximal level of sympathetic activity or heart rate achieved during severe hypotension, and the “setpoint” or the arterial pressure level associated with the midpoint of the curve. This latter change is likely mediated by resetting of baroreceptor afferents, which causes the baroreflex function curve to shift toward the lower arterial pressure

265


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CHANGE FROM PREPREGNANT (%)

50

CO

40 30

SV

20

HR

10

MAP

0 –10 –20 TPR

–30 –40 0

10

20 GESTATION (weeks)

30

40

FIGURE 55.1 Hemodynamic changes that accompany normal pregnancy in humans. Data were taken from ref [1]. CO, cardiac output; SV, stroke volume; HR, heart rate; MAP, mean arterial pressure; TPR, total peripheral resistance.

FIGURE 55.2 Pregnancy impairs baroreflex control of heart rate and renal sympathetic nerve activity (RSNA) in conscious rats. From Brooks et al. [3]. P, pregnant; NP, nonpregnant.

level during pregnancy [3]. However, the mechanisms that contribute to the decreases in the baroreflex maximums and in baroreflex gain are complex and may be largely distinct.

MECHANISMS OF PREGNANCY-INDUCED BAROREFLEX IMPAIRMENT While pregnancy could depress the function of any or all anatomical links within the baroreflex pathway, current evidence indicates that brain control is particularly impaired [3]. Within the brain, multiple sites and hormonal mediators are involved. As detailed in Chapter 33, the core brainstem baroreflex pathway begins in the nucleus tractus solitarius (NTS), which receives baroreceptor afferent information. Increases in arterial pressure activate the baroreceptors, which excite NTS second order neurons via a glutamatergic (non-NMDA) synapse. These neurons project to and excite (also via glutamate acting on NMDA receptors) interneurons in the caudal ventrolateral medulla that project to and release GABA to inhibit sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM). Thus, hormonal modulation of baroreflex function could occur at any or all of these sites, although in the case of pregnancy the RVLM appears pivotal. One hormonal mechanism that underlies decreases in the baroreflex maximum is the action of a major neurosteroid metabolite of progesterone, 3α-hydroxydihydroprogesterone (3α-OH-DHP), to enhance GABAergic suppression of RVLM premotor neurons [3] (Fig. 55.3). 3α-OH-DHP levels, as well as the enzymes responsible for the synthesis of 3α-OH-DHP from progesterone, are increased in the brain at end-gestation, the time at which baroreflex function reaches its nadir. In addition, acute systemic or RVLM administration of 3α-OH-DHP in virgin rats reduces baroreflex maximum levels of renal sympathetic activity similarly to pregnancy. The link between 3α-OHDHP and increased GABAergic tone in RVLM is supported by the well-documented ability of this neurosteroid, by binding to the GABAA receptor, to enhance its function. More importantly, evidence indicates that RVLM premotor neurons receive greater tonic GABAergic suppression during pregnancy [3]. Insulin resistance is a normal adaption of pregnancy that, by increasing circulating glucose levels, serves to enhance glucose availability into the fetus. However, several lines of evidence support the hypothesis that insulin resistance also contributes to the decrease in baroreflex gain, by decreasing brain insulin levels [3]. First, decreases in insulin sensitivity and baroreflex gain are temporally correlated in rabbits, rats and humans [3,7,8]. Second, treatment of pregnant rabbits with the insulin sensitizing drug, rosiglitazone, improves baroreflex function [8]. Third, insulin enters the brain via transport from plasma across the blood brain barrier, and insulin resistant states are associated with decreases in insulin transport. Indeed, during pregnancy, brain insulin levels fall [8,9]. Fourth, in conscious pregnant rats, intracerebroventricular infusion of insulin normalizes baroreflex gain, while in virgin rats insulin infusion is ineffective [9].


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The brain sites and circuitry by which insulin supports baroreflex function have been investigated [3]. Unlike 3α-OH-DHP, which acts in the RVLM, initial studies revealed that insulin initiates its effects in the forebrain, since lateral cerebroventricular, but not fourth ventricular, insulin infusion increased baroreflex gain. Further work identified the arcuate nucleus as the site at which insulin acts to activate the sympathetic nervous system and increase baroreflex gain, via a neural pathway that includes the paraventricular nucleus of the hypothalamus (PVN) [10]. Two major sets of neurons in the arcuate nucleus project to the PVN: proopiomelanocortin (POMC) neurons, which release alphamelanocyte-stimulating hormone, and neuropeptide Y neurons. Recently, the sympathoexcitatory response to insulin was shown to be mediated by PVN melanocortin receptors, suggesting that POMC neurons convey the signal from the arcuate nucleus to PVN [11]. From the PVN, the neuronal pathway appears to converge with brainstem baroreflex circuitry in the RVLM, since insulin’s sympathoexcitatory effect is prevented by blockade of RVLM ionotropic glutamate receptors [11]. These data provide a mechanistic explanation for why, in pregnant animals, rosiglitazone treatment to increase insulin sensitivity [8] and intracerebroventricular insulin infusion [9] each improved baroreflex gain, yet failed to improve the attenuated baroreflex maximum levels of heart rate. Given that GABAergic inhibition of RVLM premotor neurons is increased during pregnancy, this suppression would prevent insulin’s normal effect to increase baroreflex maximum levels as well (Fig. 55.3).

PREECLAMPSIA Preeclampsia is a potentially fatal hypertensive disorder of pregnancy that is initiated by reduced placental perfusion. Increased sympathetic tone may contribute to the hypertension, since basal muscle sympathetic nerve activity is clearly increased above the levels observed in normal pregnant women [4]. Moreover, baroreflex sensitivity is further decreased [3,4]. Interestingly, early gestational measurements of impaired baroreflex function, coupled with detection of reduced uterine perfusion, may noninvasively herald the subsequent development of this life-threatening disorder [3]. However, despite the clinical significance of these autonomic changes, the mechanisms are currently unknown.

SUMMARY AND CONCLUSIONS Pregnancy increases sympathetic nerve firing and decreases both basal parasympathetic activity and baroreflex gain; these changes are exaggerated in women with preeclampsia. The changes in basal autonomic tone may counteract to some degree the profound vasodilation that is a hallmark of normal pregnancy. In contrast,

BRAIN

BBB

INSULIN

BLOOD INSULIN

+

HYPOTHALAMUS

+ RVLM

_

GABA

+

3α-OH-DHP

Baroreflex Gain FIGURE 55.3 Mechanisms that contribute to impaired baroreflex function during pregnancy. Insulin in blood is transported across the blood–brain barrier (BBB) into brain, where, via actions in the hypothalamic arcuate nucleus, it enhances baroreflex function. The excitation initiated by insulin in the arcuate nucleus is conveyed via the paraventricular nucleus to a brainstem baroreflex relay, the rostral ventrolateral medulla (RVLM). During pregnancy, falls in brain insulin (which supports baroreflex function) decreases baroreflex gain. In addition, GABAergic suppression of RVLM premotor neurons mediated by the neurosteroid 3α-OH-DHP decreases maximum levels of sympathetic activity elicited by severe hypotension.

the impaired baroreflex function is not beneficial for the mother. Little is known about the mechanisms that mediate the changes in basal autonomic activity in normal pregnancy and with preeclampsia. The decrease in baroreflex function induced by normal pregnancy is mediated by at least two hormonal systems (Fig. 55.3): increased levels of 3α-OH-DHP act in the RVLM to enhance GABAergic suppression of RVLM premotor neurons and the baroreflex maximum, while reduced actions of insulin in the hypothalamus contribute to decreases in baroreflex gain.

References [1] Robson SC, Hunter S, Boys RJ, Dunlop W. Serial study of factors influencing changes in cardiac output during human pregnancy. Am J Physiol 1989;256:H1060–H1065. [2] Thornburg KL, Jacobson SL, Giraud GD, Morton MJ. Hemodynamic changes in pregnancy. Semin Perinatol 2000;24:11–14. [3] Brooks VL, Dampney RA, Heesch CM. Pregnancy and the endocrine regulation of the baroreceptor reflex. Am J Physiol Regul Integr Comp Physiol 2010;299:R439–51. [4] Fu Q, Levine BD. Autonomic circulatory control during pregnancy in humans. Semin Reprod Med 2009;27:330–7. [5] Rang S, Wolf H, Montfrans GA, Karemaker JM. Non-invasive assessment of autonomic cardiovascular control in normal human pregnancy and pregnancy-associated hypertensive disorders: a review. J Hypertens 2002;20:2111–9.


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[6] Brooks VL, Kane CM, Van Winkle DM. Altered heart rate baroreflex during pregnancy: role of sympathetic and parasympathetic nervous systems. Am J Physiol 1997;273:R960–R966. [7] Brooks VL, Mulvaney JM, Azar AS, Zhao D, Goldman RK. Pregnancy impairs baroreflex control of heart rate in rats: role of insulin sensitivity. Am J Physiol Regul Integr Comp Physiol 2010;298:R419–R426. [8] Daubert DL, Chung MY, Brooks VL. Insulin resistance and impaired baroreflex gain during pregnancy. Am J Physiol Regul Integr Comp Physiol 2007;292:R2188–R2195.

[9] Azar AS, Brooks VL. Impaired baroreflex gain during pregnancy in conscious rats: role of brain insulin. Hypertension 2011;57:283–8. [10] Cassaglia PA, Hermes SM, Aicher SA, Brooks VL. Insulin acts in the arcuate nucleus to increase lumbar sympathetic nerve activity and baroreflex function in rats. J Physiol 2011;589:1643–62. [11] Ward KR, Bardgett JF, Wolfgang L, Stocker SD. Sympathetic response to insulin is mediated by melanocortin 3/4 receptors in the hypothalamic paraventricular nucleus. Hypertension 2011;57:435–41.


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56 Aging and the Autonomic Nervous System Lewis A. Lipsitz, Vera Novak Healthy human aging is associated with several abnormalities in autonomic nervous system function that can impair an older person’s adaptation to the stresses of everyday life. Aging affects central and peripheral autonomic regulation of heart rate (HR), blood pressure (BP), temperature, and visceral organ function. However, aging, per se should not be considered a state of autonomic failure because many mechanisms compensating for demands of daily living remain intact. Orthostatic and postprandial hypotension are two common manifestations of age-associated autonomic nervous system impairment [1,2]. Both are defined as a 20 mmHg or greater decline in systolic BP on assumption of the upright posture, or within 1 hour of eating a meal, respectively. These are two distinct conditions that may or may not occur in the same patient. Orthostatic hypotension is observed in less than 7% of healthy normotensive elderly people and in as many as 30% of people older than 75 years with multiple pathologic conditions. Postprandial BP falls an average of 11 mmHg in as many as three quarters of asymptomatic community-dwelling elderly people over age 70. Postprandial hypotension is particularly common in the nursing home population where it accounts for up to 8% of cases of syncope. Orthostatic hypotension is an important symptom of autonomic failure, commonly associated with diabetes, malignancy, amyloidosis, Parkinson’s disease, multiple system atrophy, Lewy body dementia, pure autonomic failure, and other syndromes in elderly patients. A unique feature of orthostatic and postprandial hypotension in elderly people without overt autonomic failure is that they are commonly associated with supine or sitting systolic hypertension. This may be due to the adverse effects of hypertension on baroreflex sensitivity, vascular reactivity, and diastolic filling that compound age-related abnormalities in these BP regulatory mechanisms. Both hypotension and hypertension are associated with reduced cerebral perfusion, damage to gray and white matter, and cognitive decline [3]. Although it may seem counterintuitive, the gradual and judicious lowering of BP may improve, rather than worsen hypotension and cerebral perfusion in hypertensive patients. This presentation of orthostatic or postprandial hypotension in the setting of systolic hypertension must be distinguished from the supine hypertension seen in frank autonomic failure, where patients


are very symptomatic and develop hypertension later in the course, often in association with the use of pressor medications. Medications that acutely lower BP may also contribute to hypotension in elderly patients, particularly diuretics, antihypertensives, alpha-blockers used for prostatic obstruction, dopamine, tricyclic antidepressants, and neuroleptics. If clinically indicated, these medications should be given in the lowest possible dose and slowly titrated to the desired effect in order to allow BP regulatory mechanisms to adapt. Recent studies suggest that the control of hypertension in elderly people can not only reduce cardiovascular morbidity and mortality, but also reduce orthostatic hypotension [4], increase cerebral blood flow, and improve carotid distensibility [5]. Pathophysiologic mechanisms predisposing healthy elderly people to orthostatic and postprandial hypotension are summarized in Table 56.1.

CARDIAC BAROREFLEX FUNCTION Normal human aging is associated with a reduction in cardiovagal baroreflex sensitivity, which may lead TABLE 56.1 Age-Related Physiologic Changes Predisposing to Hypotension

1. Decreased baroreflex sensitivity a. Diminished HR response to hypotensive stimuli (orthostasis, meal digestion, and hypotensive medications).

b. Decreased adrenergic vascular responsiveness to orthostatic, postprandial, and medication-related BP reduction.

2. Impaired defense against reduced intravascular volume a. Reduced secretion of renin, angiotensin, and aldosterone. b. Increased atrial natriuretic peptide, supine and upright. c. Decreased plasma vasopressin response to orthostasis. d. Reduced thirst after water deprivation. 3. Reduced early cardiac ventricular filling (diastolic dysfunction) a. Increased dependence on cardiac preload to maintain cardiac output.

b. Increased dependence on atrial contraction to fill the ventricles –

leads to hypotension during atrial fibrillation. Decreased cardiac output during tachycardia when ventricular filling time is reduced. Impaired postprandial vasoconstriction a. Vasodepressor action of insulin [10].

c.

4.

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272

56. AgINg ANd THE AuToNomIC NERvouS SySTEm

to hypotension due to decreased cardiac output. This is manifest by a blunted cardioacceleratory response to hypotensive stimuli, such as upright posture, Phase II of the Valsalva maneuver, nitroprusside infusions, and lower body negative pressure; as well as the reduced bradycardic response to hypertensive stimuli, such as the administration of phenylephrine. The baroreflex may be impaired at any one of multiple sites along its arc, including carotid and cardiopulmonary pressure receptors, afferent pathways, brainstem (nucleus tractus solitarii) and higher regulatory centers, efferent sympathetic and parasympathetic neurons, postsynaptic cardiac beta receptors, or defects in intracellular signal transduction pathways within the sinoatrial node. An age-associated increase in BP also contributes to baroreflex impairment. Known agerelated abnormalities in components of the baroreflex arc are summarized below.

SYMPATHETIC ACTIVITY Studies of sympathetic nervous system activity in healthy human subjects demonstrate an age-related increase in resting plasma norepinephrine levels, muscle sympathetic nerve activity, and vascular resistance, as well as the plasma norepinephrine response to upright posture and exercise. The increase in plasma norepinephrine is primarily due to an increase in norepinephrine spillover at sympathetic nerve endings and secondarily due to a decrease in clearance. Despite apparent increases in sympathetic tone with aging, cardiac and vascular responsiveness is diminished. Infusions of beta-adrenergic agonists result in smaller increases in HR, left ventricular ejection fraction, cardiac output, and vasodilation in older compared with younger men.

PARASYMPATHETIC ACTIVITY Previous studies demonstrating age-related reductions in HR variability in response to respiration, cough, and the Valsalva maneuver suggest that aging is associated with impaired parasympathetic control of HR. Elderly patients with unexplained syncope have even greater impairments in the HR response to cough and deep breathing than healthy age-matched subjects without syncope [6]. There is evidence that physical activity may help maintain or increase vagal activity in older age.

INTEGRATION OF AUTONOMIC CONTROL NETWORKS When plotted continuously over time, the beat-to-beat HR or BP signal is highly irregular due to the interactions of multiple autonomic control systems operating over different time scales. The technique of power spectral analysis is commonly used to quantify the relative contributions

of sympathetic and parasympathetic nervous systems to this complex cardiovascular variability. The HR or BP power spectrum can be divided into low- and highfrequency components. Previous studies using betablockade, atropine, or both suggest that the low-frequency oscillations (0.05–0.1 Hz) in BP reflect sympathetic modulation of vasomotor tone and in HR reflect a combination of baroreflex-mediated sympathetic and parasympathetic influences. High-frequency components of the HR and BP power spectra (0.15–0.5 Hz) appear to be under parasympathetic control and represent the effects of respiration. Spectral analysis techniques have confirmed that healthy aging is associated with reductions in baroreflex and parasympathetic modulation of HR, with a relatively greater loss of the high-frequency parasympathetic component. The overall complexity of cardiovascular signals, which represents the integration of autonomic, endocrine, hemodynamic, and other control networks, can be quantified using a variety tools derived from nonlinear dynamics, fractals, and complex systems theory. A decline in the complexity of cardiovascular dynamics is associated with aging and is a marker of cardiovascular disease [7].

NEUROTRANSMITTERS Just as the age-related increase in plasma norepinephrine level is related to increased spillover, and decreased clearance at adrenergic nerve terminals, so must the effect of aging on any neurotransmitter be interpreted with regard to changes in its production and clearance. Other neurotransmitters that influence autonomic nervous system functions have received little attention in aging humans. In the brain, a decline in dopamine and norepinephrine is related to a loss of dopaminergic and noradrenergic neurons in the substantia nigra and locus ceruleus. The clinical implications of these changes are not fully understood, but may lead to the slowing of gait and cognitive dysfunction commonly seen in elderly people. The enzymes choline acetyltransferase and acetylcholinesterase, which are responsible for synthesis and degradation of acetylcholine, respectively, decrease in the cerebral cortex with aging. Furthermore, muscarinic and nicotinic receptors have been reported to decrease in cortical structures. These findings provide indirect evidence for a decrease in central cholinergic neurotransmission with normal aging.

CARDIAC BETA-ADRENERGIC RECEPTORS The age-related decrease in chronotropic response to sympathetic stimulation has been attributed to multiple molecular and biochemical changes in beta-receptor coupling and postsynaptic signaling. The number of beta receptors in cardiac myocytes is unchanged with advancing age, but the affinity of beta receptors for agonists is reduced. Postsynaptic changes with aging include

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a decrease in the activity of G protein and the adenylate cyclase catalytic unit, and a decrease in cAMP-dependent phosphokinase-induced protein phosphorylation. As a result of these changes, G protein-mediated signal transduction is impaired. The decrease in cardiac contractile response to sympathetic stimulation has been studied in rat ventricular myocytes, where it appears to be related to decreased influx of calcium ions through sarcolemmal calcium channels and a reduction in the amplitude of the cytosolic calcium transient after beta-adrenergic stimulation. These changes are similar to those seen in receptor desensitization caused by prolonged exposure of myocardial tissue to betaadrenergic agonists. Thus, age-associated alterations in beta-adrenergic response may be caused by desensitization of the adenylate cyclase system in response to a chronic increase in sympathetic activity.

VASCULAR REACTIVITY Aging is associated with increased vascular stiffness and peripheral resistance, and reduced vasoconstriction in response to sympathetic stimulation [8]. However, the impairment in arterial alpha-adrenergic vasoconstriction is reversible by suppression of sympathetic nervous system activity with guanadrel [8]. This remarkable observation suggests that the abnormality in alpha-adrenergic response also represents receptor desensitization caused by heightened sympathetic nervous system activity. It also indicates that some of the physiologic changes associated with aging may be reversible. Endothelium-dependent vasodilatation is progressively impaired with aging. The vasorelaxation response of both arteries and veins to infusions of the beta-adrenergic agonist isoproterenol is attenuated in elderly people. This may also be due to abnormal beta-adrenergic receptor signaling and cAMP production in vascular smooth muscle cells.

VOLUME REGULATION Aging is associated with a progressive decline in plasma renin, angiotensin II, and aldosterone levels, and increases in atrial natriuretic peptide, all of which promote salt wasting by the kidney. In many healthy elderly individuals there is also a defective plasma vasopressin response to upright posture. These physiologic changes predispose elderly people to volume contraction and hypotension. Furthermore, healthy elderly individuals do not experience the same sense of thirst as younger subjects when they become hyperosmolar during water deprivation or hypertonic saline infusion. Consequently, dehydration may develop rapidly during conditions such as an acute illness, preparation for a medical procedure, diuretic therapy, or exposure to a warm climate when fluid losses are increased and access to oral fluids is limited.

273

CEREBRAL AUTOREGULATION The process of cerebral autoregulation maintains a relatively constant cerebral blood flow during changes in perfusion pressure. This is accomplished by active vasodilatation of the cerebral microvasculature during hypotensive stimuli and by vasoconstriction during increases in BP. Cerebral autoregulation is generally effective within a mean arterial pressure range of 80 to 150 mmHg and is well-preserved in healthy aging. However, the lower pressure threshold for cerebral hypoperfusion may be shifted higher in patients with hypertension. There is some evidence that the treatment of hypertension may restore this threshold to a more normal range. Cerebral blood flow is also dependent on the arterial concentration of carbon dioxide, such that CO2 inhalation causes vasodilatation and hyperventilation causes vasoconstriction. This CO2 vasoreactivity is mediated by nitric oxide and is an indicator of endothelial function in the cerebral vasculature. Vasoreactivity is reduced with aging and cardiovascular risk factors, such as hypertension and diabetes. Elderly patients with these vascular risk factors are therefore vulnerable to symptomatic cerebral hypoperfusion if BP falls below the autoregulated range. Recent studies suggest that chronic cerebral hypoperfusion is associated with the development of periventricular white matter hyperintensities and their associated impairments in gait and cognition [3,9].

References [1] Gupta V, Lipsitz LA. Orthostatic hypotension in the elderly: diagnosis and treatment. Am J Med Oct 2007;120(10):841–7. [2] Jansen RW, Lipsitz LA. Postprandial hypotension: epidemiology, pathophysiology, and clinical management. Ann Intern Med Feb 15 1995;122(4):286–95. [3] Novak V, Hajjar I. The relationship between blood pressure and cognitive function. Nat Rev Cardiol 2010;7:686–98. [4] Masuo K, Mikami H, Ogihara T, Tuck ML. Changes in frequency of orthostatic hypotension in elderly hypertensive patients under medications. Am J Hypertens Mar 1996;9(3):263–8. [5] Lipsitz LA, Gagnon M, Vyas M, et al. Antihypertensive therapy increases cerebral blood flow and carotid distensibility in hypertensive elderly subjects. Hypertension Feb 2005;45(2):216–21. [6] Maddens M, Lipsitz LA, Wei JY, Pluchino FC, Mark R. Impaired heart rate responses to cough and deep breathing in elderly patients with unexplained syncope. Am J Cardiol Dec 1 1987;60(16): 1368–72. [7] Goldberger AL, Amaral LA, Hausdorff JM, Ivanov P, Peng CK, Stanley HE. Fractal dynamics in physiology: alterations with disease and aging. Proc Natl Acad Sci USA Feb 19 2002;99(Suppl 1):2466–72. [8] Hogikyan RV, Supiano MA. Arterial alpha-adrenergic responsiveness is decreased and SNS activity is increased in older humans. Am J Physiol May 1994;266(5 Pt 1):E717–724. [9] ten Dam VH, van den Heuvel DM, de Craen AJ, et al. Decline in total cerebral blood flow is linked with increase in periventricular but not deep white matter hyperintensities. Radiology Apr 2007;243(1):198–203. [10] Kearney MT, Cowley AJ, Stubbs TA, Evans A, Macdonald IA. Depressor action of insulin on skeletal muscle vasculature: a novel mechanism for postprandial hypotension in the elderly. J Am Coll Cardiol Jan 1998;31(1):209–16.

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57 Exercise Qi Fu, Benjamin D. Levine INTRODUCTION Physical activity is a key component of a healthy lifestyle. Increased physical activity or exercise training is not only protective against cardiovascular disease, type II diabetes, and obesity, but also effective in improving functional capacity in patients with autonomic disorders. Exercise training also improves mental health, helps to prevent depression, and promotes or maintains positive self-esteem. Endurance or “dynamic” exercise, such as running, jogging, cycling, swimming, rowing, or walking, and strength training, such as weight-lifting are two major forms of exercise though many activities include components of both. In this chapter, we predominately focus on endurance exercise. There is now convincing evidence that the protective and therapeutic effects of exercise training are related, in a substantive fashion, to effects on the autonomic nervous system [1]. In addition, training-induced improvement in vascular endothelial function, blood volume expansion, cardiac remodeling, insulin resistance, and renaladrenal function may also contribute to the protection and treatment of cardiovascular, metabolic, and autonomic disorders.

ACUTE EXERCISE The cardiovascular response during exercise is initiated by a feed-forward mechanism, termed “central command”, which involves higher brain centers such as the motor cortex, hypothalamic and mesencephalic locomotor regions that activate parallel circuits controlling locomotor, cardiovascular, and ventilatory functions [2]. As exercise continues, both mechanical and metabolic signals from active skeletal muscle provide feedback to cardiovascular centers in the brain through group III and IV muscle afferents, the so-called “exercise pressor reflex”, to precisely match systemic oxygen delivery with metabolic demand [2,3]. Vascular resistance decreases (via local metabolic factors) to facilitate increases in muscle perfusion and cardiac output increases proportionate with oxygen uptake, thus allowing the maintenance of or even increase in mean arterial pressure. An overview of the neural regulation of the cardiovascular system during exercise is shown in Figure 57.1.


During exercise, the uptake and transport of oxygen is required for oxidative phosphorylation and the efficient production of adenosine triphosphate (ATP) to support the metabolic demands of the body [4]. One of the most inviolate relationships in all of exercise physiology is that between oxygen uptake and cardiac output. Regardless of age, sex, or the presence of various disease states, in general, about 56 liters of cardiac output are required for every liter of oxygen uptake above rest (Fig. 57.2) [4,5]. When this relationship is depressed, it may be a sign of severe underlying disease with impending decompensation. Conversely, when it is exaggerated, like in patients with metabolic myopathies, it gives strong clues to the processes regulating cardiac output. Oxygen uptake is a function of the triple-product of heart rate and stroke volume (i.e., cardiac output) and arterial-mixed venous oxygen difference (the Fick principle, Fig. 57.2) [5]. The degree to which each of the variables can increase determines the upper limit for whole-body oxygen consumption, and this limit is called the maximal oxygen uptake (VO2max) [6]. Maximal heart rate and maximal arteriovenous difference are usually relatively similar among individuals of similar ages despite large differences in physical fitness; therefore, the factor most commonly accounting for the different values of VO2max in different individuals is stroke volume [5]. At rest arteriovenous oxygen difference is normally 4.5 ml/100 ml/min (approximately 23% extraction), and at VO2max this difference is commonly close to 16 ml/100 ml/min (about 80 to 85% extraction) [5]. Cardiovascular responses, such as heart rate and blood pressure, are more closely related to the relative metabolic demands than to the absolute demands. At low levels of exercise, heart rate increases almost exclusively via vagal withdrawal, with little evidence for systematic increases in sympathetic activity until the intensity of exercise is at or above the maximal steady state (ventilatory or lactate thresholds) [5]. The key determinant of the magnitude of the heart rate response to exercise is the relative intensity as well as the absolute amount of muscle mass engaged, while central command plays an essential role in the increase in heart rate during exercise [4]. Gravity plays a critical role in determining the distribution of blood within the cardiovascular system, and body posture markedly affects the relative importance of changes in stroke volume [4]. In the upright position,

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FIGURE 57.1 Autonomic neural control of the cardiovascular response during exercise. Central command initiates the exercise pressor response, which is maintained and augmented via feedback from baroreceptors, as well as by stimulation of skeletal muscle mechanically and metabolically sensitive receptors. After integration in the brain, efferent responses via the parasympathetic (vagal) and sympathetic nervous systems result in increased heart rate and contractility, vasoconstriction in non-exercising (inactive) muscle, and vasodilation in exercising (active) muscle beds mediated by release of local vasodilating substances (“functional sympatholysis”). Adapted with permission from Levine [4], originally developed by J. Mitchell.

stroke volume is only about one-half its value in the supine position due to blood pooling in the legs and a reduction in left ventricular end-diastolic volume. At the onset of exercise, the pumping action of skeletal muscle acts to augment venous return substantially, and stroke volume normally increases 50% via the Starling mechanism [4]. Maximal stroke volume in non-athletic individuals is achieved at relatively low levels of exercise intensity (approximately 50% of maximal oxygen uptake), as pericardial constraint serves to limit left ventricular end-diastolic volume. In general, patients with autonomic disorders have low levels of VO2max and blunted cardiovascular response during exercise. It has been found that patients with pure autonomic failure and multiple system atrophy have an abnormal fall in blood pressure during exercise [7,8], which is presumably attributable to reduced sympathetic nerve activity and blunted α-adrenergic vasoconstriction in nonexercising skeletal muscle in the context of exercise induced vasodilation [9]. Conversely, patients with baroreflex failure have an excessive increase in blood pressure during exercise, which is probably due to impaired baroreflex buffering [10].

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FIGURE 57.2 Relationship between the increase in oxygen uptake (VO2) and the corresponding increase in cardiac output (Qc) during exercise in humans, which in most cases is 6/1. VO2 is a function of the triple-product of heart rate (HR), stroke volume (SV), and arterial-mixed venous oxygen difference (A-V O2 Difference). Adapted with permission from Levine [4].

Both central command and the exercise pressor reflex are important in determining the cardiovascular response during exercise, while dynamic interactions between these

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Sympathetic tone Baroreflex sensitivity

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Endothelial function Hemoglobin mass Insulin resistance

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FIGURE 57.3 Effects of exercise training on the autonomic nervous system, cardiovascular system, and renal-adrenal system in humans.

feed-forward and feed-back circuits are associated with beneficial adjustments in the sympathetic and parasympathetic nervous systems. The beneficial effects can be observed soon after the initiation of exercise training and are sustained as long as activity is continued. Since the sympathetic nervous system is activated during each bout of exercise, repeated activation of this system may result in an attenuation of sympathetic nerve activity. Numerous studies have shown that regular exercise can improve cardiac autonomic balance (i.e., increasing parasympathetic while decreasing sympathetic regulation of the heart), as well as increase the sensitivity of the baroreflex [11]. Additionally, exercise training can result in cardiac remodeling (i.e., increased cardiac size and mass, and improved cardiac function). It has been demonstrated that training increases the release of nitric oxide through shear stress during exercise, and chronic increases in nitric oxide lead to functional and histological alterations of vascular endothelium, causing enhanced vascular structure and function. Greater glucose delivery occurs with exercise training due to increased muscle blood flow and capillary density. An improvement in insulin resistance has been found after exercise training in humans. Training-induced muscle adaptations appear to be important in attenuating insulin mediated sympathetic activation, and may be especially enhanced by strength training which increases overall muscle mass [12]. Exercise training has also been shown to improve aerobic capacity and vascular conductance, and

lower body fat, each of which could contribute to a reduction in blood pressure. Many human studies have found that exercise training increases total hemoglobin mass, red blood cell volume, plasma volume, and blood volume. Recent research has reported that training may also improve renal-adrenal function and decrease circulating levels of angiotensin II [13]. Figure 57.3 depicts the beneficial effects of exercise training in humans.

EXERCISE AS A NON-DRUG THERAPY There is abundant evidence showing that regular exercise decreases the risk of cardiovascular disease, hypertension, colon and breast cancer, type II diabetes, and obesity. Numerous clinical studies have proven that exercise training can be used therapeutically to restore the autonomic function towards normal, and thus, contribute to an improvement in outcome. For example, it has been reported that exercise training is effective in treating patients with chronic heart failure, myocardial infarction, or after coronary artery bypass surgery by increasing vagal modulation and decreasing sympathetic tone. Additionally, exercise training seems to be effective in the prevention of sudden cardiac death by augmenting baroreflex sensitivity and heart rate variability [14]. Many clinical investigations have shown that exercise training improves functional capacity in patients with

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autonomic disorders, such as Parkinson’s disease, stroke, multiple sclerosis, spinal cord injury, Guillain–Barré syndrome, muscular dystrophy, or metabolic myopathies. Although the effects of exercise training on orthostatic tolerance in healthy individuals are controversial, increased orthostatic tolerance after mild to moderate training has been found in patients with unexplained syncope or orthostatic hypotension [15]. Increased baroreflex sensitivity and decreased frequency of syncopal episodes after exercise training were observed in patients with neurally mediated syncope [16]. In addition, it was reported that exercise training had a beneficial effect on physiological and subjective parameters in patients with chronic fatigue syndrome [17]. Fu et al. [18] have found that short-term (i.e., 3 months) exercise training improves or even cures the postural orthostatic tachycardia syndrome (POTS, also called chronic orthostatic intolerance) in most patients; more importantly, patient quality of life assessed by the 36-Item Short-Form Health Survey is significantly improved in all the patients after training. Recent research has demonstrated that exercise training improves older adults’ cognitive function. Exercise has also been found to be neuroprotective in many neurodegenerative and neuromuscular diseases in humans. Given the beneficial effects of exercise training, the American Heart Association, the US Surgeon General, the Centers for Disease Control and Prevention, and the American College of Sports Medicine recommend at least 30 minutes per day of at least moderate-intensity exercise, including brisk walking, jogging, cycling, swimming, or running on most, and preferably all, days of the week [19]. Physicians’ advice to increase physical activity can be a strong motivator to patients, and advice conveyed as a written prescription may enhance success. Supervised exercise training is preferable to maximize functional capacity. Heart rate can be used as an easily measured estimate of relative exercise intensity, and the target training heart rate is usually set at approximately 75% of the maximal heart rate [(take 220–age) 5 beats/min]. However, it is important to emphasize that these are only guidelines that in some patients, such as those who are taking beta-blockers or other medications or with underlying autonomic disorders, may affect the heart rate response to exercise and may not accurately reflect exercise intensity. For patients with autonomic disorders, heat and body temperature during exercise may compromise blood pressure further (by further increases in vascular conductance); cooling the skin or semi-recumbent exercise is recommended. For patients with POTS or orthostatic intolerance, exercise training should be initiated by using a recumbent bike, rowing, or swimming [18]. The use of only semi-recumbent exercise at the beginning is a critical strategy, allowing patients to exercise while avoiding the upright posture that elicits their symptoms. As the patients become relatively fit, the duration and intensity of exercise should be progressively increased, and upright exercise (e.g., upright bike, walking on the treadmill, or jogging) can be gradually added as tolerated [18].

CONCLUSION A sedentary lifestyle is considered to be one of the most important modifiable risk factors for morbidity and mortality in humans. Physical activity or exercise training is necessary to maintain overall health and functional capacity, and it plays a crucial role in the prevention of cardiovascular disease, sudden cardiac death, hypertension, type II diabetes, colon and breast cancer, and obesity. Exercise training can be therapeutic for patients with orthostatic intolerance, syncope, or POTS. In addition, exercise training improves mental health, helps to prevent depression, and promotes or maintains positive self-esteem. Adaptations involving the autonomic nervous system play a large role in the protective and therapeutic effects of exercise training. Moderate-intensity exercise at least 30 minutes per day and at least 5 days per week is recommended for the vast majority of people. Supervised exercise training is preferable to maximize functional capacity, and may be particularly important in patients with autonomic disorders.

References [1] Joyner MJ, Green DJ. Exercise protects the cardiovascular system: effects beyond traditional risk factors. J Physiol 2009;587:5551–8. [2] Mitchell JH. Wolffe JB memorial lecture. Neural control of the circulation during exercise. Med Sci Sports Exerc 1990;22:141–54. [3] Michelini LC, Stern JE. Exercise-induced neuronal plasticity in central autonomic networks: role in cardiovascular control. Exp Physiol 2009;94:947–60. [4] Levine BD. 2001 Exercise physiology for the clinician. In: Exercsie and sports cardiology. Medical Publishing Division: McGraw-Hill. p. 3–29. [5] Rowell LB. Central circulatory adjustments to dynamic exercise. Human Cardiovascular Control 1993:162–203. [6] Levine BD. VO2max: what do we know, and what do we still need to know? J Physiol 2008;586:25–34. [7] Humm AM, Mason LM, Mathias CJ. Effects of water drinking on cardiovascular responses to supine exercise and on orthostatic hypotension after exercise in pure autonomic failure. J Neurol Neurosurg Psychiatry 2008;79:1160–4. [8] Smith GD, Mathias CJ. Differences in cardiovascular responses to supine exercise and to standing after exercise in two clinical subgroups of Shy-Drager syndrome (multiple system atrophy). J Neurol Neurosurg Psychiatry 1996;61:297–303. [9] Schrage WG, Eisenach JH, Dinenno FA, Roberts SK, Johnson CP, Sandroni P, et al. Effects of midodrine on exercise-induced hypotension and blood pressure recovery in autonomic failure. J Appl Physiol 2004;97:1978–84. [10] Ziegler MG, Ruiz-Ramon P, Shapiro MH. Abnormal stress responses in patients with diseases affecting the sympathetic nervous system. Psychosom Med 1993;55:339–46. [11] Okazaki K, Iwasaki K, Prasad A, Palmer MD, Martini ER, Fu Q, et al. Dose-response relationship of endurance training for autonomic circulatory control in healthy seniors. J Appl Physiol 2005;99:1041–9. [12] Church TS, Blair SN, Cocreham S, Johannsen N, Johnson W, Kramer K, et al. Effects of aerobic and resistance training on hemoglobin A1c levels in patients with type 2 diabetes: a randomized controlled trial. JAMA 2010;304:2253–62. [13] Rush JW, Aultman CD. Vascular biology of angiotensin and the impact of physical activity. Appl Physiol Nutr Metab 2008;33:162–72.

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[14] La Rovere MT, Bersano C, Gnemmi M, Specchia G, Schwartz PJ. Exercise-induced increase in baroreflex sensitivity predicts improved prognosis after myocardial infarction. Circulation 2002;106:945–9. [15] Mtinangi BL, Hainsworth R. Increased orthostatic tolerance following moderate exercise training in patients with unexplained syncope. Heart 1998;80:596–600. [16] Gardenghi G, Rondon MU, Braga AM, Scanavacca MI, Negrao CE, Sosa E, et al. The effects of exercise training on arterial baroreflex sensitivity in neurally mediated syncope patients. Eur Heart J 2007;28:2749–55. [17] Joosen M, Sluiter J, Joling C, Frings-Dresen M. Evaluation of the effects of a training programme for patients with prolonged fatigue

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on physiological parameters and fatigue complaints. Int J Occup Med Environ Health 2008;21:237–46. [18] Fu Q, Vangundy TB, Galbreath MM, Shibata S, Jain M, Hastings JL, et al. Cardiac origins of the postural orthostatic tachycardia syndrome. J Am Coll Cardiol 2010;55:2858–68. [19] Marcus BH, Williams DM, Dubbert PM, Sallis JF, King AC, Yancey AK, et al. Physical activity intervention studies: what we know and what we need to know: a scientific statement from the american heart association council on nutrition, physical activity, and metabolism (subcommittee on physical activity); council on cardiovascular disease in the young; and the interdisciplinary working group on quality of care and outcomes research. Circulation 2006;114:2739–52.

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58 Effects of High Altitude Luciano Bernardi INTRODUCTION Ascent to high altitude reduces the inspired partial pressure of O2, leading to hypobaric hypoxia. This requires a complex adaptive process (acclimatization) which, in its early phases is largely influenced by the autonomic nervous system. This process is complex and the integrated response depends upon a number of factors, including the extent, rate of ascent, and duration of hypoxic exposure. Acute responses are modified by chronic adaptations that restore circulatory function towards normoxic levels over time periods that may range from a few days or weeks for the sea level sojourner, to years for the high altitude native. Tolerance to hypoxia varies greatly amongst individuals, and subjects with a particular susceptibility may develop inappropriate responses leading to acute mountain sickness and even to life threatening conditions, such as high altitude cerebral and pulmonary edema (HAPE). Sympathetic over activity seems to play an important role in HAPE.

EFFECTS OF ACUTE HYPOXIA The main oxygen sensors involving the autonomic nervous system response are the peripheral chemoreceptors, located in the carotid body and in the arch of the aorta. The carotid sensors respond mainly to a lowering in PaO2 (aortic sensors respond mainly to CaO2). Peripheral chemoreceptor afferents synapse in a primary cardiovascular control center located within nucleus tractus solirarii (NTS) of the dorsomedial medulla. The NTS relays this input via projections terminating in the rostral ventrolateral medulla. Stimulation of neurons in the ventrolateral medulla by hypoxic stimulation of peripheral chemoreceptors leads to hyperventilation, and excitation of both sympathetic and parasympathetic neurons. However, this response is modified by numerous secondary influences such as hyperventilation and hypocapnia [1]. The hyperventilation, which is proportional to the decreases in PaO2, stimulates lung stretch receptors (during inspiration), whose final response is an inhibition of cardiac vagal tone. In addition, hyperventilation induces hypocapnia, which attenuates the sympathetic activation associated with the peripheral chemoreflex, and also reduces the stimulus from the central chemoreflex – so called hypocapnic


braking effect [2]. Due to the increase in ventilation, the respiratory changes in stroke volume are also increased, and this contributes to increase baroreceptor loading and unloading during expiration and inspiration, respectively. The baroreflexes remain able to maintain adequate cardiovascular modulation during chemoreflex activation [3]. Thus arterial baroreceptors, central/peripheral chemoreceptors and pulmonary stretch receptors all may modulate the sympathetic activation associated with peripheral chemoreflex excitation [1]. Although complete inability to increase ventilation in response to hypoxia (causing dyspnea) is generally admitted as a contraindication to travel to high altitude, the extent of these autonomic/ventilatory responses are not directly proportional to performance at high altitude. Contrary to general belief, climbers able to reach extreme altitudes (Everest and K2 summits) without oxygen supplementation were characterized by a high respiratory efficiency, but only moderate increase in ventilatory responses, and only moderate sympathetic activation during acclimatization at 5200 m. Other climbers unable to reach extreme altitudes were characterized by higher ventilatory responses and higher sympathetic activation. This suggested that better respiratory efficiency was associated, in part, with a reduction in the cardiorespiratory autonomic reactions at intermediate altitudes thereby maintaining a higher reserve for the more demanding tasks of extreme altitudes [4]. The purpose of this combined cardiovascular and autonomic response is to maintain systemic and regional oxygen despite the fall in PaO2 [5]. This is achieved by an increase in cardiac output (proportional to the altitude), mainly due to an increase in heart rate: tachycardia results from increased sympathetic activity due to chemoreceptor stimulation, and probably also from vagal withdrawal, as a consequence of hyperventilation (central pathways, feedback from pulmonary stretch receptors, and hypocapnia) [2]. Acute hypoxia provokes vasodilation in all vascular beds, except the lung (where hypoxic vasoconstriction in susceptible subjects is often exaggerated and may be a crucial factor leading to HAPE). Peripheral vasodilation, in addition to increased in heart rate and cardiac output causes a remarkably effective redistribution of flow to vascular beds with the greatest metabolic demand, similar to the response seen during physical exercise [6]. The increment in blood flow associated with hypoxia, at rest and during exercise,

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precisely matches the decrease in arterial oxygen content, keeping O2 delivery to tissues constant. Both endothelial and neurally derived NO may be involved in this integrated autonomic and cardiovascular response to hypoxia [1,7]. Initially, the level of blood pressure depends on the balance between hypoxic-induced vasodilation and the vasoconstrictor effect of peripheral chemoreceptor reflex sympathetic activation; in this acute phase it shows little change. When peripheral vasodilation is not effectively compensated by sympathetic activity syncope may occur; this is more frequent after very rapid ascent to high altitude. Plasma catecholamines are also involved in the regulation of blood pressure at altitude, but their changes are still not fully understood. At the moment it remains accepted that adrenaline increases predominantly in the early phases of exposure to high altitude, whereas noradrenaline predominates after several days of exposure. These modifications are compatible with the delayed increase in blood pressure, observed in the majority of subjects during sojourn to high altitude [8]. Hypoxia also causes cerebral vasodilation, which is partially counteracted by hypocapnia, but can lead to a pulsating headache, which is a common symptom upon arrival at high altitude.

EFFECTS OF CHRONIC HYPOXIA Over a period of days to weeks, reduction in plasma volume and later increase in red cell mass increase hemoglobin concentration and CaO2 (also with a contribution of hyperventilation). The increased CaO2 reduces the hyperdynamic circulation and both cardiac output and peripheral blood flow returns towards normal. However, the still low PaO2 maintains peripheral chemostimulation and hence sympathetic activation. This results in a progressive increase in vascular resistance and blood pressure, as the increase in CaO2 reduces the vasodilatory effect of hypoxia. The arterial baroreflexes, although diminished, continue to function at high altitude despite the marked increase in chemoreflexes [3], suggesting a central resetting of the baroreflex with exposure to hypoxia [1]. Cardiac output is consistently depressed after acclimatization to high altitude, often below values measured at sea level; this is due to a decrease in stroke volume (with preserved contractile function) due, in turn, to a reduction in left ventricular end-diastolic volume, which results from a 20–30% reduction in plasma volume. Sympathetic activation and slower acting neurohormones, such as angiotensin, aldosterone or vasopressin may be important in sustaining the reduction in plasma volume in chronic hypoxia. This relative dehydration unloads low-pressure cardiopulmonary receptors as well as arterial baroreceptors (by reducing aortic dimensions, and by decreasing pulsatile flow through arterial baroreceptors [1]). Chronic sympathetic hyperactivity leads to downregulation of peripheral β-adrenoceptors, with a gradual diminution of the heart rate response to sympathetic

activation [9]. After a prolonged sojourn to high altitude sympathetic activity diminishes and blood pressure normalizes. After years of acclimatization, high altitude natives appear similar to sea level natives. While this response is typically observed at altitudes varying from 3000 to 5000 m, at extreme altitudes the acclimatization process cannot normalize arterial oxygen content; as a result, peripheral vasodilation and reduction in peripheral resistance persist as in acute hypoxia.

AUTONOMIC NERVOUS SYSTEM AND ALTITUDE ILLNESS HAPE is associated with severe pulmonary hypertension probably with uneven distribution, thus allowing pulmonary areas of hyperperfusion and hypertension; this leads to pulmonary capillary leak, endothelial dysfunction, possibly a late inflammation and alveolar edema. Although the origin of HAPE is still debated, recent studies in the Italian/Swiss Alps, on patients with HAPE susceptibility, have confirmed the essential role of pulmonary hypertension. Such patients have a marked increase in sympathetic activity during acute exposure to hypoxia, even before the development of HAPE. Sympathetic activation may thus play at least a facilitatory role in HAPE, presumably by contributing to the development of pulmonary hypertension in susceptible individuals [10].

References [1] Levine BD. Mountain Medicine and the autonomic nervous system. In: Appenzeller O, editor. Handbook of clinical neurology Vol 75 (31): the autonomic nervous system. Amsterdam: Elsevier; 2000. p. 259–80. Part II [2] Somers VK, Mark AL, Zavala DC, Abboud FM. Influence of ventilation and hypocapnia on sympathetic nerve responses to hypoxia in normal humans. J Appl Physiol 1989;67:2095–100. [3] Bernardi L, Passino C, Spadacini G, Calciati A, Robergs R, Greene ER, et al. Cardiovascular autonomic modulation and activity of carotid baroreceptors at altitude. Clin Sci 1998;95:565–73. [4] Bernardi L, Schneider A, Pomidori L, Paolucci E, Cogo A. Hypoxic ventilatory response in successful extreme altitude climbers. Eur Respir J 2006;27:165–71. [5] Wolfel EE. Sympatho-adrenal and cardiovascular adaptation to hypoxia. In: Sutton JR, Houston CS, Coates G, editors. Hypoxia and molecular medicine. : Queen City Burlington; 1993. p. 62–80. [6] Cerretelli P, Marconi C, Deriaz O, Giezendanner D. After effects of chronic hypoxia on cardiac output and muscle blood flow at rest and exercise. Eur J Appl Physiol 1984;53:92–6. [7] Thomas GD, Victor RG. Nitric oxide mediates contraction-induced attenuation of sympathetic vasoconstriction in rat skeletal muscle. J Physiol (Lond) 1998;506:817–26. [8] Hainsworth R, Drinkhill MJ, Rivera-Chira M. The autonomic nervous system at high altitude. Clin Auton Res 2007;17:13–19. [9] Voelkel NF, Hegstrand L, Reeves JT, McMurtry IF, Molinoff PB. Effects of hypoxia on density of beta-adrenergic receptors. J Appl Physiol 1981;50:363–6. [10] Scherrer U, Allemann Y, Jayet PY, Rexhaj E, Sartori C. High altitude, a natural research laboratory for the study of cardiovascular physiology and pathophysiology. Progr Cardiovasc Dis 2010;52:451–5.

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59 Space Physiology Gilles Clément, Scott Wood INTRODUCTION

SPACE MOTION SICKNESS

Space medicine and space physiology are often viewed as two aspects of space life sciences, with the former being more operational, and the latter being more investigational. Space medicine tries to solve medical problems encountered during space missions. These problems include some adaptive changes to the space environment, including weightlessness, radiation, the absence of the 24-hour day/night cycle; as well as some non-pathologic changes that become maladaptive on return to Earth, such as muscle atrophy and bone demineralization. Space physiology tries to characterize body responses to space, especially weightlessness, reduced activity, and stress. It provides the necessary knowledge required for an efficient space medicine [1]. Space physiology is as old as the first flight of humans in a hot air balloon, when the symptoms of hypoxia were first discovered (at the expenses of the life of the pilot). The interest in this field of research kept growing along with the space program and the opportunities it provided for flying more and more humans in space on board capsules, shuttles, space stations, and soon suborbital space planes. The future of human space flight will inevitably lead to human missions to Mars. These missions will be of long duration (30 months) in isolated and somewhat confined habitats, with the crew experiencing several transitions in levels of gravity, dangerous radiation, and the challenges of landing and living on their own on another planet. Many research questions must be addressed before safely sending humans to explore Mars, when our current knowledge on humans in space does not exceed 14 months in only one individual. A human research roadmap for tackling these research questions has been recently detailed by NASA [http://humanresearchroadmap.nasa .gov/] [2]. The effects of space flight conditions on the autonomic nervous system could be at the origin of two medical issues experienced by a significant number of astronauts. These issues are space motion sickness immediately after entering weightlessness or after returning to Earth’s gravity, and post-flight orthostatic intolerance. As discussed below, due to shared neural pathways, clinical treatment of one condition often interacts with the other condition.

Space motion sickness is a special form of motion sickness, which include such symptoms as depressed appetite, a nonspecific malaise, gastrointestinal discomfort, nausea, and vomiting. Symptoms have their onset from minutes to hours after orbital insertion. Excessive head movement early on-orbit generally increases these symptoms. Symptom resolution usually occurs between 30 and 48 hours, with a reported range of 12 to 72 hours, and recovery is rapid (Fig. 59.1). Even if someone doesn’t literally get sick to their stomach, they may feel a less dramatic motion-sickness effect known as “sopite syndrome”, characterized by lethargy, mental dullness, and disorientation. Many astronauts have noticed this effect, which they call “mental viscosity” or “space fog”. About two-thirds of the Space Shuttle astronauts and Soyuz cosmonauts experience some symptoms of space motion sickness. There are no statistically significant differences in symptom occurrence between pilots versus non-pilots, males versus females, different age groups, or first time flyers versus veterans repeat flyers. An astronaut’s susceptibility to space motion sickness on his/her first flight correctly predicted susceptibility on the second flight in 77% of the cases. In other words, astronauts who have been sick during their first flight are likely to be sick again during their subsequent flights [3]. Many astronauts returning to Earth after long-duration stays on board the International Space Station now experience symptom recurrence at landing (Fig. 59.1). The severity of the symptoms and the functional recovery after the flight seem to be directly proportional to the time on orbit [4]. Head or full body movements made upon transitioning from microgravity to a gravitational field less than that on Earth, and vice versa, may not be as provocative. It is interesting to note that of the twelve Apollo astronauts who walked on the Moon, only three reported mild symptoms, such as stomach awareness or loss of appetite prior to extravehicular activities. None reported symptoms while in the one-sixth gravity of the lunar surface, and no symptoms were noted upon return to weightlessness after leaving the Moon surface. There are considerable individual differences in susceptibility to space motion sickness, and currently it is not


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FIGURE 59.1 Space motion sickness incidence (dashed line) and drug treatment (continuous line) during early in-flight and post-flight periods. The incidence (y-axis on the left) is measured on a scale from 0 (no symptoms) to 10 (vomiting). The drug treatment (y-axis on the right) is measured as the percentage of motion sickness medication taken by the crew compared to the other medication used during the same period. Note that flight rules restrict docking of spacecraft before flight day two and extravehicular activities before flight day three to allow for space motion sickness symptoms resolution.

possible to predict with any accuracy those who will have some difficulty with sickness while in space. Although anti-motion sickness drugs offer some protection, they may interfere with the adaptation process, and symptoms controlled by these drugs are experienced again once treatment ceases. This was observed for scopolamine, which resulted in a shift towards the use of promethazine. There have been anecdotal reports of medication usage prior to extravehicular activities, with concerns about cognitive and performance side effects associated with this usage. Other issues related to the adaptation of the central nervous system through the vestibular pathways include: (a) the perceptual effects and illusions of free-falling, visual reorientation illusions, and acrophobia episodes during extravehicular activity; (b) decreased sensorimotor performance and oscillopsia during re-entry; (c) disequilibrium and ataxia when standing and walking after landing; and (d) g-state flashbacks during unusual stimulation of the vestibular system during the re-adaptation period following landing [5]. A sensory conflict theory of motion sickness postulates that motion sickness occurs when patterns of sensory inputs to the brain are markedly re-arranged, at variance with each other, or differ substantially from expectations of the stimulus relationships in a given environment. In orbit and during re-entry, sensory conflict can occur in several ways. First, there can be conflicting information regarding tilt transmitted by the otoliths and the semicircular canals. Sensory conflict may also exist between the visual and vestibular systems during motion; the eyes transmit information to the brain indicating body movement, but no corroborating impulses are received from the otoliths, such as during car sickness. A third type of conflict may exist in space because of differences in perceptual habits and expectations. On Earth, we develop a neural

store of information regarding the appearance of the environment and certain expectations about functional relationships, e.g., the concepts of “up” and “down”. In space, these perceptual expectations are at variance, especially during the illusions described above.

ORTHOSTATIC INTOLERANCE It is conceivable that in the same way the central nervous system elaborates an internal representation of gravity for spatial orientation using several sensory inputs, the sympathetic activation and maintenance of blood pressure during orthostatism would be based on an internal representation of the intravascular pressure, previously built based on the same information as used for the internal representation of gravity. On return to Earth, post-flight orthostatic intolerance is characterized by a variety of symptoms that follow standing: lightheadedness, increase in heart rate, decrease in blood pressure, and pre-syncope or syncope. Both orthostatic intolerance and diminished exercise capacity become more severe with longer exposure to microgravity and require more lengthy recovery times after returning to Earth. Orthostatic intolerance affects approximately 30% of short-duration and 80% of long-duration crewmembers. This may pose a problem for “space tourists” during suborbital flight. They will be exposed to re-entry forces even higher that those experienced in the Space Shuttle or Soyuz capsules by well cardiovascular fit professional astronauts. Another threat is whether a debilitated crew can respond to an emergency upon landing [6]. The extent of orthostatic intolerance post-flight is variable and depends on the duration of the flight, individual differences in cardiovascular function among the

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astronauts, and the elapsed time after landing and method of post-flight testing. Recovery to the preflight level of orthostatic tolerance occurs within a day or so following flights of less than one-month's duration, but longer recovery is associated with longer duration flights. In contrast to space motion sickness, there is a well-known gender difference in orthostatic tolerance (see Chapters 106–108). Post-flight orthostatic intolerance is probably induced by multiple factors, including changes in hemodynamics, alterations in baroreceptor reflex gain, decreases in exercise tolerance and aerobic fitness, hypovolemia, and altered sensitivity of β-adrenergic receptors in the periphery [7]. Astronauts in orbit experience a headward shift of fluid at the onset of weightlessness resulting in facial puffiness and distended neck veins. This fluid shift causes distension of heart chambers, which in turn activates mechanisms associated with rapid blood volume reduction. The effects of a reduced blood volume are probably amplified by changes in the low-pressure venous system of the lower limbs and by impaired baroreflex function. Changes in the otolith signals, or their reinterpretation, may also be involved. Indeed, the removal of the normal head-to-foot gravity vector acts not only on fluid, because of the loss of hydrostatic pressure gradient, but also on the otolithic system. A central reinterpretation of otolith signals is presumably taking place during re-adaptation to Earth gravity. However, no data are available yet on the possible role of non-cardiovascular inputs controlling the activity of the sympathetic nervous system during or after space flight. Because the otolithic control of the cardiovascular system is supposed to compensate for head tilt coupled with the observation that otolith tilt reflexes generally vanish during adaptation to microgravity, it can be hypothesized that the otolithic control of the cardiovascular system will be altered after space flight. This alteration would then participate in cardiovascular deconditioning. If this hypothesis is confirmed, it could have potential consequences for the design of countermeasures preventing cardiovascular deconditioning. In this context, providing artificial gravity by using centrifugation in supine subjects with the head off-center might be an effective means for maintaining otolith sensitivity and preserving vestibulosympathetic reflexes [8].

CLINICAL INTERACTIONS The pharmacological intervention for motion sickness and orthostatic intolerance has been an issue after returning from space flights. As stated above, promethazine has been the preferred medication by space medicine for treatment of motion sickness. However, promethazine significantly increases the incidence of orthostatic hypotension, presumably via inhibition of sympathetic responses [9]. Midrodrine has been successful in preventing orthostatic hypotension post-flight. However, its effects are negated by promethazine and this combined usage can also lead to akathisia. Other countermeasures, such as fluid loading and compression garments, have provided successful nonpharmaceutical approaches for orthostatic intolerance. The unique challenges provided by adaptation to the conditions of space flight highlight the interactions in the autonomic nervous system, particularly for those physiological mechanisms that contribute to cardiorespiratory regulation and spatial awareness during changes in posture.

References [1] Clément G. Fundamentals of space medicine, 2nd ed. New York: Springer; 2011. [2] Clément G, Reschke MF. Neuroscience in space. New York: Springer; 2008. [3] Bacal K, Bilica R, Bishop S. Neurovestibular symptoms following space flight. J Vestib Res 2003;13:93–102. [4] Ortega HJ, Harm DL. Space and entry motion sickness. In: Barratt M, Pool S, editors. Principles of clinical medicine for space flight. New York: Springer; 2008. p. 211–22. [Chapter 10] [5] Paloski WP, Oman CM, Bloomberg JJ, Reschke MF, Wood SJ, Harm DL, et al. Risk of sensory-motor performance failures affecting vehicle control during space missions: A review of the evidence. J Gravit Physiol 2008;15:1–29. [6] Hamilton D. Cardiovascular disorders. In: Barratt M, Pool S, editors. Principles of clinical medicine for space flight. New York: Springer; 2008. p. 317–60. [Chapter 16] [7] Yates BJ, Kerman IA. Post-spaceflight orthostatic intolerance: Possible relationship to microgravity-induced plasticity in the vestibular system. Brain Res Rev 1998;28:73–82. [8] Antonutto G, Clément G, Ferretti G, Linnarsson D, Pavy-Le Traon A, Di Prampero P. Physiological targets of artificial gravity: The cardiovascular system. In: Clément G, Bukley A, editors. Artificial gravity. Hawthorne and Springer: New York: Microcosm Press; 2007. p. 137–62. [Chapter 5] [9] Shi SJ, Platts SH, Ziegler MG, Meck JV. Effects of promethazine and midodrine on orthostatic tolerance. Aviat Space Environ Med 2011;82:9–12.

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60 Hypothermia and Hyperthermia Nisha Charkoudian INTRODUCTION The human body is remarkably capable of maintaining core temperature within a few tenths of a degree of 37°C over a very wide range of environmental exposures and activity levels. Sympathetic neural mechanisms controlling skin blood flow and sweating are central to these abilities, in addition to shivering which increases heat production during cold exposure.

CENTRAL NEURAL CONTROL OF THERMOREGULATION The main area which controls body temperature within the central nervous system is the preoptic area of the anterior hypothalamus (PO/AH). This region contains temperature-sensitive and temperature-insensitive neurons which interact to regulate the systemic thermoregulatory response to a given environment or situation (for review, see Boulant [1]). For example, when body temperature increases, “warm-sensitive” neurons in the PO/AH region are activated. This then initiates a series of neural events which activates heat dissipation via cutaneous vasodilation and sweating. Changes in the activity of neurons in the PO/AH region are linked to appropriate increases or decreases in heat dissipation, as discussed below, as well as to increases in heat generation (shivering) during body cooling [1]. Thermoregulatory control at the PO/AH occurs via both directly sensed temperature and via integration with input from peripheral thermosensory afferents which give information about peripheral body temperature, in particular surface temperature [1,8]. Thus, skin temperature can have a significant influence on the rate of a thermoregulatory response (e.g., sweating) for a given level of core temperature. A practical example is that exercising in a cool environment might require a lower sweating rate compared to the same exercise in a warmer environment, even if core temperature is similar in the two conditions. Thus, the skin temperature influence can contribute to the optimization of the integrated sweating response. The relative contributions of core and surface temperature can vary, but in general the relative influence of core to mean skin temperature is about 9:1 [1,8].


The neural network involving the PO/AH and other inputs results in a set-point for thermoregulation, and changes in the activity of the network when body temperature goes above or below that set-point result in activation of efferent neuronal activation of appropriate thermoregulatory pathways [1]. The set-point concept is related to the concept of a threshold for activation of a certain thermoregulatory effector. The temperature (most often core temperature) at which a certain thermoregulatory response (e.g., sweating) is initiated is referred to as the threshold for the activation of that response. The gain or sensitivity of the response refers to the incremental changes (slope) of the response relative to body temperature with further changes in body temperature beyond the threshold [1,2,8]. The integrated control of body temperature acts as a classical negative feedback loop, as summarized in Figure 60.1. Changes in body temperature are sensed peripherally and centrally, and appropriate changes in heat dissipation and/or heat generation act to minimize or reverse those changes. New information subsequently feeds back to the PO/AH which then appropriately modulates the integrated thermoregulatory response.

REGULATION OF BODY TEMPERATURE IN THERMONEUTRAL ENVIRONMENTS During most daily activities in healthy people, only minor changes in heat dissipation are required to maintain normothermia during changes in activity or when one moves to a slightly warmer or cooler environment. This range of environment/activity combinations has been referred to as the “neutral zone” of thermoregulation [7]. The minor changes in thermoregulation required for this “zone” are brought about primarily by small changes in skin blood flow mediated by modulations in the activity of sympathetic noradrenergic vasoconstrictor nerves innervating the cutaneous circulation [2,3,7]. These cutaneous vasoconstrictor nerves exhibit tonic activity in normothermia, and are responsible for keeping normothermic skin blood flow relatively low (whole body skin blood flow during normothermia is ~250 mL/min). In a slightly warmer environment, small decreases in vasoconstrictor activity cause small “passive” vasodilation (and increased heat loss), and conversely in a slightly cooler environment,

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60. HyPoTHERmIA And HyPERTHERmIA

increases in vasoconstrictor activity decrease skin blood flow (and conserve heat).

HYPOTHERMIA During exposure to cold environments, the goal of the thermoregulatory response is to maintain core body temperature, or to minimize any drops in body temperature that may occur (Fig. 60.1, right side). The two physiological “strategies” for the maintenance of core temperature are (1) to decrease dissipation of heat from the body to the environment; and (2) when necessary, to increase metabolic generation of body heat to offset loss of heat to the environment. The initial response to exposure to a cold environment is sympathetic neurally-mediated vasoconstriction in the skin, which decreases convective heat transfer from the core to the surface of the skin and thus decreases heat loss from the body [2,3,9]. During mild body cooling, cutaneous vasoconstriction may be sufficient as a thermoregulatory response. During more severe or prolonged cooling, however, the physical gradient for heat loss (difference in temperature between the body and the environment) is so great that cutaneous vasoconstriction alone is insufficient to maintain body temperature. In this case, an increase in metabolic heat production is required to offset the heat lost, and maintain internal temperature. Shivering involves involuntary, rhythmic muscle contractions which occur solely for the purpose of generating heat; i.e., not for locomotion or to accomplish some other activity. The increase in metabolic rate that occurs during shivering is substantial, in the order of the increase in metabolic rate that occurs during mild to moderate exercise [9]. Because heat is a byproduct of increased

PO/AH

increased body temperature (–) cutaneous vasodilation sweating

decreased body temperature (–) cutaneous vasoconstriction shivering

FIGURE 60.1 Schematic summary of the negative feedback loops involved in human physiological thermoregulation. Responses to hyperthermia are shown on the left side of the figure, where increased body temperature elicits increased heat dissipation via cutaneous vasodilation and sweating. Responses to hypothermia are shown on the right side of the figure, where decreased body temperature elicits cutaneous vasoconstriction (to decrease heat loss) and shivering (to increase metabolic heat production). These responses then minimize or reverse the initial change in body temperature.

skeletal muscle metabolism (i.e., muscle contraction), shivering results in an increase in heat generation. This increased heat production protects against hypothermia by helping to maintain core temperature. In neonatal humans, and in adults of other species such as rats, a phenomenon exists called “non-shivering thermogenesis”. Non-shivering thermogenesis occurs via sympathetic neurogenic activation of brown adipose tissue, in which uncoupling protein-1 (UCP1) uncouples metabolic activity from the generation of ATP. Therefore, activation of brown adipose tissue results in the generation of heat in the absence of shivering. It is generally accepted that nonshivering thermogenesis does not exist in adult humans [9]. Thermoregulatory responses to environment which involve specific activity or behaviors are referred to as “behavioral thermoregulation”. This is in contrast to the physiological thermoregulatory responses outlined above, and include huddling, putting on warmer clothing or going indoors when one is uncomfortable outdoors. In general, humans tend to rely to a greater extent on behavioral thermoregulation in the cold, since human physiological responses to a cold environment are limited compared to other species [9].

HYPERTHERMIA Human exposure to hyperthermia is usually due to some combination of environmental heat exposure and/or exercise. It is relevant in this context that metabolic heat production (from exercising skeletal muscle) can increase as much as 10 to 20-fold with intense exercise. Thus even mild to moderate exercise represents a substantial, “endogenous” heat stress, and would result in dangerous increases in body temperature were it not for appropriate physiological heat loss responses. Even resting heat exposure can increase body temperature substantially, so when the two stresses are combined, the increased thermal load requires substantial heat dissipation. Physiological thermoregulatory responses to heat in humans are much more efficient than responses to cold; therefore, physiological thermoregulation is primary during hyperthermia (although behavioral responses contribute as well, such as seeking out cooler environments or wearing lighter clothing). Thermoregulation during hyperthermia involves increasing the dissipation of heat from the core to the environment. This is done in two ways: (1) by increasing the convective transfer of heat from the core to the skin via increased skin blood flow; and (2) by cooling the skin with evaporative heat loss by sweating.

Skin Blood Flow The sympathetic innervation of the human skin circulation involves two distinct branches. Sympathetic noradrenergic vasoconstrictor nerves, mentioned above, are tonically active and release norepinephrine

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ovERAll SummARy

and cotransmitters which cause vasoconstriction. The sympathetic active vasodilator system is less well understood, but appears to function via cholinergic nerve co-transmission [5]. A specific neurotransmitter(s) for the active vasodilator system has not been identified, although several substances appear to contribute to the reflex vasodilator response (for review, see [2]). During increases in body temperature due to environmental heat exposure or exercise, the initial vasodilator response in the skin is caused by withdrawal of vasoconstrictor neural activity, and is therefore referred to as “passive” vasodilation. Further increases in body temperature result in activation of the active vasodilator system, which is responsible for 80–90% of the very large vasodilation that can occur during whole body heating [4]. During severe hyperthermia, skin blood flow can increase from its low baseline values to as much as 6–8 L/min or 60% of cardiac output [6].

Sweating Sweating works in concert with cutaneous vasodilation to optimize heat dissipation during hyperthermia. The cooler skin accomplished by the sweating response allows the heat transfer from skin blood flow to be more efficient: when the warmer blood arrives at the skin surface, a cool skin results in a larger thermal gradient and therefore more efficient heat loss from the body. The process of sweating involves the secretion of a hypotonic saline solution onto the surface of the skin, which then evaporates, cooling the skin. Particularly when environmental temperature is warmer than body temperature, evaporative heat loss is necessary to cool the skin surface and facilitate heat transfer away from the body core. Sweating is caused by activation of sympathetic sudomotor nerves innervating eccrine sweat glands, which cover most of the body surface (for review, see [8]). These nerves release acetylcholine, which interacts with muscarinic cholinergic receptors at the sweat gland and triggers the onset of sweating. Sweating can therefore be blocked by local or systemic administration of atropine. Because sweat evaporation is essential to the process of cooling the skin, sweating itself will not cause heat dissipation if evaporation does not occur. Therefore, in dry environments, where evaporation of sweat can occur easily, evaporative skin cooling (and therefore overall heat

dissipation) is much more efficient than in humid environments of similar absolute temperature. Sweat which does not evaporate but simply drips off the skin is “wasted” from the perspective of heat dissipation and overall thermoregulation.

OVERALL SUMMARY As summarized in Figure 60.1, human thermoregulation is a complex, integrative process which relies on multiple organ systems working together in a coordinated fashion to optimize body temperature for a variety of environmental and activity conditions. Responses to changes in body temperature are coordinated centrally at the preoptic area of the anterior hypothalamus. Major physiological responses during hypothermia in humans are cutaneous vasoconstriction and shivering. During hyperthermia, heat is dissipated from the body via cutaneous vasodilation and sweating.

References [1] Boulant JA. Neuronal basis of Hammel's model for set-point thermoregulation. J Appl Physiol 2006;100:1347–54. [2] Charkoudian N. Mechanisms and modifiers of reflex induced cutaneous vasodilation and vasoconstriction in humans. J Appl Physiol 2010;109:1221–8. [3] Hodges GJ, Johnson JM. Adrenergic control of the human cutaneous circulation. Appl Physiol Nutr Metab 2009;34:829–39. [4] Johnson JM, Proppe DW Cardiovascular adjustments to heat stress. In: Handbook of physiology environmental physiology. New York: Oxford University Press; 1996. p. 215–44. [5] Kellogg Jr DL, Pergola PE, Piest KL, Kosiba WA, Crandall CG, Grossmann M, et al. Cutaneous active vasodilation in humans is mediated by cholinergic nerve cotransmission. Circ Res 1995;77:1222–8. [6] Rowell L.B. Cardiovascular adjustments to thermal stress. In: Handbook of physiology the cardiovascular system: peripheral circulation and organ blood flow. Bethesda: American Physiological Society; 1983. p. 967–1023. [7] Savage MV, Brengelmann GL. Control of skin blood flow in the neutral zone of human body temperature regulation. J Appl Physiol 1996;80:1249–57. [8] Shibasaki M, Wilson TE, Crandall CG. Neural control and mechanisms of eccrine sweating during heat stress and exercise. J Appl Physiol 2006;100:1692–701. [9] Young AJ, Castellani JW. Exertion-induced fatigue and thermoregulation in the cold. Comp Biochem Physiol A Mol Integr Physiol 2001;128:769–76.

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61 Psychological Stress and the Autonomic Nervous System Michael G. Ziegler INTRODUCTION

Patterns of Autonomic Response to Stress

Autonomic responses to psychological stress prepare the body for fight or flight. Cannon and Selye described stereotypic responses to stress that involved activation of sympathetic nerves and adrenocortical hormone release. Corticotropin-releasing factor (CRF) in the central nervous system activates autonomic and adrenocortical responses to stressors. CRF injected into the brain increases arousal and responsiveness to stressful stimuli. On the other hand, CRF antagonists can reverse behavioral responses to many stressors. CRF alters the discharge of locus coeruleus neurons in the brainstem and these effects are mimicked by some stressors. Locus coeruleus noradrenergic neurons project into the cerebral cortex. In the paraventricular nucleus of the hypothalamus, norepinephrine stimulates further CRF release. Other central nervous system pathways also mediate stress-induced activation of the sympathetic nervous system but they have generally not been studied as well as the CRF pathways [1].

Normal Psychological Stresses and Autonomic Activity There is a 24-hour rhythm in the plasma levels of norepinephrine and epinephrine which tend to be lowest at 3 a.m., and rise rapidly until a peak at 9 a.m. similar to the cortisol rhythm. Sleep decreases sympathetic nerve activity. Arousals during sleep cause a rapid increase in muscle sympathetic nerve electrical activity which falls back to baseline when sleep resumes. Muscle sympathetic nerve activity nearly doubles when going from sleep to wake and doubles yet again when going from recumbent to standing posture. Exercise, pain, and cold lead to even more dramatic increases in sympathetic nerve activity which is detailed in Chapters 80 and 81 of this book. These are normal responses to the stresses of day to day life which may help explain why there is a preponderance of myocardial infarction and sudden death between 6:00 a.m. and noon. The autonomic responses to stress might also help explain early mortality during both unemployment and bereavement.


The general response to both physical and psychological stress is activation of the sympathetic nervous system with inhibition of the parasympathetic nervous system. When stress becomes severe or uncontrolled then adrenomedullary release of epinephrine ensues. As stress increases even further then CRF not only activates the sympathetic nervous system but leads to the release of adrenocorticotropic hormone (ACTH) and adrenocortical steroids. Sympathetic nervous stimulation constricts muscle vasculature and increases peripheral vascular resistance. Sympathetic nerves that supply the skin both vasoconstrict and supply sweat glands through sympathetic cholinergic innervation. Activation of this skin sympathetic pathway can precipitate a “cold sweat” or perspiration and flushing of the skin. During sleep, muscle sympathetic nervous activity and skin sympathetic nervous activity have similar firing intensities and frequencies. However, during stress, muscle and skin sympathetic nerve activities diverge. When a doctor took patients’ blood pressure, skin sympathetic nerve activity increased by 38% while muscle sympathetic nerve activity decreased by 25%. This was accompanied by an apparent increase in sympathetic nerve activity to the heart as heart rate and blood pressure increased [2]. Some stimuli such as hypoglycemia elicit a fairly specific activation of adrenomedullary epinephrine release without a marked increase in sympathetic nerve activity. When medical residents climbed stairs, their plasma norepinephrine increased; however, when they presented a public speech, epinephrine levels showed an even greater increase [3]. Psychological stress not only tends to increase epinephrine disproportionately, it also tends to increase sympathetic nerve activity to the heart, leading to increased cardiac output. As we age, norepinephrine release in response to the cold pressor test increases. Less epinephrine is released from the adrenal medullae in the elderly; however, epinephrine blood levels do not decrease because of diminished clearance of epinephrine from the circulation with advancing age.

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CARDIAC DISEASE Psychological stress affects the heart in many ways. Stress increases heart rate by withdrawal of parasympathetic input and increased sympathetic stimulation. This is commonly accompanied by increased blood pressure leading to an increased rate–pressure product and myocardial oxygen consumption. Although the increased oxygen demand requires increased blood flow through the coronary arteries, studies of patients during mental stress showed increased norepinephrine spillover from the heart and a reduction of blood flow through regions of high stenosis by an average of 27% [4]. The combination of hypoxia and sympathetic stimulation can cause arrhythmias. Pigs with an experimentally induced coronary occlusion developed ventricular fibrillation when restrained unless they were adapted to the restraint [5]. Implanted cardioverters discharge in response to dangerous ventricular arrhythmias. Patients experiencing intense anger have an estimated seven times greater risk of cardioverter discharge during both mental and physical stress. There is epidemiological evidence that psychological stress can cause arrhythmias and fatal human cardiac events. After the Northridge earthquake, coroners’ records for Los Angeles County found that sudden deaths from cardiac causes increased from an average of 4.6 per day in the week before to 24 on the day of the earthquake [6]. Studies of the 1995 Hanshin-Awaji earthquake in Japan showed similar results [7]. Psychological stress can precipitate angina pectoris, indicating that it causes myocardial ischemia in patients with coronary artery disease. It can also cause cardiac arrhythmias, triggering cardioverter discharge. Emotional stress can also increase blood levels of fibrinogen, von Willebrand factor, factors VII and VIIII and fibrin D-dimer and activate platelets, all of which can predispose to coronary occlusion [7]. These acute cardiac consequences of stress may be exacerbated if chronic stress is associated with high blood pressure (see Chapter 71). Extreme sympathetic nervous system activation in stress probably plays a role in stress (takotsubo) cardiomyopathy as well as in panic attacks accompanied by coronary artery spasm. The high level of sympathetic firing in these disorders is accompanied by release of neuropeptide Y (NPY), which can lead to prolonged vasoconstriction [8].

GASTROINTESTINAL (GI) CONTROL The thought of food can elicit salivation, gastric motility and acid secretion. Stress inhibits GI motility when it activates the sympathetic nervous system, primarily through release of norepinephrine at its synaptic interface with the enteric nervous system. Postganglionic projections from sympathetic nerves terminate in myenteric submucous ganglia of the enteric nervous system where they suppress motility and secretion. Alpha adrenergic stimulation inhibits both GI secretion and GI blood flow. In animal

models, cold water immersion is associated with an inhibition of gastric emptying. While stress decreases gastric emptying and intestinal motility it does not decrease colonic motility. Sacral parasympathetic stimulation of the large bowel during stress is accompanied by degranulation of enteric mast cells triggered by postganglionic sympathetic nerve release of corticotrophin releasing hormone (CRH) [5]. This releases inflammatory mediators and is postulated to underlie the secretory diarrhea and abdominal discomfort associated with stress.

PSYCHOSOMATIC DISORDERS AND THE AUTONOMIC NERVOUS SYSTEM Autonomic responses to stress frequently lead to medical care. Feelings of warmth and cold, palpitations, tachycardia, nausea, abdominal pain, diarrhea, and constipation can all be the consequence of autonomic stress responses. Twenty per cent of patients with borderline hypertension in the doctor’s office have entirely normal home blood pressures. Sympathetic nervous stimulation of the heart increases heart rate, cardiac output and blood pressure in novel or stressful environments. This cardiovascular stress response increases myocardial oxygen consumption and can precipitate angina pectoris in patients with coronary artery disease. A vasovagal response can be triggered by a stressful situation that makes a person want to run away even though social constraints prevent the person from leaving. When this happens, hypothalamic activation of medullary cardiovascular responses triggered by emotional stress can lead to increased inotropic stimulation to the heart. This can stimulate ventricular mechano-receptors and promote vasodilation, bradycardia, and fainting.

POST-TRAUMATIC STRESS DISORDER, PANIC, AND ANXIETY Post-traumatic stress disorder (PTSD) occurs when intrusive thoughts elicit memories of an unusually stressful event. Nineteenth century descriptions of “soldier’s heart” noted abnormal excitability of the cardiac nervous system in the absence of serious cardiac disease. PTSD is often accompanied by tachycardia, palpitations, and high blood pressure [4,10]. In panic disorder, a psychological stimulus elicits an autonomic response characterized by flushing, tachycardia, palpitations, hypertension, and gastrointestinal symptoms. The autonomic response can sometimes be extinguished by repeated exposure to the stimulus under reassuring circumstances. In anxiety disorder, similar autonomic symptoms occur with no inciting stimulus. In all three of these psychological disorders baseline norepinephrine and epinephrine are normal but plasma and urinary catecholamines increase when symptomatic reactions

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PoST-TRAumATIC STRESS dISoRdER, PANIC, ANd ANxIETy

Autonomic responses to psychological stress serve the useful function of preparing us for action by increasing muscle blood supply and slowing vegetative functions. However, inappropriate stress responses are the basis for many psychosomatic disorders. Familiarity with the patterns of autonomic response to psychological stress is essential to understanding human disease.

TABLE 61.1 Sympathetic Nerve Responses to Stress Organ

Response

Receptor

Cardiac atrium

Heart rate

β1

Cardiac ventricle

Inotropism

β1

Eye

Pupil dilation

α1

Skin blood vessel

Constriction

α1

Hand sweat glands

Sweat

Cholinergic

Blood vessels

Constriction

α1

Salivary glands

Constriction, dry mouth

α1, α2

Gut

Decrease motility

α1, α2, β2

Gut sphincters

Contraction

α1

Kidney

Renin release

β1

Bladder

Relaxation

β2

Bladder sphincter

Contraction

α1

Hair

Piloerection

α1

Muscle cells

Glycogenolysis

β2

Muscle cells

K uptake

β2

Muscle blood vessels

Dilation

β2

293

References

are triggered [5,11]. Beta adrenergic blocking drugs tend to diminish subjective symptoms of palpitations and tremor and they often eliminate episodes of tachycardia.

[1] Koob GF. Corticotropin-releasing factor, norepinephrine, and stress. Biol Psychiatry 1999;46:1167–80. [2] Grassi G, Turri C, Vailati S, Dell'Oro R, Mancia G. Muscle and skin sympathetic nerve traffic during the “white-coat" effect. Circulation 1999;100:222–5. [3] Dimsdale JE. Psychological stress and cardiovascular disease. J Am Coll Cardiol 2008;51:1237–46. [4] Steptoe A, Brydon L. Emotional triggering of cardiac events. Neurosci Biobehav Rev 2009;33:63–70. [5] Chrousos GP. Stress and disorders of the stress system. Nat Rev Endocrinol 2009;5:374–81. [6] Appels CW, Bolk JH. Sudden death after emotional stress: a case history and literature review. Eur J Intern Med 2009;20:359–61. [7] Steptoe A, Brydon L. Emotional triggering of cardiac events. Neurosci Biobehav Rev 2009;33:63–70. [8] Esler M. Pathophysiology of the human sympathetic nervous system in cardiovascular diseases: the transition from mechanisms to medical management. J Appl Physiol 2010;108:227–37. [9] Plourde V. Stress-induced changes in the gastrointestinal motor system. Can J Gastroenterol 1999;13(Suppl A):26A–31A. [10] Lamprecht F, Sack M. Posttraumatic stress disorder revisited. Psychosom Med 2002;64:222–37. [11] Hoehn-Saric R, McLeod DR. Anxiety and arousal: physiological changes and their perception. J Affect Disord 2000;61:217–24.

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62 Mind–Body Interactions Calvin Carter, Daniel Tranel INTRODUCTION Mind–body interactions are a two-way street. For example, perceptions and emotions (mind) influence many bodily functions, such as sweating, heart rate, blood pressure, and gastrointestinal smooth muscle contractions (body). Conversely, different physiological states (e.g., as in health and disease) influence an individual’s mental state and mood. The study of these mind–body interactions has been greatly enhanced by three techniques which quantify arousal of the autonomic nervous system following stimulation: the electrodermal skin conductance response (SCR), the electrogastrogram (EGG), and cardiovascular parameters (heart rate and blood pressure; HR and BP). The SCR is a remarkably powerful and informative psychophysiological index. Because SCRs are relatively easy to measure, and provide reliable indices of a wide variety of psychological states and processes, SCRs have been arguably the most popular aspect of the autonomic nervous system (ANS) activity used to study human cognition and emotion [1]. The EGG records the stomach’s smooth muscle contractions, called peristalsis, measuring the activity of the digestive and gastrointestinal (GI) systems. The primary function of the GI system is to process ingested foods and liquids, digesting and absorbing nutrients and water and excreting wastes. These functions are highly regulated by the enteric nervous system, the only branch of the ANS that can function independently of the sympathetic (SNS) and parasympathetic nervous system (PNS) [2]. Finally, cardiovascular (CV) responses, including changes in heart rate (HR) and blood pressure (BP), are regulated by the SNS and PNS. Cardiovascular responses are particularly useful in ethnic studies due to robust differences in CV arousal between ethnic groups [3]. Our group and others have used these techniques to study mind–body interactions in patient, meditation and ethnic based studies.

SKIN CONDUCTANCE RESPONSE (SCR)

The Somatic Marker Hypothesis

Decision-Making Making advantageous decisions in everyday situations requires conscious reasoning; however, non-conscious


biases can also come into play, and can influence many aspects of decision-making. Using SCRs, we found that healthy individuals generate non-conscious autonomic responses (SCRs) while pondering a decision that later turned out to be risky. These processes are mediated by the ventromedial (VM) prefrontal sector of the brain. In our experiment, participants were asked to win the most and lose the least amount of money in a card game, using a loan of $2000. Study participants chose to play from two different sets of card decks, one set of decks was advantageous in the long run (the “good deck” because participants won more money than they lost) and the other set was disadvantageous (the “bad deck” because participants lost more money than they won). The participants had no way to predict which of the decks was advantageous or disadvantageous. Healthy participants began to generate SCRs before choosing a card from the “bad deck” and eventually avoided these decks whereas VM patients could do neither (Fig. 62.1) [4]. Furthermore, these SCRs preceded conscious awareness of these decks being disadvantageous (“pre-hunch” period) as determined by questioning the participants throughout the course of the experiment. After playing further, healthy participants became aware of the “good and bad” decks (“hunch period”). Later in the game, healthy participants figured out the nature of the game and expressed “why” certain decks were more advantageous than others (“conceptual period”). During all three periods healthy participants continued to generate SCRs whenever they considered choosing a card from a “bad” deck and subsequently chose cards from the good decks (Fig. 62.1). By contrast, none of the VM patients generated anticipatory SCRs nor expressed a hunch and only few became aware of why the decks were good or bad. Yet, VM patients continued to choose from the “bad deck” despite their overt knowledge of what was going on in the game (Fig. 62.1) [4], suggesting that the VM plays a role in pre-monitory decision-making.

The findings reviewed here, and other related observations, have led to the development of a framework that is termed the somatic marker hypothesis. In a nutshell, the theory posits that feelings and emotions give rise to “somatic

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62. MInd–Body InTERACTIons

FIGURE 62.1 Aberrant decision-making in patients with VM damage. The magnitude of anticipatory SCRs is significantly lower in VM patients compared to controls during all experimental periods (pre-hunch, hunch and conceptual period). Healthy controls chose more and more cards from the good decks as they gained more insight into the experiment; in contrast, VM patients continued to choose cards from bad decks despite their increasing knowledge of what was going on in the task. Experimental periods: Pre-hunch  no knowledge of the task; Hunch  Insight into task; Conceptual  Verbalized why the task worked the way it did (adopted with permission from [4]).

markers,” which serve as guideposts that help steer behavior in an advantageous direction. Deprived of these somatic markers, VM patients lose the ability to experience appropriate emotional responses to various stimuli and events. We have proposed that the absence of these emotional responses – evidenced, for example, by the missing SCRs in the experiments described above – leads to defective planning and decision-making; this, in turn, leads to socially inept and inappropriate behavior that is characteristic of VM patients [5].

ELECTROGASTROGRAM (EGG) Emotion One great avenue for studying mind–body interactions is the brain–gut axis. Have you ever felt butterflies in your stomach before a big interview? The brain–gut connection is the likely culprit as both organs are intimately linked via the vagus nerve. The process begins with an external stimulus (e.g. a traumatic life event) causing anxiety which in turn activates hypothalamic outputs to the pituitary and pontomedullary nuclei. Both structures mediate the neuroendocrine and autonomic outputs to the body and the gut via the vagus nerve [5]. The study of this mind–body interaction has been greatly improved with the introduction of the electrogastrogram in recent years [6]. It is wellestablished that psychological factors influence the activity of the gastrointestinal system, including the stomach and intestine, however, the role of the GI system on a person’s

psychological and emotional state has not been welldocumented [6]. Using EGGs, our lab investigated the role of the gastrointestinal system in one’s subjective emotional experiences. We studied patients with Crohn’s disease, an inflammatory disease of the gastrointestinal system resulting in increased gut sensitivity, in the active phase (Crohn’s-active, CA) or silent/remissive phase (Crohn’ssilent, CS) and healthy participants (HP). We hypothesized that the gut influences emotions and feelings and consequently, participants with an actively abnormal GI (CA participants) would have aberrant feelings of emotion compared with participants with near-normal or normal GIs (CS and NC participants). To test this, we measured participant’s EGG activity while watching emotionally charged clips (happy, disgust, fear and sad), baseline measurements were obtained prior to viewing film clips. Following each presentation of a film clip, each participant completed a self-report questionnaire that assessed the intensity of their gastrointestinal sensations and their subjective emotional experience in response to each film clip. We found that patients with CA demonstrated increased gastrointestinal activity before stimuli were introduced (Fig. 62.2A) and self-reported greater feelings of negative emotions (disgust, fear and sad) after viewing emotionladen film clips compared to CS patients and healthy participants (Fig. 62.2B). Additionally, none of the participants from any group stated a change in gastrointestinal feelings after viewing film clips [7]. These results suggest a cause and effect relationship between the body (GI activity) and one’s subjective emotional experience (self-report) in which abnormal GI activity pre-disposed CA patients to

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297

(C)

(D)

Self-reported emotion ratings

(B) 7

*

6 5 4

Disgust

3

Fear

2

Sad

1 0 HP

CA

0.5 1

2

3

4 5 Arousal

6

7

1

2

3

4 5 Arousal

6

7

CS

CA

1

NC

–1.5 –1.0 –0.5 0.0

z-score peak EGG

10

*

z-score peak EGG –1.5 –1.0 –0.5 0.0 0.5 1.0

EGG Activity

(A)

0e.00 1e.07 2e.07 3e.07 4e.07 5e.07 6e.07

CARdIovAsCulAR ARousAl (HR And BP)

CS

FIGURE 62.2 Mind-gut axis. (A) Active Crohn’s patients (CA) demonstrated significant increases in gastrointestinal activity before emotional stimuli were introduced and (B) self-reported greater feelings of negative emotions after viewing emotion-laden film clips compared to healthy participants (HP) and silent Crohn’s patients (CS) suggesting that abnormal GI activity pre-disposed CA patients to a greater emotional experience (* represents a significant difference between groups (p7 6.5-7

6-6.5

6-6.5

5.5-6

5-5.5

4.5-5

3.5-4 4-4.5

3-3.5

2.5-3

2-2.5

1.5-2

1-1.5

0.5-1

7

5.5-6

5-5.5

4.5-5

4-4.5

3.5-4

3-3.5

2.5-3

2-2.5

1.5-2

1-1.5

0.5-1

7

6.5-7

6-6.5

5.5-6

5-5.5

4.5-5

4-4.5

3.5-4

3-3.5

2.5-3

2-2.5

1.5-2

1-1.5

0.5-1

1.21

HR response to standing

HR is measured as the subject moves Ratio of >1.04 from a resting supine position to standing. A normal tachycardic response is maximal around the 15th beat after rising. A relative bradycardia follows, that is most marked around the 30th beat after standing. The response to standing is expressed as a 30:15 ratio and is the ratio of the longest R-R interval around the 30th beat to the shortest R-R interval around the 15th beat

HR response to deep breathing

The subject takes six deep breaths in 1 minute. The maximum and minimum heart rates during each cycle are measured, and the mean of the differences (maximum HR – minimum HR) during three successive breathing cycles is taken as the maximum-minimum HR

Sympathetic BP response to standing

The subject moves from resting Difference supine to standing, and standing SBP 15 beats/min

Difference >16 mmHg

BP, blood pressure; DBP, diastolic blood pressure; HR, heart rate; SBP, systolic blood pressure. (Reprint from David B. Glick, 2009 Miller Anesthesiology, 7th edn. Churchill Livingstone.)

ventilation may result in disastrous hypotension in patients and should be avoided [7].

SPECIAL CONSIDERATION FOR ANESTHESIA Airway Management Several features related to the pathophysiology of the autonomic disorder may complicate airway management in the anesthetic care setting. The combination of impaired gastrointestinal motility and laryngeal dysfunction make

XIV. EXPERIMENTAL AUTONOMIC NEUROSCIENCE

SPECIAL CONSIDERATION fOR ANESTHESIA

Noradrenergic inhibition Common

Rare

Prescribed drugs Neurocardiogenic syncope Drabetic autonomic neuropathy Alcohol Parkinson disease Hyperthyroidism Multiple system atrophy Multiple myeloma Quadriplegia Amyloidosis Pure autonomic failure Chagas disease Familial dysautonomia Dopamine β-hydrpxylase deficiency

Noradrenergic activation Common

Essential hypertension Congestive heart failure Myocardial infarction Postural tachycardia syndrome Panic disorder Carotid endarterectomy Intracranial bleeding Hyperdynamic circulation syndrome Renovascular hypertension Guillain-Barré syndrome Baroreflex failure “Autonomic epilepsy” Norepinephrine transporter deficiency

Rare

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pressure ventilation on venous return and cardiac output. In patients with autonomic failure, hyperventilation causes a rapid fall in blood pressure. Conversely, hypercapnea (such as hypoventilation or increased dead space ventilation) can rapidly raise blood pressure.

Hepatic Blood Flow and Lidocaine The liver blood flow can decrease as much as 30% with upright posture in patients with autonomic failure. In consequently, a hepatically cleared drug like lidocaine may display plasma levels that are dramatically posturedependent owing to their blood pressure dependence. When patients with orthostatic hypotension are receiving intravenous lidocaine, the plasma drug levels are almost twice as high when they are seated vs. supine, and this change in posture can result in a toxic seizure despite an unchanged infusion rate during anesthetic management.

Temperature Dysregulation Impaired perspiration can lead to an inability to dissipate heat adequately. This could also contribute to a persistent high fever postoperatively. With aggressive warming measures, there is sometimes also a thermally induced hypotension. Conversely, the inability of autonomic disorder to vasoconstrict in response to a cool environment may result in excessive heat loss and hypothermia and should be watched cautiously in anesthetic care.

Response to Infection

FIGURE 140.1 Dysautonomias featuring altered sympathetic noradrenergic function. (Reprint from David, S., Goldstein et al. Ann Intern Med. 2002;137:753–763.)

aspiration a particular concern in these patients. Rapid sequence induction should be considered. Mask induction with volatile anesthetics technique should be encouraged for those patients with autonomic disorders complicated by compromised cardiopulmonary reserve. Apnea in autonomic failure patients, either due to upper airway obstruction (such as that seen in obstructive sleep apnea) or due to impaired central regulation of respiration, may be aggravated postoperatively. In such instances it is likely due to disordered central control of respiration during anesthesia rather than extreme respiratory center sensitivity to the anesthetic agent [8].

Ventilatory Management The inability of patients with autonomic disorders to increase cardiac output through sympathetic activation makes them exquisitely sensitive to the effects of positive

Patients with autonomic disorder often have impaired capacity to manifest a fever in response to infection. Instead an infection may present with minimal fever or sometimes none at all. If a patient with autonomic failure experiences greater than usual hypotension or blood pressure lability, new-onset recurrent syncope, or an acute decrease in performance status, a search should be undertaken for occult infection, with emphasis on evaluation for urinary tract infection or aspiration pneumonia.

Diabetic Neuropathy Diabetes mellitus is the most commonly recognized cause of autonomic dysfunction. Unexplained tachycardia has been attributed to damage of parasympathetic fibers and it is thought to represent early cardiac involvement by diabetic peripheral neuropathy. When in the late stages, sympathetic impairment of cardiac innervation also occurs. Unexpected intraoperative bradycardia in patients with diabetic autonomic neuropathy may not respond to atropine (since vagal tone is already diminished in these patients), and alternatives such as isoproterenol or a temporary pacemaker can be life-saving.


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Uremia Uremic patients are sometimes encountered with afferent baroreflex impairment, and the patient’s blood pressure and heart rate are no longer buffered. In this circumstance, large excursions of blood pressure and heart rate may occur in response to stress (very high blood pressures) and sedation (low blood pressures).

SUMMARY Patients with autonomic disorders usually have significantly perturbed homeostatic responses to normal physiological and environmental stresses. Severe autonomic disorders increase in prevalence with age; aging is associated with reduced baroreflex responsiveness, decreased cardiac compliance, and attenuation of the vestibulosympathetic reflex. Most anesthetics remove an important compensatory mechanism to maintain blood pressure and exaggerate the response to vasoactive and cardiac depressant agents in human subjects [9]. Preoperatively, cardiovascular autonomic function should be carefully evaluated and should facilitate meticulous control of blood volume in patients with autonomic disorders. The pharmacologic complexities of anesthetic management in patients with

autonomic disorders suggest that all anesthetics should be administered judiciously. Postoperatively, orthostatic hypotension control necessitates the use of volume expansion, postural training and occasionally vasoconstrictors.

References [1] Goldstein DS, Sharabi Y. Neurogenic orthostatic hypotension: A pathophysiological approach. Circulation 2009;119:139–46. [2] Grenier Y, Drolet P. Asystolic cardiac arrest: An unusual reaction following iv metoclopramide. Can J Anaesth 2003;50:333–5. [3] Malan MD, Crago RR. Anaesthetic considerations in idiopathic orthostatic hypotension and the Shy-Drager syndrome. Can Anaesth Soc J 1979;26:322–7. [4] Freeman R. Clinical practice. Neurogenic orthostatic hypotension. N Engl J Med 2008;358:615–24. [5] Gunther A, Witte OW, Hoyer D. Autonomic dysfunction and risk stratification assessed from heart rate pattern. Open Neurol J 2010;4:39–49. [6] Tokuda K, Motoyama Y, Kai Y, Sakaguchi Y, Hoka S. Anesthetic management for a patient with significant orthostatic hypotension probably due to pure autonomic failure. Masui 2009;58:1010–3. [7] Shimazu T, Tamura N, Shimazu K. Aging of the autonomic nervous system. Nippon Rinsho 2005;63:973–7. [8] Drury PM, Williams EG. Vocal cord paralysis in the Shy-Drager syndrome. A cause of postoperative respiratory obstruction. Anaesthesia 1991;46:466–8. [9] Klein CM. Evaluation and management of autonomic nervous system disorders. Semin Neurol 2008;28:195–204.


C H A P T E R

141 Evolution of the Cardiovascular Autonomic Nervous System in Vertebrates Tobias Wang The need for coordinated control of visceral functions to maintain homeostasis has undoubtedly been of paramount importance since the early evolution of animals. Thus, a functional analogy of the autonomic nervous system (ANS) is likely to have an ancient evolutionary history, and must have been essential as soon as multicellular animals divided specific physiological function to particular organs. Because of this evolutionary history, studies on different animal groups can provide fundamental insight to the foundations of both anatomy and function of the ANS in mammals, including humans. Many of the basic functions of the ANS were originally discovered in ectothermic vertebrates. As prominent examples, Gaskell’s demonstrations of the vagal inhibitory role on the heart were performed on turtles and frogs and Loewi’s demonstration of acethylcholine being the postganglionic neurotransmitter within the parasympathetic nervous system (“vagus stoff”) was based on studies in amphibians. These animals were chosen because of their enormous tolerance to hypoxia and low temperatures, rendering these animals resilient to experimentation and hence suitable for physiological studies before methods for anesthesia and mechanical ventilation of mammals. More recently, studies on ANS functions in ectothermic vertebrates were crucial for the discovery of non-adrenergic-non-cholinergic neurotransmitters within the sympathetic and parasympathetic nervous systems.

THE AUTONOMIC NERVOUS SYSTEM IN VERTEBRATES A phylogeny depicting extant (living) groups of chordates (animals that have a notochord during some stage within their life cycle) is shown in Figure 141.1, where the different vertebrate groups, i.e., animals with skeletal elements surrounding the spinal cord and notochord, are highlighted within the grey box [1]. While tunicates and amphioxus are endowed with a nervous system that may resemble the enteric nervous system, the equivalent of an ANS is not present [2,3]. Within hagfishes and lampreys the ANS is considered rudimentary, because either some organs are devoid


of innervation or most organs lack dual innervation. In both groups, the left and right vagi unite to form a ramus intestinalis impar that innervates the intestine and the gallbladder. In hagfishes, the ramus intestinalis impar does not innervate the heart, whereas the heart of lampreys does receive vagal innervation. In both hagfishes and lampreys, spinal “sympathetic” nerves leave the dorsal as well as well as ventral spinal nerves, but there are no sympathetic chains or segmental ganglia. The sympathetic nerves innervate several visceral organs, but not the heart. It must be emphasized that hagfishes and lampreys have evolved over the past several hundred millions years and are unlikely to represent exact copies of their ancestors. The eyes of both groups, for example, are degenerated, which may relate to the poor innervation by the ocular nerve. Nevertheless, it seems very reasonable to conclude that the ANS was poorly developed in early vertebrates [2–4]. Sharks and rays (cartilaginous fishes) have segmentally arranged paravertebral ganglia that are linked by a loose plexus of nerve fibers [2–4]. The vast majority of the fibers arise from the ventral roots of the spinal nerves. These sympathetic nerves innervate most visceral organs with the notable exception of the heart, and do not appear to enter the head. Cranial autonomic fibers, i.e. the parasympathetic nervous system, occur within the oculomotor (III), facial (VII), glossopharyngeal (IX) and vagus (X) nerves. The vagal fibers reach the stomach and the anterior parts of the intestine. The vagus also innervates the heart and exerts both negative chronotropic and inotropic effects [2,3]. In bony fishes (teleosts), the sympathetic chain ganglia are well developed and form distinct sympathetic chains [2,3]. Some of these sympathetic fibers join the vagus, creating a vagosympathetic trunk. This is also the case in amphibians and reptiles. The overall pattern of innervation and the actions of the autonomic nervous system in bony fishes resemble that found amphibians, reptiles, birds and mammals (i.e., the tetrapods) is rather similar. Therefore, while the ANS of early vertebrates can be considered simple in comparison to subsequent groups of vertebrates, the basic foundations of the ANS were established by the time of fishes and evolved prior to the invasion of terrestrial habitats. The swimbladder of bony

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141. EVoluTIoN of THE CARdIoVASCulAR AuToNomIC NERVouS SySTEm IN VERTEbRATES

FIGURE 141.1 A phylogeny of the major

Cardiac innervation Vagal

groups of extant (i.e., living) craniates and vertebrates. Note that only birds and mammals are endothermic and that this trait appears to have evolved independently from two different groups of reptilian and ectothermic ancestors (modified from Liem et al., 2001 [1]). The figure includes a synopsis of the parasympathetic and sympathetic innervations of the heart and vasculature.

Sympathetic

Tunicates Amphioxus Hagfishes Excitatory

Lampreys

Coelacanth Lungfishes Amphibians Birds Crocodilians Lizards & snakes Turtles

A-and β adrenrgic innervation

Bony fishes

Excitatory sympathetic innervation of the heart

Inhibitory parasympathetic innervation of the heart

Sharks & rays

Mammals

fishes is used to control buoyancy, but probably evolved originally as a gas exchange organ. Therefore, the swimbladder can be considered homologous to the lungs of lungfish and tetrapods. Both organs form embryologically as an outpocketing of the gut; the swimbladder as a ventral extension, while the lungs derive from a dorsal extension. Both organs receive a dual innervation form the ANS in most species.

COMPARATIVE ASPECTS OF THE AUTONOMIC REGULATION OF THE CARDIOVASCULAR SYSTEM Anatomy of the Cardiovascular System in Vertebrates The morphology and the function of the heart and cardiovascular systems of vertebrates have undergone large evolutionary changes associated with the transition between water and air-breathing and during the evolution of endothermy within mammals and birds [5,6]. A schematic representation of these cardiovascular designs is shown in Figure 141.2, where all the different groups of

fish (hagfishes, lampreys, cartilaginous fishes, teleosts and the coelacanth) are presented as having a similar “piscine” circulatory design. Thus, while the cardiac morphology, the number of gills arches and other characters differ considerable between these groups, they all rely on gills for gas exchange [7]. With some variations, the hearts of all groups consist of a sinus venosus, a single atrium and a single ventricle as well as an outflow tract that may be both muscular and contractile as the conus arteriosus in sharks. This heart ejects the oxygen poor-blood that has returned from the body towards the gills, where the blood is oxygenated before it is delivered to the body. The branchial circulation and the perfusion of the systemic vascular beds of fish, therefore, occur in series and the emergence of parallel circulations arose with evolution of the lungs. In lungfishes (Dipnoi), the first group of vertebrates with real lungs, the common pulmonary vein draining the lungs empties into the sinus venosus in close proximity to the atrium. The single trabeculated atrium is partially divided by a fold that prevents admixture of the oxygenrich blood from the lungs and oxygen-poor blood from the systemic circulation. The oxygen-poor blood is preferentially directed through the posterior gill arches and can either enter the pulmonary artery or the dorsal aorta after having perfused the gills. The oxygen-rich blood primarily


ComPARATIVE ASPECTS of THE AuToNomIC REgulATIoN of THE CARdIoVASCulAR SySTEm

671

Body Body

Lungfish

Body

Amphibians

Lungs

Body

Birds

Body

Crocodiles

Body

Turtles, lizards and snakes

Body

Lungs Lungs Lungs

Skin

Lungs

Heart

Gills

Gills

“Piscine”

Lungs

Autonomic Regulation of the Heart

Mammals

FIGURE 141.2 The evolution of the cardiovascular system amongst the major groups of extant vertebrates. In this representation, the cardiovascular design of hagfish, lampreys as well as the cartilaginous and bony fishes are presented as a general “piscine design”. Note that only mammals and crocodiles have a complete division of the ventricle and that lungfishes, amphibians and reptiles have the capacity to mix oxygen-rich and oxygen-poor blood within the ventricle (modified from Jensen et al., 2010 [6]).

enters the anterior and degenerated gill arches to enter the dorsal aorta directly [7]. In amphibians, snakes, lizards and turtles there are two separate atria receiving the venous blood from the systemic and pulmonary circulations. While the ventricular anatomy differs enormously amongst these groups, they are characterized by having a single ventricle, but distinct outflows to either the systemic or pulmonary circulations [6]. In crocodiles, which in some ways are more closely related to birds than the other reptiles, a complete septum divides the ventricle, but because the left aorta arises from the right ventricle, blood can bypass the pulmonary circulation. Birds have a cardiac anatomy that closely resembles mammals, but an independent evolution of the divided ventricle is indicated by birds retaining the right aortic arch during ontogenetic development, whereas mammals retain the left aortic arch [1].

The aneural myogenic hagfish heart is devoid of any innervation and is remarkably insensitive to acetylcholine or cholinergic antagonists [4,7]. The hagfish heart, nevertheless, is under a tonic paracrine β-adrenergic tone that seems to derive from catecholamine stores within the heart [4,7]. It remains uncertain whether the cardiac release of catecholamines and hence the adrenergic tone is regulated. Heart rate increases markedly upon increased filling, a response that is independent of adrenergic stimulation and this may be important to increase cardiac output when venous return is elevated. The mechanism for this response still remains to be determined. Hagfish inhabit hypoxic sediments and exhibit a marked tolerance to oxygen deprivation, associated with a pronounced reduction in heart rate that may be direct effects of oxygen lack on the pacemaker cells. Consistent with the lack of innervation, hagfish exhibit only small changes in heart rate during exercise [7]. Lampreys represent the first group of chordates with a vagal innervation of the heart, where the vagus travels along the jugular vein to the sinus venosus where the primary pacemaker region is located. This innervation, however, differs fundamentally from the rest of the vertebrates by being excitatory and that cardiac acceleration can be blocked by nicotinic cholinoceptor antagonists. Although there is no sympathetic innervation, the lamprey heart, nevertheless, exhibit positive inotropic and chronotropic responses to β-adrenergic stimulation and heart rate. As in hagfish, catecholamines are released in a paracrine fashion from cardiac stores in lampreys and increased filling of the heart also causes tachycardia [4,7]. The typical inhibitory action of the vagus nerve did not appear until the evolution of cartilaginous fishes, while the opposing role of an excitatory sympathetic innervation evolved later with the emergence of bony fishes. This cardiac innervation has remained the same within all subsequent groups of vertebrates, although the lungfish heart lacks sympathetic innervation although it does respond with increased rate and contractility to β-adrenergic stimulation originating from catecholamines stored within the heart [7].

Innervation of the Systemic Vasculature In tunicates and Amphioxus, the endothelium within the blood vessel is either very poorly developed or absent, and there is no endothelium in innervation amongst protostomes. Also there is no evidence for adrenergic receptors affecting vascular tone. Sympathetic nerves appear to be present in both hagfish and lampreys and the systemic vasculature responds to adrenergic agonists as well as acetylcholine. In all other groups of vertebrates, there are both α- and β-adrenergic receptors causing constriction and dilatation, respectively. This innervation is present on both the arterial and venous side of the circulatory system, but


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FIGURE 141.3 The cardiovascular changes in a fresh-

ventilation

120 100 80 60 40 20 0 100 80 60 40 20 0 50 40 30 20 10 0

water turtle (Trachemys scripta) during the transition from apnea to ventilation of the lungs. Heart rate and pulmonary blood flow are low during breath-holding (apnea), but increase markedly during ventilation associated with a change in the cardiac shunt pattern (modified from Wang and Hicks, 1996 [9]).

pulmonary blood flow (ml min–1)

systemic blood flow (ml min–1)

hart rate (min–1) 2 min

very little is known about putative difference between the different vascular beds to specific organs. The sympathetic innervation therefore is involved in both blood pressure regulation and regulation of venous tone and cardiac filling. The endothelium of ectothermic vertebrates releases nitric oxide as in mammals, but endothelial nitric oxide synthase (eNOS) only seems to have evolved in tetrapods, so neural NOS is responsible for NO production in the endothelium of fish.

Cardiovascular Responses to Altered Pressure, Exercise and Hypoxia A barostatic regulation of heart rate mediated through the ANS is present in fishes. Being aquatic, the circulatory system of fishes is not influenced by gravity and blood pressure control therefore seems to have evolved prior to any orthostatic pressure changes. The ancestral evolutionary benefit of an inhibitory vagal effect on heart rate and an opposing positive chronotropic and inotropic responses from sympathetic stimulation (or release of catecholamines) is likely to have been the ability to accommodate changes in metabolism with appropriate changes in heart rate and stroke volume, while maintaining a stable perfusion pressure. In all of the ectothermic vertebrates, metabolism changes in accordance with altered body temperatures; typically metabolism increases two-tothreefold when temperature increases 10°C. In these cases, heart rate increases proportionally, primarily because of a direct effect of temperature on the cardiac pacemaker and the scope for autonomic regulation normally remains intact. Therefore, the animals are able to increase heart

rate during exercise while the capacity to increase aerobic metabolism remains intact over a broad range of temperatures. Similarly the ectothermic animals retain the capacity for heart rate to respond to altered blood pressure over a broad temperature range. Hypoxia is common in aquatic habitats. Given that the foundations of autonomic regulation evolved in vertebrates relying on aquatic respiration over the gills, it is interesting that cartilaginous and bony fishes typically respond to hypoxia with a reduction in heart rate. Cardiac output typically remains unchanged, because the bradycardia is accompanied by increased stroke volume. The hypoxic bradycardia does not appear to enhance gas exchange efficiency across the gills, and may have evolved to protect the heart from oxygen lack. Regardless of the functional benefit of the hypoxic bradycardia, it is interesting that mammals also exhibit bradycardia during hypoxic stimulation of peripheral chemoreceptors but only when afferent feedback from pulmonary stretch receptors is ablated. Hence, this somewhat “counter-intuitive” cardiovascular response to hypoxia is likely to reflect a piscine condition that evolved prior to pulmonary gas exchange.

Autonomic Regulation of the Pulmonary Circulation Lungs and the pulmonary circulation evolved prior to the separation of the ventricle by a complete ventricular septum that is a characteristic of mammals and birds (Fig. 141.2). The ventricle in lungfish, amphibians, snakes, lizards and turtles, accordingly, is not fully divided and systolic blood pressures in the systemic and


ComPARATIVE ASPECTS of THE AuToNomIC REgulATIoN of THE CARdIoVASCulAR SySTEm

pulmonary circulations are identical [6]. The lack of division also implies that oxygen-rich blood from the lungs will, at least to some extent, be mixed with the oxygenpoor blood returning from the systemic circulation. As in the example presented in Figure 141.3, where blood flows were recorded in a freshwater turtle, the degree of mixing of oxygen-rich and oxygen-poor blood within the ventricle as well as the distribution of blood flows between the systemic and pulmonary circulations changes consistently with pulmonary ventilation [9]. Thus, during apnea, which in turtles and other diving species can last for more than one hour, heart rate and pulmonary blood flow are low and much of the oxygen poor blood returns directly into the systemic circulation (i.e., a large right-to-left cardiac shunt). As soon as pulmonary ventilation resumes pulmonary blood flow and heart rate then increase abruptly, while systemic blood flow is less affected. In several species, pulmonary blood flow exceeds systemic blood flow during ventilation, implying that a significant fraction of the blood returning from the lungs is returned directly to the pulmonary circulation (i.e., a left-to-right cardiac shunt).While the changes in cardiac shunt pattern act to provide a temporal matching of pulmonary ventilation and perfusion, the functional significance of these cardiovascular changes remain to be understood. The degree of oxygenated and deoxygenated blood mixing and the distribution of blood flows between the systemic and pulmonary circulations are dictated by the vascular resistance in the lungs and body [8]. Thus, when pulmonary vascular resistance is low compared to the systemic circulation, blood will primarily be distributed towards the lungs and vice versa [6,8]. The resistance of

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the pulmonary circulation is regulated by the ANS, where vagal innervation of smooth muscle in the pulmonary artery can cause constriction and reduce pulmonary blood flow. In mammals and birds, this vagal regulation of pulmonary blood vascular resistance is not present.

References [1] Liem KF, Bemis WE, Walker WF, Grande L. Functional anatomy of the vertebrates An evolutionary perspective. Philadelphia: Harcourt; 2001. [2] Burnstock G. Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol Rev 1969;21:247–324. [3] Gibbins I. Comparative anatomy and evolution of the autonomic nervous system. In: Nilsson S, .Holmgren S, editors. Comparative physiology and evolution of the autonomic nervous system.: Harwood Academic Publishers; 1994. p. 1–67. [4] Nilsson S, Holmgren S. The autonomic nervous system and chromaffin tissue in hagfishes. In: Jørgensen JM, Lomholt JP, Weber RE, Malte H, editors. Biology of Hagfishes. London: Chapman & Hall; 1998. p. 480–95. [5] Morris JL, Nilsson N. The circulatory system. In: Nilsson S, Holmgren S, editors. Comparative physiology and evolution of the autonomic nervous system. : Harwood Academic Publishers; 1994. p. 193–246. [6] Jensen B, Nielsen JM, Axelsson M, Pedersen M, Löfman C, Wang T. How the python heart separates pulmonary and systemic blood pressures and blood flows. J Exp Biol 2010;213:1611–7. [7] Farrell AP. Cardiovascular systems in primitive fishes. In: McKenzie DJ, Farrell AP, Brauner C J, editors. Fish physiology: Primitive fishes.: Elsevier; 2007. p. 53–120. [8] Taylor EW, Andrade D, Abe AS, Leite Cleo AC, Wang T. The unequal influences of the left and right vagi on the control of the heart and pulmonary artery in the rattlesnake, Crotalus durissus. J Exp Biol 2009;212:145–51. [9] Wang T, Hicks JW. Cardiorespiratory synchrony in turtles. J Exp Biol 1996;199:1791–800.


C H A P T E R

142 Human Physiome Peter Hunter INTRODUCTION Computational biology and computational physiology are playing an increasingly important role in helping scientists understand and quantify the complex multiscale structure-function relations of biological systems. Interpreting the wealth of quantitative data now available on tissue and subcellular processes requires a new level of international collaboration between biological, physical (including engineering), mathematical, computer and computational scientists. Furthermore, biological processes operate primarily at the molecular scale (ligand/ protein/DNA/RNA interactions) but are influenced by, or in turn create an influence on, the physiological systems of cells, tissues, organs and whole body organ systems. This influence therefore encompasses a 109 range of spatial scales (from nm at the molecular scale to m at the human organ system level) and a 1015 range of temporal scales (from μs for molecular interactions to the 109 s of a human lifespan). The VPH/Physiome project is facilitating the multidisciplinary contributions and addressing the challenges of modeling the enormous range of spatial and temporal scales involved in physiological processes. In this brief overview of the human physiome we discuss the development of modeling standards, databases and computational tools by the VPH/Physiome project and illustrate these with reference to the heart. We then suggest how the framework could be applied to the autonomic nervous system and describe some initial steps that have been taken.

PHYSIOME STANDARDS As the computational models inevitably become more complex, it is increasingly difficult for anyone other than the author(s) of the publication describing the model to decipher, code and run the model in order to reproduce the results claimed in a publication. It can also be very

difficult to then use this model as one component of a more complex model. To address these challenges several groups have developed standards for encoding models over the past 10 years. These modeling standards typically use the eXtensible Markup Language (XML) developed by the w3c1 as well as a variety of other standards based on XML, such as MathML for encoding mathematics, as well as various metadata standards. Two XML-based model encoding standards are currently being developed under the IUPS Physiome Project [1,2] and the European Virtual Physiological Human (VPH) project (www.vph-noe.eu). CellML (www.cellml. org) is designed to encode lumped parameter biophysically based systems of ordinary differential equations (ODEs) and nonlinear algebraic equations (together called differential algebraic or DAE systems). FieldML (www. fieldml.org) is designed to encode spatially and temporally varying field information such as anatomical structure, the spatial distribution of protein density or computed fields such as the electrical potential or oxygen concentration throughout a tissue. A third markup language called the “systems biology markup language” or SBML (www.sbml. org) has been developed by the systems biology community. This is similar to CellML but targeted more specifically to representing models of biochemical reactions. CellML maintains a clean separation between the syntax of a model (e.g., the mathematical equations encoded in MathML) and the semantics (the biological and biophysical meaning of the model components and parameters) defined in the model metadata by reference to suitable ontologies. This facilitates building complex models by importing modular components defined in libraries. SBML is more closely tied to the concept of a biochemical “reaction”, and does not maintain a separation between the model syntax and the biological semantics. FieldML deals with the encoding of anatomy at multiple spatial scales by allowing hierarchies of material coordinate systems that preserve anatomical relationships (e.g., coronary arteries embedded in a deforming myocardial tissue that is itself part of a heart contained within a torso).

1

“w3c” refers to the worldwide web consortium that is developing and maintaining standards for information exchange via the internet – see www.w3c.org.


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FIGURE 142.1 The multi-scale cardiac physiome modeling hierarchy from genes to the whole organism. Parameters used in a model at one scale can often be derived from a more detailed model at a lower spatial scale.

These three standards are supported by the US National Institutes of Health (NIH) and the European Commission’s funding agency (currently operating under Framework 7). Model repositories are available for all three markup languages – PMR2 (Physiome Model Repository 2, see models.cellml.org) for CellML and FieldML models and Biomodels (www.biomodels.net) for SBML models. Various minimum information standards are also available including MIRIAM (www.ebi.ac.uk/miriam) and MIASE (www .ebi.ac.uk/compneur-srv/miase). An illustration of the use of these standards, together with the computational software and model repositories associated with the CellML and FieldML standards, is given in Fig. 142.1. The heart physiome project is the most developed example of multi-scale modeling as it has been able to draw on decades of measurements and modeling of cardiac ion channels [3], myofilament mechanics [4] and myocardial structure [5]. The models link the biomechanics of the myocardial tissue and blood flow (ventricles and coronaries) to cellular processes that include the ion channel based electrophysiology, intracellular calcium transport, myofilament mechanics, control of pH and bicarbonate, metabolic pathways and signal transduction pathways. A key feature of physiome models is the incorporation of anatomical structure into the models in order to

accurately represent structure–function relations at the various spatial scales. For example, the 3D structure of tissue that gives rise to continuum properties (conductivity, elasticity, etc) is shown as part of the multi-scale framework in Figure 142.2. Note that there are characteristic material directions in the tissue that define structural features, such as the fiber direction or sheet orientation, and that the continuum properties are different in each direction (the tissue is said to be “anisotropic”). Note also that these characteristic material directions and the values of the continuum properties vary throughout the tissue (the tissue properties are said to be “inhomogeneous”). Thus the electrical and mechanical properties along the fiber direction in cardiac muscle are different from those properties measured transversely to the fibers and these properties are different in different material locations. Anisotropy and inhomogeneity are characteristics of all biological tissues. The mechanics equations are usually solved using finite element techniques and the electrical activation equations are solved using finite volume or similar techniques based on grid points defined as material points of the deforming mechanics grid. Other organ systems that have been modeled with anatomically and biophysically based multi-scale approaches have been the lungs [6], the digestive system [7], the neuro-musculo-skeletal system [8]. In fact there are now


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modElIng THE AuTonomIC nERvous sysTEm

Torso model

Organ model

Myocardial activation Ventricular mechanics Ventricular blood flow & heart valve mechanics

Continuum tissue model

Calcium transport models Myofilament mechanics

Composite lumped parameter

Discrete tissue structure model

Ion channel model of the Hodgkin-Huxley type

Signal pathway models

3D protein model

Metabolic pathway models Gene regulation models

Markov ion channel model

Coarse grained MD model 3D cell model Molecular dynamics model Quantum mechanics model

FIGURE 142.2 Types of model used in the multiscale modeling hierarchy. Models based on systems of ODEs and algebraic equations are shown in blue (so called ‘lumped parameter’ models) and these are encoded in CellML. Models that require the solution of partial differential equations are shown in pink and are encoded in FieldML. The FieldML models link to CellML models at material points in the tissue. The arrows above are shown as unidirectional but, in fact, information flows both ways. The models shown in gray will be linked into the cardiac modeling hierarchy in the future.

nascent physiome modeling efforts on some aspects of all of the body’s 12 organ systems.

MODELING THE AUTONOMIC NERVOUS SYSTEM There is of course a large neuroscience community involved in modeling many aspects of the brain but there has been surprisingly little engagement between neuroscientists and the physiome community over the challenges of connecting organ system models to their neural control or sensory systems. The autonomic nervous system would be a good place to start. Two steps that could be taken are as follows: 1. Develop a FieldML-encoded finite element model of the spinal chord and associated neural pathways throughout the body (not the brain) – see Figure 142.3. These would need to be defined anatomically with respect to the musculo-skeletal system so that they would be, for example, carried with the tissue during movement. 2. Encode, curate and annotate all published models of neural processes in the CellML model repository. These can then be used to create more integrated models that can be linked to organ system physiology. This process has been started and the models that are currently in the CellML model repository at

models.cellml.org/neurobiology are listed below. Literature references are also given for all of these models: Bertram, Rhoads, Cimbora, 2008 [9]. A phantom bursting mechanism for episodic bursting: (a) Original model. (b) Modified to include channel noise in the leak current. Butera, Rinzel, Smith, 1999 [10,11]. Models of respiratory rhythm generation in the pre-Botzinger complex: (a) Bursting Pacemaker Neurons: model 1 (does not include a slow potassium current). (b) Bursting Pacemaker Neurons: model 2 (includes a slow potassium current). (c) Populations of Coupled Pacemaker Neurons: 5 Cell Model. (d) Populations of Coupled Pacemaker Neurons: 10 Cell Model. (e) Populations of Coupled Pacemaker Neurons: Single Cell Model. (f) Populations of Coupled Pacemaker Neurons: Single Cell Model. Cloutier, Bolger, Lowry, Wellstead, 2009 [12]. An integrative dynamic model of brain energy metabolism using in vivo neurochemical measurements. Friel, 1995 [13]. [Ca2]i oscillations in sympathetic neurons: an experimental test of a theoretical model.


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FIGURE 142.3 (a) Neural pathways, and (b) a schematic mapping to organ systems. Figure 3a is from Rita Carter, ‘The Human Brain Book’, by permission Dorling Kindersley Ltd.

Li, Bertram, Rinzel, 1996 [14]. Modeling N-methyl-Daspartate-induced bursting in dopamine neurons: (a) Simple model based on the equations in the published paper. (b) Complex model based on the equations in the published paper. (c) Simple model based on the equations in the original code. Ostby, Omholt, Oyehaug, Einevoll, Nagelhus, Plahte, Zeuthen, Voipio, Lloyd, Ottersen, 2008 [15]. Astrocytic processes explaining neural-activity-induced shrinkage of extraneuronal space. Phillips, Robinson, 2007 [16]. A quantitative model of sleep-wake dynamics based on the physiology of the brainstem ascending arousal system.

Phillips, Robinson, 2008 [17]. Sleep deprivation in a quantitative physiologically based model of the ascending arousal system: (a) Baseline model. (b) Sleep deprivation model. Plant, 1981 [18]. Bifurcation and resonance in a model for bursting nerve cells. Purvis, Butera, 2005 [19]. Ionic current model of a hypoglossal motoneuron. Purvis, Smith, Koizumi, Butera, 2007 [20]. Intrinsic bursters increase the robustness of rhythm generation in an excitatory network: (a) Single pacemaker cell. (b) Single non-pacemaker cell.


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FIGURE 142.4 Schematic of the computational model of the brain stem respiratory network. The model includes interacting neuronal populations within the major brain stem respiratory compartments (Pons, BotC, pre-BotC, and rVRG). Spheres represent neuronal populations (excitatory, red; inhibitory, blue; motoneuronal, brown); green triangles represent sources of tonic excitatory drives (in pons, RTN/BotC, and pre-BotC compartments) to different neural populations. Excitatory and inhibitory synaptic connections are indicated by arrows and small circles, respectively. Simulated 'transections' (dashed lines) mimic those performed in situ.

Rubin, Shevtsova, Ermentrout, Smith, Rybak, 2009 [21]. Multiple rhythmic states in a model of the respiratory central pattern generator. Smith, Abdala, Koizumi, Rybak, Paton, 2007 [22]: (a) (b) (c) (d) (e)

Synaptic coupling. Preinspiratory neuron. Early inspiratory neuron. Augmenting expiratory neuron. Postinspiratory neuron.

Tabak, Mascagni, Bertram, 2010 [23]. Mechanism for the universal pattern of activity in developing neuronal networks. Vasalou, Henson, 2010 [24]. A multiscale model to investigate circadian rhythmicity of pacemaker neurons in the suprachiasmatic nucleus. The model by Smith et al. (2007) [21] provides an example of the use of the CellML framework. A schematic of the model is given in Figure 142.4 which shows interacting neuronal populations within the major brain stem respiratory compartments. The model deals with the brain stem respiratory central pattern generator (CPG) that produces rhythmic movements and shows how the normal

three-phase respiratory rhythm transforms to a two-phase and then to a one-phase rhythm as the network is reduced. Expression of the three-phase rhythm required the presence of the pons, generation of the two-phase rhythm depended on the integrity of Botzinger and pre-Botzinger complexes and interactions between them, and the onephase rhythm was generated within the pre-Botzinger complex. The model shows that the respiratory network has rhythmogenic capabilities at multiple levels of network organization, allowing expression of motor patterns specific for various physiological and pathophysiological respiratory behaviors.

CONCLUSIONS The VPH/Physiome project is providing a framework for multi-scale modeling of physiological processes, typically involving anatomically and biophysically based models that capture structure/function relations at multiple spatial (and sometimes temporal) scales. The framework has processes for ensuring model reproducibility, including markup languages for encoding models, data standards


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and model repositories, as well as open source software for authoring, visualizing and solving models. In this brief overview, we have described the markup languages CellML, SBML and FieldML and have illustrated the use of the physiome framework for modeling the heart. We have also proposed two steps that could now be taken to extend the physiome approach to neural processes, and in particular the autonomic nervous system.

References [1] Hunter PJ, Borg TK. Integration from proteins to organs: The physiome project. Nat Rev Mol Cell Biol 2003;4:237–43. [2] Hunter PJ, Nielsen PMF. A strategy for integrative computational physiology. Physiology 2005;20:316–25. [3] Noble D, Rudy Y. Models of cardiac ventricular action potentials: Iterative interaction between experiment and simulation. Phil Trans R Soc Lond A 2001;359(1783):1127–42. [4] Nash MP, Hunter PJ. Computational mechanics of the heart. J Elasticity 2001;61(1–3):113–41. [5] LeGrice IJ, Hunter PJ, Smaill BH. Laminar structure of the heart: a mathematical model. Am J Physiol 1997;272:H2466–H2476. [6] Tawhai M, Hoffman EA, Lin CL. The lung physiome: Merging imaging-based measures with predictive computational models of structure and function. Wiley Interdiscip Rev Syst Biol 2009;1(1): p. 61–72. [7] Cheng LK, O'Grady GB, Du P, Egbuji JU, Windsor JA, Pullan AJ. Gastrointestinal system. Wiley Interdiscip Rev Syst Biol Med 2010;2(1):p65–79. [8] Fernandez JW, Mithraratne P, Thrupp SF, Tawhai MH, Hunter PJ. Anatomically based geometric modeling of the musculoskeletal system and other organs. Biomech Model Mechanobiol 2004;2(3):139–55. [9] Bertram R, Rhoads J, Cimbora WP. A phantom bursting mechanism for episodic bursting. Bull Math Biol 2008;70:1979–93. [10] Butera RJ, Rinzel J, Smith JC. Models of respiratory rhythm generation in the pre-Botzinger complex. I. Bursting pacemaker neurons. J Neurophysiol 1999;81:382–97. [11] Butera RJ, Rinzel J, Smith JC. Models of respiratory rhythm generation in the Pre-Botzinger complex. II. Populations of coupled pacemaker neurons. J Neurophysiol 1999;82:398–415.

[12] Cloutier M, Bolger FB, Lowry JP, Wellstead P. An integrative dynamic model of brain energy metabolism using in vivo neurochemical measurements. J Comput Neurosci 2009;27:391–414. [13] Friel DD. [Ca2]i oscillations in sympathetic neurons: an experimental test of a theoretical model. Biophys J 1995;68:1752–66. [14] Li YX, Bertram R, Rinzel J. Modeling N-methyl-D-aspartateinduced bursting in dopamine neurons. Neuroscience 1996;71:397–410. [15] Ostby I, Oyehaug L, Einevoll GT, Nagelhus EA, Plahte E, Zeuthen T, Lloyd CM, Ottersen OP, Omholt SW. Astrocytic mechanisms explaining neural-activity-induced shrinkage of extraneuronal space. PLoS Comput Biol 2009;5:1. [16] Phillips AJK, Robinson PA. A quantitative model of sleep-wake dynamics based on the physiology of the brainstem ascending arousal system. J Biol Rhythms 2007;22:167–79. [17] Phillips AJK, Robinson PA. Sleep deprivation in a quantitative physiologically based model of the ascending arousal system. J Theor Biol 2008;255:413–23. [18] Plant RE. Bifurcation and resonance in a model for bursting nerve cells. J Math Biol 1981;11:15–32. [19] Purvis LK, Butera RJ. Ionic current model of a hypoglossal motoneuron. J Neurophysiol 2005;93:723–33. [20] Purvis LK, Smith JC, Koizumi H, Butera RJ. Intrinsic bursters increase the robustness of rhythm generation in an excitatory network. J Neurophysiol 2007;97:1515–26. [21] Rubin JE, Shevtsova NA, Ermentrout GB, Smith JC, Rybak IA. Multiple rhythmic states in a model of the respiratory central pattern generator. J Neurophysiol 2009;101:2146–65. [22] Smith JC, Abdala AP, Koizumi H, Rybak IA, Paton JF. Spatial and functional architecture of the mammalian brain stem respiratory network: A hierarchy of three oscillatory mechanisms. J Neurophysiol 2007;98:3370–87. [23] Tabak J, Mascagni M, Bertram R. Mechanism for the universal pattern of activity in developing neuronal networks. J Neurophysiol 2010;vol. 103:2208–21. [24] Vasalou C, Henson MA. A multiscale model to investigate circadian rhythmicity of pacemaker neurons in the suprachiasmatic nucleus. PLoS Comput Biol 2010;6


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143 Modeling the Autonomic Nervous System Ilya A. Rybak, Yaroslav I. Molkov, Julian F.R. Paton, Ana P.L. Abdala, Daniel B. Zoccal Sympathetic nerve activity normally exhibits respiratory modulation that suggests the existence of central interactions between the respiratory and sympathetic networks within the brainstem. A large-scale computational model of interacting respiratory and sympathetic circuits has been developed and used to investigate the possible mechanisms of sympatho-respiratory interactions and their role in the baroreceptor reflex control of sympathetic activity and in the elevated sympathetic activity following chronic intermittent hypoxia. Several model predictions have been formulated and tested experimentally. The model provides important insights into the role of sympatho-respiratory interactions in the control of sympathetic outflow and arterial blood pressure under different physiological and patho-physiological conditions.

LARGE-SCALE COMPUTATIONAL MODEL OF THE BRAINSTEM SYMPATHO-RESPIRATORY NETWORK A large-scale computational model of the brainstem sympatho-respiratory network has been developed to simulate the respiratory and sympathetic neural circuits interacting within the brainstem (Fig. 143.1A). The major circuits critically involved in generation of the respiratory rhythm and pattern are located in the ventral respiratory column (VRC) and include (rostral-to-caudal) the Bötzinger (BötC) and pre-Bötzinger (pre-BötC) complexes and the rostral (rVRG) and caudal (cVRG) ventral respiratory groups [1,2]. The core of the respiratory central pattern generator was proposed to include (i) an excitatory pre-inspiratory/inspiratory (pre-I/I) population of neurons with intrinsic bursting properties located in the pre-BötC and (ii) a ring of three mutually inhibiting neural populations: the post-inspiratory (post-I) and augmenting-expiratory (aug-E) populations of BötC, and the early-inspiratory (early-I(1)) population of pre-BötC (Fig. 143.1A, see [2,3]). The respiratory circuitry in the model also incorporates two neural populations within the rVRG, the bulbospinal ramp-inspiratory (ramp-I) neurons, projecting to phrenic motoneurons in the spinal cord that send their axons to the


phrenic nerve (PN), and the inhibitory early-I(2) neurons shaping the firing pattern of ramp-I neurons, and a population of bulbospinal premotor expiratory neurons of cVRG (bs-E) projecting to the abdominal motoneurons that define activity of the abdominal nerve (AbN) (Fig. 143.1A). The sympathetic circuits in the model include neurons located in the rostral (RVLM) and caudal (CVLM) ventrolateral medulla (VLM). Specifically, the RVLM neurons define the activity in the thoracic sympathetic nerve (tSN). In addition, the following populations were included in the model: two populations of 2nd order baroreceptor neurons in the nucleus tractus solitarii (NTS) receiving baroreceptor afferents and a population of phase-spanning inspiratory-expiratory neurons (IE) in the ventrolateral pons. (Fig. 143.1A, see also [4]). The model also incorporates a compartment known as the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), containing neurons performing central chemoreceptor function whose activity is sensitive to CO2. This compartment includes a population of neurons with intrinsic bursting properties termed the late-expiratory (late-E) population (Fig. 143.1A). The inclusion of this population in the RTN/pFRG is based on the multiple experimental data that the late-E activity emerging in AbN during hypercapnia originates in this region [3,5]. In addition to multiple mutual interactions all respiratory neural populations in the model receive excitatory drives from the pons, RTN/pFRG and raphé [2,3].

MODELING THE EFFECTS OF BARORECEPTOR ACTIVATION ON THE RESPIRATORY PATTERN: INSIGHTS INTO RESPIRATORY–SYMPATHETIC INTERACTIONS The baroreceptor reflex is an important negative feedback mechanism controlling sympathetic outflow. The classical baroreflex controls tSN via 2nd order barosensitive neurons in the NTS that receive the direct excitatory inputs from baroreceptor afferents. It is suggested that excitatory NTS neurons project to CVLM neurons, which

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FIGURE 143.1 The computational model of sympatho-respiratory brainstem network. (A) Schematic of the model showing interactions between different populations of respiratory neurons within major brainstem compartments involved in the control of breathing and sympathetic activity. Abbreviations: brainstem compartments: BötC – Bötzinger complex; CVLM – caudal ventrolateral medulla; cVRG – caudal ventral respiratory group; NTS – nucleus tractus solitarii; pre-BötC – pre-Bötzinger complex; RTN/pFRG – retrotrapezoid nucleus/parafacial respiratory group; RVLM – rostral ventrolateral medulla; rVRG – rostral ventral respiratory group; VLM – ventrolateral medulla; VRC – ventral respiratory column; neural populations: aug-E – augmenting expiratory; early-I – early-inspiratory; IE – phase-spanning inspiratory-expiratory; late-E – late-expiratory; post-I – postinspiratory; post-I(e) – post-inspiratory (excitatory); pre-I/I – pre-inspiratory/inspiratory; ramp-I – ramp-inspiratory; motor outputs: PN – phrenic nerve; AbN – abdominal nerve; tSN – thoracic sympathetic nerve. Keys are shown in the right-bottom corner. Each population (large sphere) consists of 20–50 neurons modeled in the Hodgkin–Huxley style. All tonic drive sources (gray triangles) provide constant drive, except for RTN/pFRG which is CO2-dependent (see panel C). Not all connections from pontine and RTN/pFRG drive sources are shown. For details see [2,3]. (B) Conceptual model of interaction between VRC, PONS, NTS, and RVLM/CVLM. The sympathetic baroreceptor reflex operates via two pathways: one direct pathway (black solid arrows) includes baroreceptors, NTS (2nd order barosensitive cells) and CVLM, which inhibits RVLM and tSN; the other pathway goes from baroreceptors through NTS and VRC (black dashed arrows), whose post-inspiratory neurons inhibit RVLM and tSN. Gray dashed arrows show interactions between PONS and medullary compartments VRC and RVLM/CVLM. (C) RTN/pFRG tonic drive as function of CO2 for the control (solid curve) and CIH-conditioning (dashed curve) cases.


ModElINg THE EFFECTS oF BARoRECEPToR ACTIvATIoN oN THE RESPIRAToRy PATTERN

inhibit RVLM neurons hence lowering the tSN activity (Fig. 143.1B). On the other hand, tSN has an obvious respiratory modulation that persists after vagotomy and decerebration, supporting the idea of central coupling between respiratory and sympathetic networks. In turn, the respiratory activity is known to be modulated by baroreceptor afferents through the same 2nd order barosensitive neurons of NTS. This suggests that central sympathorespiratory interactions may contribute to the dynamic control of sympathetic activity. Specifically, it has been hypothesized [4] that the sympathetic baroreceptor reflex has two major pathways (Fig. 143.1B): one direct path mentioned above, that is independent of the respiratorysympathetic interactions, and the other, operating via the baroreceptor modulation of the respiratory activity and respiratory-sympathetic interactions and hence dependent on the respiratory modulation of tSN activity. At rest, the tSN activity typically exhibits a well expressed positive modulation during inspiration and a negative modulation during post-inspiration (see Fig. 143.2A1–A3 before applied stimulations). The tSN respiratory modulation was significantly suppressed or eliminated after removal of the pons, when phrenic nerve (PN) activity transformed to an apneustic pattern with prolonged inspiratory bursts and shortened expiration durations (see Fig. 143.2A4 before applied stimulation), underlying a critical role of the pons in the tSN respiratory modulation [6]. In Figure 143.2A1–A5, the transient increases in the arterial pressure were induced in the arterially perfused in situ rat preparation [6]. Stimuli were delivered during inspiration, post-inspiration or late expiration and produced phase-dependent effects on both the phrenic nerve (PN) activity and the respiratory modulation of tSN (Fig. 143.2A1–A3). With pons intact, the applied barostimulation had almost no effect on the amplitude and duration of the phrenic bursts even when stimuli were delivered during inspiration (Fig. 143.2A1). At the same time, these stimuli suppressed or abolished inspiratory modulation of tSN activity. In contrast, the same stimuli delivered during post-inspiration (Fig. 143.2A2) or late expiration (Fig. 143.2A3) produced an increase in the expiration period and decreased the tSN activity. The barostimulation-evoked prolongation of expiration was greater if stimulation was applied later during the expiratory phase (compare Figs 143.2A2 and A3). Importantly, after pontine transection the respiratory modulation of tSN activity was greatly reduced [6]. In all cases, however, the sympathetic baroreflexinduced lowering of tSN persisted and the barostimulation shortened the apneustic inspiratory burst (see Fig. 143.2A4). Figure 143.2B1–B4 shows the results of our simulation of the effects of transient barostimulation during different phases of the respiratory cycle using the computational model described above (Fig. 143.1A) before (Fig. 143.2B1– B3) and after (Fig. 143.2B4) removal of the pontine compartment. The model generates a normal three-phase respiratory pattern with augmenting PN bursts (Fig. 143.2A1–A3). The sympathetic output in the model (tSN) exhibits a positive

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inspiratory modulation provided by the pontine IE population to RVLM and a negative post-inspiratory modulation resulting from the inhibitory inputs from the post-I population of BötC to the RVLM. Transient barostimulation applied to the barosensitive 2nd order NTS population produces a temporal reduction of tSN output via direct activation of the CVLM population that inhibits the activity of RVLM population. Stimulus application during inspiration (Fig. 143.2B1) does not affect respiratory (PN) activity. In contrast, stimuli applied during post-inspiration (Fig. 143.2B2) and late expiration (Fig. 143.2B3) prolong expiration via activation of post-I neurons of BötC that inhibit both the aug-E population and the RVLM. These interactions represent a second component of the sympathetic baroreflex involving interactions between the respiratory and sympathetic circuits. Similar to our experimental data (Fig. 143.2A2,A3), stimulation-evoked prolongation of expiration is greater if stimulation is applied later during the expiratory phase (Fig. 143.2B2,B3). Removing the pontine compartment in the model converts the normal eupnea-like respiratory pattern to the apneustic pattern characterized by prolonged PN busts (see Fig. 143.2B4). As demonstrated previously [2], this pattern is characterized by a lack of post-I activity that is strongly dependent on pontine drive. Therefore, with the pons removed, the respiratory modulation of tSN (formed by inputs from pontine IE and BötC’s post-I populations to RVLM) is abolished. Simultaneously, the central suppression of the baroreflex gain by the rVRG early-I(2) population, whose activity in the model is also dependent on the pontine drive, is eliminated with pontine removal. Therefore the applied barostimulation can activate post-I population during inspiration and produce an advanced termination of the apneustic inspiratory bursts hence shortening inspiration (compare Fig. 143.2B4 with Fig. 143.2A4). Figure 143.2B5 illustrates the neural mechanism by which the transient barostimulation applied during expiration prolongs this expiration in the intact model (see Fig. 143.2B3 for comparison). The post-I neurons of BötC when activated inhibit all inspiratory (and aug-E) neurons and initiate the post-inspiratory phase of expiration. During expiration, the activities of these neurons decrement hence releasing aug-E neurons form inhibition and allowing for their gradual activation (see unperturbed breathing cycles in Fig. 143.2B5). When a barostimulus occurs during expiration, the post-I population is activated and inhibits the aug-E population, hence producing a “resetting of expiration”. This resetting of expiration by the transient barostimulation provides a mechanistic explanation for expiratory period prolongation. To test this model prediction, extracellular recordings of post-I and aug-E neurons were made within the BötC of in situ rat preparations [4] (see example in Fig. 143.2A5). With the transient increase in perfusion pressure during expiration, the activity of post-I neurons increased and the activity of aug-E neurons decreased in full accordance with the model prediction (compare Fig. 143.2A5 with Fig. 143.2B5). In most cases,


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FIGURE 143.2 Effects of transient, respiratory phase-dependent baroreceptor stimulation on phrenic (PN) and sympathetic (tSN) nerve activities in situ and in the model. (A1–A5) Experimental results from the arterially perfused in situ preparation A1-A3: Stimulation was applied to the intact preparation during inspiration (A1), post-inspiration (A2) and late expiration (A3). After pontine transection (A4), the applied stimulus shortened the apneustic inspiratory (PN) burst. Traces from top to bottom: integrated sympathetic (tSN) activity, integrated phrenic (PN) activity; perfusion pressure (PP). In A5, top two traces show an extracellular recording from a post-I neuron in BötC and the histogram of its activity; the next pair of traces show a simultaneous extracellular recording from an aug-E neuron of BötC and the corresponding histogram; the remaining traces show the integrated activities of PN and tSN) and perfusion pressure (PP). (B1–B5) Corresponding simulation results. In simulations shown, the stimulus was applied during inspiration (B1), post-inspiration (B2) and late expiration (B3), and also after removal of the pontine compartment in the model (B4). In B5, the top pair of traces show membrane potential of a randomly chosen neuron from the post-I population of BötC and integrated spike histogram of the entire post-I population. Second pair of traces: membrane potential of a randomly chosen neuron from the aug-E population of BötC and spike histogram of the entire aug-E population.

the barostimulation applied during expiration resulted in prolongation of expiration, and this prolongation was greater when stimulation was applied later in expiration. These studies have clarified the role of baroreceptor input in activating post-I neurons and inhibiting aug-E neurons and demonstrated that even weak excitatory input from baroreceptors to the post-I neurons can account for the prolongation of expiration and the corresponding effect on tSN activity. In general, this demonstrates an important contribution of central sympatho-respiratory interactions to the baroreceptor control of arterial pressure.

SYMPATHETIC NERVE ACTIVITY FOLLOWING CHRONIC INTERMITTENT HYPOXIA-INDUCED SENSITIZATION OF CENTRAL CHEMORECEPTORS Recurrent episodes of hypoxia, such as observed in obstructive sleep apnea lead to the development of hypertension. It was shown that rats exposed to chronic intermittent hypoxia (CIH) exhibited higher levels of arterial pressure associated with an elevated sympathetic vasomotor tone [7,8] and an enhanced respiratory modulation


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143.3 Sympathetic and respiratory responses to hypercapnia before and after CIH-conditioning: experimental records and simulations. (A1– A5) Activity of phrenic (PN), abdominal (AbN), and thoracic sympathetic (tSN) nerves in the representative control preparation at 5% CO2 (base level, A3), during hypercapnia (7% CO2 in A4 and 10% CO2 in A5) and during hypocapnia (3% CO2 in A2 and 1% CO2 in A1). Note the skipping of some late-E bursts in both AbN and tSN at 7% CO2 (in A4). (B1–B5) PN, AbN, and tSN activities in the representative CIH-conditioned preparation at 5% CO2 (base level, B3), during hypercapnia (7% CO2 in B2 and 10% CO2 in B3) and hypocapnia (3% CO2 in B2 and 1% CO2 in B1). Note the presence of late-E bursts in both AbN and tSN at 5% CO2 (in B1). The activity of each nerve is represented by raw recording (bottom trace) and integrated activity (upper trace). (C–E) Model performance in simulated control (C) and CIH (E) cases. In both cases, integrated activity for phrenic (PN), abdominal (AbN) and thoracic sympathetic (tSN) outputs are shown. The CO2 level was changed in a step-wise manner from 1% (hypocapnia) to 10% (hypercapnia) which is shown in panel D. The dashed vertical arrows indicate CO2 levels for emerging late-E activity and for hypocapnic apnea, respectively.

of sympathetic activity [7]. This suggests that central coupling between respiratory and sympathetic circuits may contribute to hypertension in CIH-conditioned animals [8]. Typical patterns of respiratory (PN and AbN) and sympathetic (tSN) activities in the naïve rat (arterially perfused in situ preparation) are shown in Fig. 143.3A1–A5.

Under normal conditions (5% CO2), the integrated PN burst has an augmenting profile, AbN shows low-amplitude activity, and the tSN expresses an augmenting inspiratory modulation (Fig. 143.3A3). Hypercapnia (increase in CO2 level from 5%) evokes high-amplitude lateexpiratory (late-E) AbN discharges, which are phase-locked


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to PN bursts (Fig. 143.3A4,A5; see also [3,5]). With progressive development of hypercapnia, the ratio of AbN late-E burst frequency to PN burst frequency quantally (step-wise) increases from about 1:4/1:3 to 1:2 (at 7% CO2), when approximately each second respiratory cycle was skipping in AbN late-E activity (Fig. 143.3A4), and finally, to 1:1 as the CO2 level increases to 10% (Fig. 143.3A5), when the AbN late-E discharges preceding PN bursts are observed in every respiratory cycle [3]. This CO2-induced AbN late-E activity appears to originate in the RTN/ pFRG region [3,5,9] that is also known to be a major site for central chemoreception [10]. The CO2-censitive RTN/ pFRG neurons project to both VRC and RVLM. The latter is important for the sympatho-excitation and the corresponding increase of arterial pressure observed during hypercapnia. The hypercapnia-evoked late-E activity, quantally accelerated with an increase in CO2, has been observed in tSN (Fig. 143.3A4,A5). Moreover, the tSN lateE activity coincides with the AbN late-E bursts, suggesting a common source of late-E activities in AbN and tSN located the RTN/pFRG. The interesting effect of CIH conditioning is that it alters the AbN and tSN activities in both normocapnia and hypercapnia [7,8]. Figure 143.3B3 shows that the juvenile rats submitted to CIH for 10 days exhibits an expressed late-E activity in both AbN and tSN (with a frequency ratio to PN of about 1:3/1:2) in the baseline conditions (5% CO2), and at 7% CO2 this ratio already reaches 1:1, i.e. full synchronization (Fig. 143.3B4). We suggest that CIH conditioning augment the CO2 sensitivity of RTN/pFRG neurons, and hence reduces CO2 threshold for the emergence of late-E oscillations seen in both AbN and tSN. The computational model shown in Figure 143.1A was used for simulating the effect of CIH-induced sensitization of RTN/pFRG neurons on the respiratory and sympathetic activities and the sympatho-respiratory response to hyperand hypocapnic conditions. The model includes excitatory tonic drives from several sources including RTN/ pFRG that is considered to be a major central chemoreceptor site sensitive to CO2 [10]. In the model, we consider RTN/pFRG tonic drive to be not constant but dependent on the CO2 level as shown in Figure 143.1C (solid curve). As hypothesized above, the CO2 sensitivity of RTN/pFRG increases as a result of CIH exposure. This is simulated by the horizontal shift of the CO2-dependent RTN drive by 2% CO2 to the direction of lower CO2 values (to the left, see dashed curve in the Fig. 143.1C). Figure 143.3C shows the results of our simulations with CO2 step-wise increasing from 1% CO2 (hypocapnia) through 5% CO2 (normocapnia) to 10 % CO2 (hypercapnia) as illustrated in Figure 143.3D. In our simulations, progressive hypercapnia (Fig. 143.3C,D, right part of the graph) leaded to the emergence and quantal acceleration of late-E bursts in both AbN and tSN, which was consistent with experimental records (see panels A3– A5). Specifically, the late-E discharges in AbN and tSN

appeared at 7% CO2 and reached 1:1 ratio to the PN bursts at 9% CO2. Note also (see Fig. 143.3C,D, left part) that a reduction of CO2 below 3% caused “hypocapnic apnea” (a lack of PN activity). To simulate CIH conditions, the curve reflecting the CO2 dependence of RTN/pFRG drive was shifted to the left (Fig. 143.1C). As a result of this shifting, the late-E bursts in AbN and tSN emerged at 4% CO2, and in the normocapnic state (5% CO2) they showed a stable 1:2 ratio to the PN bursts (Fig. 143.3E); at 7% CO2 this ratio reached 1:1 (Fig. 143.3B4), which was consistent with our experimental observations (see Fig. 143.3B). The second observation from the above simulation is that “CIH conditioning” reduced the apneic threshold for hypocapnia by at least 2% CO2, since the PN bursts were still generated even at 1% CO2 (see Fig. 143.3E, left). To check this modeling prediction, the control (naïve) and CIH-conditioned rat preparations were exposed to progressive hypocapnia (from normal 5% CO2 to 3% and then to 1%). The naïve rat preparations exhibited a reduction in the integrated PN burst amplitude at 3% CO2 and a hypocapnic apnea at 1% CO2 (Fig. 143.3A2,A1). Importantly, these preparations never expressed late-E activity in AbN or tSN in either normocapnia or hypocapnia, and the respiratory modulation of tSN was reduced at 3% CO2 and absent at 1% during hypocapnic apnea (Fig. 143.3B2,B1). In CIH rat preparations, the expressed late-E activity in both AbN and tSN was already present during normocapnia (at 5% CO2) and disappeared from both nerves at 3% CO2 (Fig. 143.3B2). At the same time, PN activity with a reduced amplitude (and respiratory modulation of tSN) was still present even at 1% CO2 (Fig. 143.3B1), hence confirming modeling prediction on a reduction of apneic threshold for hypocapnia in CIH-conditioned rats. Our multidisciplinary investigation suggests that the arterial blood pressure elevation associated with CIH may result from an increased CO2 sensitivity of central chemoreceptors and early emergence of late-E oscillations in the RTN/pFRG.

References [1] Alheid GF, McCrimmon DR. The chemical neuroanatomy of breathing. Respir Physiol Neurobiol 2008;164:3–11. [2] Smith JC, Abdala APL, Koizumi H, Rybak IA, Paton JFR. Spatial and functional architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory mechanisms. J Neurophysiol 2007;98:3370–87. [3] Molkov YI, Abdala APL, Bacak BJ, Smith JC, Paton JFR, Rybak IA. Late-expiratory activity: Emergence and interactions with the respiratory CPG. J Neurophysiol 2010;104:2713–29. [4] Baekey DM, Molkov YI, Paton JFR, Rybak IA, Dick TE. Effect of baroreceptor stimulation on the respiratory pattern: Insights into respiratory-sympathetic interactions. Respir Physiol Neurobiol 2010;174:135–45. [5] Abdala APL, Rybak IA, Smith JC, Paton JFR. Abdominal expiratory activity in the rat brainstem-spinal cord in situ: Patterns, origins and implications for respiratory rhythm generation. J Physiol 2009;587:3539–59.


SyMPATHETIC NERvE ACTIvITy FollowINg CHRoNIC INTERMITTENT HyPoxIA-INduCEd SENSITIzATIoN oF CENTRAl CHEMoRECEPToRS

[6] Baekey DM, Dick TE, Paton JFR. Pontomedullary transection attenuates central respiratory modulation of sympathetic discharge, heart rate and the baroreceptor reflex in the in situ rat preparation. Exp Physiol 2008;93:803–16. [7] Zoccal DB, Simms AE, Bonagamba LG, Braga VA, Pickering AE, Paton JFR, Machado BH. Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity. J Physiol 2008;586:3253–65. [8] Zoccal DB, Paton JFR, Machado BH. Do changes in the coupling between respiratory and sympathetic activities contribute

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to neurogenic hypertension?. Clin Exp Pharmacol Physiol 2009;36:1188–96. [9] Janczewski WA, Onimaru H, Homma I, Feldman JL. Opioidresistant respiratory pathway from the preinspiratory neurones to abdominal muscles: In vivo and in vitro study in the newborn rat. J Physiol 2002;545:1017–26. [10] Guyenet PG, Stornetta RL, Bayliss DA. Retrotrapezoid nucleus and central chemoreception. J Physiol 2008;586:2043–8.


C H A P T E R

144 Optogenetics Sergey Kasparov Optogenetic Reporters for Ca2

INTRODUCTION Central regulation of autonomic outflows is a highly complex process coordinated by a network of neurons distributed throughout the whole extent of the brainstem. The spectrum of neuronal phenotypes involved in various autonomic responses is remarkably diverse and includes glutamatergic, GABA-ergic, acetylcholinergic, peptidergic, catecholaminergic, serotonergic and others. In many cases it is essential to be able to selectively study and manipulate these diverse groups of cells in order to reveal their respective contributions to the physiological process in question. Optogenetics provides a unique opportunity for this type of enquiries. The term “optogenetics” means experimentation using a combination of genetic manipulation and optics. Genetic engineering enables targeted expression of reporters and effectors (or actuators) in mammalian cells. Optogenetics can be used for studies of the autonomic areas of the brain and provides tools for specific control of phenotypically identified groups of neurons and more recently, astrocytes.

OPTOGENETIC REPORTERS Optical monitoring of various processes in cells of interest is advantageous compared to electrical recordings because it is non-invasive, allows much better spatial resolution, not limited to electrogenic processes and typically provides information from more than one cell, thus greatly increasing the throughput. Optogenetic reporters can be easily used in vitro in brain slices. Their in vivo application for studies of the autonomic centers of the brain has been hampered by the poor optical access to these areas which are all located in deep brain structures. Nevertheless, in vivo application of this approach is possible [1]. Multiphoton excitation is of little help here because its depth of penetration (typically 100–150 μm) is only sufficient for the upper layers of cortex. In the near future fluorescent imaging of the autonomic areas will be facilitated by the introduction of new fiber-based microscopes which use needle-like “objectives” and reveal fluorescence in any brain structure irrespective of its depth. Optogenetic reporters can also be effectively used in slices and slice cultures which contain autonomic nuclei [2,3].


Genetically encoded calcium indicators (GECI) consist of one or two fluorescent protein(s) (FP) and a Ca2sensitive domain. GECI respond to Ca2 by altering their fluorescence intensity or by a wavelength shift. The most common single GFP-based biosensor platform relies on circularly permuted GFP. The N and C-termini of these GECI contain “sticky ends” – the Ca2 binding motif from calmodulin and its target binding protein, M13 (derived from myosin light chain kinase) which are attracted in the presence of Ca2. The most recently published GECI of that family is GCaMP3, which has an improved dynamic range, good maturation in mammalian neurons and is suitable for in vivo Ca2 imaging [4]. Another cpGFP indicator – Case 12 [5] has an exceptionally high contrast ratio (12 times) and proven very effective for imaging of [Ca2]i in astrocytes in vitro [3] and in vivo [1]. Förster resonance energy transfer (FRET) is a different principle applied to design of GECI. In this design two fluorescent proteins are used, typically cyan and yellow and Ca2 binding results in a spectral shift [6,7]. The recent constructs from this subfamily were reported to detect Ca2 transients triggered by single action potentials [8]. In combination with appropriate genetic targeting these and other newer probes will allow enquiries into the Ca2 signaling in identified populations of cells located in the autonomic centers of the brain. Optical monitoring using genetic probes is by no means limited to studies of [Ca2]i concentration. Optogenetic sensors for cAMP, Cl and other important indicators of cellular activity have been developed. Probes for membrane potential are of particular interest although their dynamic range is still limited [9].

Optogenetic Effector Proteins Controlling activity of neurons and astrocytes using optogenetic effectors is a new and very exciting technology which circumvents the limitations of all the previous methods used for the same purpose. In contrast to electrical current, light will only affect cells which specifically express optogenetic effectors and this will have no effect on other cells even if they are located nearby or on the fibers en-passant. Many optogenetic effectors operate with millisecond precision and therefore permit a high degree

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144. OPTOgEnETICs

of temporal control over the activity of the neurons under study. Light-Sensitive Cation Channel Channelrhodopsin-2 and its Derivatives Channelrhodopsins (ChRs) are the only currently known family of ion channels that are directly gated by light. These proteins have seven transmembrane spanning domains and have been cloned from algae which use them for detection of light. ChRs are non-selective cation channels permeable to Na, K and Ca2 and when opened upon illumination depolarize the membrane [10]. ChRs have been extensively used in neuroscience as tools to depolarize neuronal membranes but they can be used to control other excitable cells such as cardiac myocytes or skeletal muscles. Presently used variants of ChR2 differ in their kinetics, dynamics of inactivation and recovery, channel conductance, spectral characteristics and membrane trafficking [11]. In almost all studies they are used as fusions with a fluorescent protein, such as yellow or red fluorescent protein to assist visualization [12]. Fusions with red fluorescent proteins are advantageous because they are visualized using green or yellow light to which ChRs are only minimally sensitive thus avoiding an unsolicited activation of ChRs. Because of the relatively modest conductance of ChRs, their successful application requires high level of expression in target cells which needs to be taken into account and controlled for its non-specific effects. Nevertheless, it proves a means to optogenetically control the excitability of various populations of neurons in vitro and in vivo [13,14]. Optogenetic excitation of the retrotrapezoid nucleus has recently been used to address its role in respiratory control [15]. Expression of ChR2 in astrocytes allows one to induce increases in [Ca2]i in these cells, the effect which has recently been used to demonstrate their involvement in central chemosensitivity [1]. All currently used variants of ChR are most sensitive to blue light (450–480 nm) which can be delivered from pulsing sources of light to deep structures via “optrodes” (Fig. 144.1). High levels of light (more than a few mW) and especially, continuous (not pulsing) light can evoke artifacts in vitro and in vivo and it is therefore essential to carefully control the power [14]. Optogenetic “Silencers” Two families of genes are currently being used for “silencing” of neurons with light on demand. The first one is the microbial halorhodopsin NpHR, a fast lightactivated electrogenic Cl pump and its mutants and derivatives [16]. The second is the light-activated proton pump “Arch” from Halorubrum sodomense [17]. Both genes are able to significantly hyperpolarize neurons and within milliseconds curtail their action potential activity. Interestingly, both NpHR and Arch are sensitive to yellow/orange light (550–590 nm) which makes it possible to combine them with ChR in the same experiment.

FIGURE 144.1 Optogenetic stimulation of a brainstem autonomic center combined with chronic direct measurement of arterial blood pressure in a rat. Viral vectors were used to introduce ChR into one of the autonomic nuclei of the brainstem. The animal was then implanted with a light guide (optrode) which is mounted into the skull and connected to the black light guide. Light guide is connected to a solid state pulsing laser controlled by a computer (not shown). Additionally, the rat was implanted with an arterial catheter (exteriorized on its back) to monitor blood pressure directly. The animal is kept in a small box to limit its activity and the light guide and catheter are kept out of its sight to prevent damage. This approach can be used to study the roles of diverse neuronal populations in acute and chronic control of blood pressure. Photo supplied by Dr. A.P.L. Abdala.

Light-Sensitive G-protein Coupled Receptors (GPCRs) Many essential processes in the autonomic centers of the brain are mediated via receptors coupled via G-proteins. Moreover, all known receptors for noradrenaline and the “M” type receptors for acetylcholine are GPCRs. These receptors belong to rhodopsin superfamily and have the same 7-transmembrane topology. This closeness has allowed generation of chimeras where the extracellular and membrane-spanning part of the receptor is taken from rhodopsin and the intracellular part from one of the “classic” G-protein coupled receptors, such as α1 or β2 adrenergic receptors [18]. These interesting optogenetic tools enable control of intracellular signaling by mimicking G-protein-mediated events. Using “optoGPCR” it is now possible to “switch on” with light all three main pathways employed by these ubiquitous regulators of cellular metabolism, including the signaling cascades affected by GαS, GαQ and GαI subunits [19]. It is difficult to overestimate the potential use of this technology for studies into the roles of specified populations of neurons and astrocytes in the autonomic nuclei of the brain.

MEANS OF GENE DELIVERY Successful gene delivery is the key to application of optogenetics. There are two basic technologies which can


COnClusIOn

be used for that purpose: viral gene delivery and germline transgenesis (almost exclusively mice at present). Both approaches can also be combined.

Cell-Specific Viral Targeting Viral vectors are extremely powerful and versatile scientific tools. Their advantages are speed and flexibility, fairly low cost, applicability to any strain of rodents, ease of sharing between investigators. Targeting specified cell types with viral vectors always relies on isolation of a relatively short (typically 1–3 kilobases) DNA sequence which may be used as a cell-specific promoter. In some cases this approach has been highly successful, while for some cell types no such short promoters are known. The most commonly used types of vectors at present are lentiviral vectors (usually derived from HIV), adeno-associated virus vectors and adenoviral vectors [2].

Germline Transgenics Review of transgenic technology is outside of the scope of this chapter. Briefly, transgenic animals may also have cassettes with short promoters driving optogenetic transgenes incorporated in their genomes. However, for the most specific targeting bacterial artificial chromosomes (BAC) are used, because this allows incorporation of the whole transcriptional unit with all the elements potentially important for the correct expression of the transgene. BAC can either directly control expression of any of the optogenetic tools mentioned above or express Cre recombinase to allow activation of the target genes only in specified cells. This last approach is favored at present because it is highly flexible and usually gives higher level of gene expression as well as site-specificity.

CONCLUSION Almost all areas of the brain implicated in autonomic control contain heterogeneous populations of neurons with diverse and in some cases opposing functions, connectivity and activity patterns. In addition, astroglia have recently been recognized as another potentially important component of central information processing machinery. Optogenetics offers new opportunities for selective studies of genetically targetable autonomic cells in vitro and in vivo. The range of available optogenetic tools is rapidly expanding.

Acknowledgements

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References [1] Gourine AV, Kasymov V, Marina N, Tang F, Figueiredo MF, Lane S, et al. Astrocytes control breathing through pH-dependent release of ATP. Science 2010;329:571–5. [2] Teschemacher AG, Paton JFR, Kasparov S. Imaging living central neurones using viral gene transfer. Adv Drug Deliv Rev 2005;57:79–93. [3] Guo F, Liu B, Tang F, Lane S, Souslova EA, Chudakov DM, et al. Astroglia are a possible cellular substrate of angiotensin(1-7) effects in the rostral ventrolateral medulla. Cardiovasc Res 2010;87:578–84. [4] Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 2009;6:875–81. [5] Souslova EA, Belousov VV, Lock JG, Strömblad S, Kasparov S, Bolshakov AP, et al. Single fluorescent protein-based Ca2  sensors with increased dynamic range. BMC Biotechnol 2007;7:37. [6] Lutcke H, Murayama M, Hahn T, Margolis DJ, Astori S, Zum Alten Borgloh SM, et al. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice. Front Neural Circuits 2010;4:9. [7] Mank M, Santos AF, Direnberger S, Mrsic-Flogel TD, Hofer SB, Stein V, et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat Methods 2008;5:805–11. [8] Horikawa K, Yamada Y, Matsuda T, Kobayashi K, Hashimoto M, Matsu-Ura T, et al. Spontaneous network activity visualized by ultrasensitive Ca(2) indicators, yellow Cameleon-Nano. Nat Methods 2010;7:729–32. [9] Mutoh H, Perron A, Akemann W, Iwamoto Y, Knopfel T. Optogenetic monitoring of membrane potentials. Exp Physiol 2010;96:13–8. [10] Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci U S A 2003;100:13940–13945. [11] Lin JY. A user’s guide to channelrhodopsin variants: features, limitations and future developments. Exp Physiol 2010;96:19–25. [12] Figueiredo M, Lane S, Tang F, Liu B, Hewinson J, Marina NG, et al. Optogenetic experimentation on astrocytes. Exp Physiol 2010;96:40–50. [13] Gradinaru V, Zhang F, Ramakrishnan C, Mattis J, Prakash R, Diester I, et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 2010;141:154–65. [14] Zhang F, Gradinaru V, Adamantidis AR, Durand R, Airan RD, de LL, et al. Optogenetic interrogation of neural circuits: Technology for probing mammalian brain structures. Nat Protoc 2010;5:439–56. [15] Abbott SB, Stornetta RL, Fortuna MG, Depuy SD, West GH, Harris TE, et al. Photostimulation of retrotrapezoid nucleus phox2bexpressing neurons in vivo produces long-lasting activation of breathing in rats. J Neurosci 2009;29:5806–19. [16] Gradinaru V, Thompson KR, Deisseroth K. eNpHR: a Natronomonas halorhodopsin enhanced for optogenetic applications. Brain Cell Biol 2008;36:129–39. [17] Chow BY, Han X, Dobry AS, Qian X, Chuong AS, Li M, et al. Highperformance genetically targetable optical neural silencing by lightdriven proton pumps. Nature 2010;463:98–102. [18] Airan RD, Thompson KR, Fenno LE, Bernstein H, Deisseroth K. Temporally precise in vivo control of intracellular signalling. Nature 2009;458:1025–9. [19] Masseck OA, Rubelowski JM, Spoida K, Herlitze S. Light- and drug-activated G-protein coupled receptors to control intracellular signaling. Exp Physiol 2010;96:51–96.

Financial support from the British Heart Foundation and Royal Society is gratefully acknowledged.


Index

A AA, see Arachidonic acid AAADC, see Aromatic amino acid decarboxylase AAG, see Autoimmune autonomic gangliopathy Abdominal pain, chronic, 598–599 Acarbose, postprandial hypotension management, 639–641 Acetylcholine (ACh) airway control, 202 autonomic neuron development role, 7 axon reflex, 409 cotransmission, 27, 29–32 metabolism, 71, 75, 631 synthesis, 71 transporters, 71 Acetylcholine receptor (AChR) autoimmunity, see Autoimmune autonomic gangliopathy; Paraneoplastic autoimmune dysfunction muscarinic receptors agonists, 76–77 antagonists, 77 overview, 71–72, 76 nicotinic receptors electrophysiology of activation, 80–81 overview, 72 structure, 79–82 subtypes, 79–80 optogenetics, 688 pharmacology, 72–73 tissue distribution, 75–76, 81 Acetylcholinesterase (AChE) functional overview, 631 inhibitors, see also Pyridostigmine carbamates, 632 organophosphates, 633 phenanthrene, 632 piperidines, 632 quaternary amines, 631–632 therapeutic applications, 633 ACh, see Acetylcholine AChE, see Acetylcholinesterase AChR, see Acetylcholine receptor ACTH, see Adrenocorticotrophic hormone Acupuncture clinical role, 656 neurological substrate, 653–655 principles, 653 prospects for study, 657 Western understanding, 653 AD, see Alzheimer's disease Adaptive immunity autonomic nervous system effects on cells, 326–328 overview, 325–326

Adenosine cardiovascular effects in nucleus of the solitary tract, 142 cardiovascular autonomic regulation, 96–97 central autonomic regulation, 96 formation, 95 metabolism, 95 neuroexcitatory actions, 96 postsynaptic anti-adrenergic effects, 95 presynaptic effects, 95–96 Adenosine receptor central autonomic regulation, 96 pharmacology, 91 ADHD, see Attention deficit hyperactivity disorder Adrenal gland, see also Pheochromocytoma autonomic control of adrenocortical function, 573 crosstalk between cortex and medulla, 571 diabetic autonomic dysfunction effects, 479 insufficiency management, 572 primary, 571–572 secondary, 572–573 sympathoadrenal axis, 20–21 Adrenaline, see Epinephrine α1-Adrenergic receptor functions, 52–54 ligand binding and activation, 51–52 regulation, 54 signaling, 54 structure, 51, 53 subtypes, 51–52 α2-Adrenergic receptor gene polymorphisms, 56–57 ligands, 55 protein–protein interactions, 56–57 signaling, 55–56 subtypes, 55–56 trafficking, 55–56 β-Adrenergic receptor aging and cardiac receptor response, 272–273 desensitization, 60 gene polymorphisms, 61 metabolism regulation, 253–254 signaling, 59–60 structure, 60 subtypes, 59 therapeutic targeting beta-agonists, 60–61 beta-blockers, 60 tissue distribution, 59 Adrenocorticotrophic hormone (ACTH), adrenal insufficiency, 571–573 Adrenomedullin (AM) apoptosis inhibition, 129

693

inflammation attenuation, 128–129 oxidative stress attenuation, 128 receptor and signaling, 128 structure, 127 synthesis, 127 tissue distribution, 127 Aging cardiac β-adrenergic receptor response, 272–273 cardiac baroreflex function effects, 271–272 cerebral autoregulation effects, 273 integration of autonomic control networks, 272 neurotransmitter changes, 272 parasympathetic activity response, 272 sympathetic activity response, 272 Airway innervation bronchodilator nerves inhibitory nerves, 203 sympathetic nerves, 202 comparative biology of pulmonary circulation autonomic regulation, 670–671 nerve types afferent nerves, 201 cholinergic efferents, 202 cholinergic nerves, 202 overview, 201 neuropeptides, 203 neurotransmitters, 201 pathology, 203 Aldosterone hypertension and excess, 117–118 hypoaldosteronism, 573 kidney effects, 117 receptor, see Mineralocorticoid receptor salt-sensitivity of blood pressure role, 314, 316 tissue injury mediation, 119 Altitude illness acute hypoxia effects, 281–282 chronic hypoxia effects, 282 high altitude cerebral and pulmonary edema, 281–282 Alzheimer's disease (AD), α-synuclein in pathology, 303 AM, see Adrenomedullin Ambenonium, 631–632 γ-Aminobutyric acid (GABA) receptors GABAA signaling, 109–110 subtypes, 110 therapeutic targeting, 110–111 GABAB, 109 synthesis, 109 transporters, 109

694 α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor, functional overview, 104 AMPA receptor, see -Amino-3-hydroxyl5-methyl-4-isoxazole-propionate receptor Amygdala, autonomic function, 9 Amyloidosis hereditary amyloidosis autonomic function testing, 486 diagnosis, 486 gene mutations, 486 pathogenesis, 486 prognosis, 486–487 treatment, 486–487 immunoglobulin amyloidosis diagnosis, 484–485 pathogenesis, 483 peripheral neuropathy, 483 prognosis, 485 treatment, 485 overview, 483 reactive amyloidosis, 486 Anemia, autonomic failure association, 643 Anesthesia, autonomic dysfunction of patients airway management, 664–665 assessment, 663–664 diabetic neuropathy, 665 infection response, 665 lidocaine and hepatic blood flow, 665 pathophysiology, 663 preoperative management, 664 temperature dysregulation, 665 uremia, 666 ventilatory management, 665 Angiotensin, see Renin-angiotensin system Anhidrosis distal anhidrosis, 556 focal anhidrosis, 557 global anhidrosis, 556–557 hemianhidrosis, 557 overview, 554–556 segmental anhidrosis, 557 treatment of heat intolerance and heat stroke, 559 Anterior cingulate cortex, autonomic function, 9 Anxiety disorder, autonomic function, 292 AP2, autonomic nervous system development and specification role, 5–6 Apraclonidine, pupil response, 240 Aquaporin, vasopressin regulation, 122 Arachidonic acid (AA), metabolites in saltsensitivity of blood pressure, 317 Arecoline, pupil response, 240 Arginine vasopressin (AVP) aquaporin regulation, 122 disorders of body water homeostasis, 122–126 orthostatic stress response, 195 receptor types, 121 secretion regulation, 121 Aromatic amino acid decarboxylase (LAAAD) deficiency clinical presentation, 427 diagnosis, 429 treatment, 429 functional overview, 38, 427

Index

functions, 427 gene therapy, 46–47 metabolomics, 42 Arrector pilorum muscle, innervation, 414 β-Arrestin, dopamine receptor signaling, 69 Asthma, neural control of airways, 203–204 Atomoxetine efficacy, 629–630 mechanism of action, 628–629 ATP cotransmission, 27–32 metabolism, 88 overview of neurotransmission, 87–89 receptors, see Purinergic receptors ATP7A, Menkes disease mutations, 435 Atrial fibrillation, obstructive sleep apnea association, 567–568 Atrioventricular (AV) node, 178–179, 181 Atropine, pupil response, 239 Attention deficit hyperactivity disorder (ADHD), norepinephrine transporter polymorphisms, 442 Autoimmune autonomic gangliopathy (AAG) clinical features, 489 course, 490–491 diagnosis, 490 pathogenesis, 489–490 Autonomic neuron, see Neuron, autonomic AV node, see Atrioventricular node AVP, see Arginine vasopressin Axon reflex, testing, 409–411

B Baroreceptor reflex, see also Bionic baroreflex system aging and cardiac baroreflex function, 271–272 arterial baroreflex, 161–162 baroreceptor activity determinants large artery compliance, 163 neuronal mechanisms mediating sensory transduction, 163 rate sensitivity, 161, 163 baroreflex sensitivity blood pressure control, 164 cardiovascular risk with decrease, 164 genetic determinants, 164, 167–169 heart rate control, 163–164 therapeutic targeting, 164–165 cardiopulmonary baroreflex, 161–162 failure autonomic failure comparison, 349–350 causes, 349 clinical presentation, 349–351 diagnosis, 351–352 treatment, 352 hypertension and baroreflex adaptation/ resetting, 163 modeling of baroreceptor activation effects on respiratory pattern, 679, 681–682 pregnancy and impairment, 265–267 stimulator therapy for hypertension, 650 BAT, see Brown adipose tissue BBS, see Bionic baroreflex system Benign joint hypermobility syndrome, see Joint hypermobility Beta-blockers, see β-Adrenergic receptor BH4, see Tetrahydrobiopterin

Bile, autonomic control of secretion, 205 Bionic baroreflex system (BBS) artificial vasomotor center algorithm, 660 efficacy, 660–661 epidural catheter approach, 661–662 implantable device, 662 rationale, 659 spinal cord injury application, 662 theory, 659–660 Biopsy, see Skin biopsy BK potassium channel, polymorphisms and baroreflex function, 168 Bladder, see Lower urinary tract Bladder pain syndrome (BPS), stress effects, 25 Blood pressure (BP), see also Baroreceptor reflex; Hypertension; Salt-sensitivity of blood pressure aging effects, 272–273 autonomic dysfunction evaluation, 377, 380 emotion studies, 297–298 head-up tilt table testing monitoring, 383 high frequency oscillation, 151–154 power spectrum analysis, 405–407 variability patterns baroreflex disorders, 357 body temperature, 356 diurnal rhythms, 355–356 emotional states, 356 exercise, 356–357 food intake and postprandial hypotension, 356 intraoperative variation, 357 medication effects, 357 postural adaptation, 356 respiratory variation, 355 salt sensitivity, 357 smoking effects, 357 white coat hypertension, 356 sympathetic nervous system outflow control, 355 Blood vessel comparative biology of innervation, 669–670 innervation, 414 BMPs, see Bone morphogenetic proteins BOLD signal, see Functional magnetic resonance imaging Bone anatomy, 257 muscle sympathetic nerve activity findings in loss, 396–397 ontogeny, 257 pathology, 258–269 remodeling effects, 257–258 sympathetic innervation Bone morphogenetic proteins (BMPs), autonomic neuron development role, 4 Bosentan, hypertension trials, 137 Botulinum toxin overactive bladder syndrome management, 228 pupil response, 239 BP, see Blood pressure BPS, see Bladder pain syndrome BQ-788, blood pressure response, 137 Bradykinin receptor, polymorphisms and baroreflex function, 168

Index

Brain autonomic output parasympathetic outputs, 11–12 sympathetic preganglionic units, 11 brainstem components in autonomic control caudal raphe nuclei, 11 caudal ventrolateral medulla, 11 functional magnetic resonance imaging, 13–16 nucleus of the solitary tract, 11 parabrachial complex, 11 periaqueductal gray, 10–11 rostral ventrolateral medulla, 11 circulation, see Cerebral blood flow computational modeling of brainstem sympatho-respiratory network, 679–680 forebrain components in autonomic control amygdala, 9 anterior cingulate cortex, 9 hypothalamus, 9–10 insular cortex, 9 leptin receptors, 132–133 Bromocriptine, neuroleptic malignant syndrome management, 543 Brown adipose tissue (BAT), thermoregulation, 243 BuChE, see Butylcholinesterase Butylcholinesterase (BuChE), functional overview, 631

C Calcitonin gene-related peptide (CGRP) airway control, 203 apoptosis inhibition, 129 cotransmission, 29 inflammation attenuation, 128–129 migraine role, 545 oxidative stress attenuation, 128 receptor and signaling, 128 release, 127–128 structure, 127 synthesis, 127 tissue distribution, 127 vasomotor control, 187, 190 Calcium, fluorescent genetic reporters, 687 CAN, see Cardiac autonomic neuropathy; Central autonomic nucleus Capacitance vessels, orthostatic reflex adjustment, 194–195 Carbachol, pupil response, 240 Carbon monoxide (CO), carotid body hypoxic sensing, 332 Carcinoid tumor carcinoid syndrome features, 589–590 epidemiology, 589 serotonin metabolism, 590 sites, 589 treatment, 590–591 Cardiac autonomic neuropathy (CAN), diabetic autonomic dysfunction, 476, 479–480 Cardiac norepinephrine spillover (CNES), heart failure studies, 367–369 Cardiac vagal ganglia abundance, 182 atrial ganglionated plexuses, 182 autonomic nervous system integration, 183–185

ventral ganglionated plexuses, 182–183 Cardiovascular-respiratory coupling, see Respiratory-cardiovascular coupling Carotid body gas messengers in hypoxic sensing, 332 hypoxic stimulus transduction, 331–332 molecular determinants of oxygen sensing, 332–333 morphology, 331 pathology, 333 unique aspects of oxygen sensing, 331 Carotid sinus hypersensitivity, 357 Carpal tunnel syndrome (CTS), amyloidosis, 483 Catechol-O-methyltransferase (COMT), functional overview, 41–42 Caudal pressor area (CPA), functional magnetic resonance imaging, 16 Caudal raphe nuclei, autonomic function, 11 Caudal ventrolateral medulla (CVLM) autonomic function, 11, 13 functional magnetic resonance imaging, 15–16 CBF, see Cerebral blood flow CCHS, see Congenital central hypoventilation syndrome CCK, see Cholecystokinin Central autonomic nucleus (CAN), sympathetic nervous system, 17 Central sleep apnea, see Sleep apnea Cerebral blood flow (CBF) aging effects, 273 autonomic innervation, 199 autoregulation, 197–199 imaging, 197 neurovascular coupling, 197 CFS, see Chronic fatigue syndrome CGRP, see Calcitonin gene-related peptide Channelrhodopsins, optogenetics, 688 Charcot–Marie–Tooth disease (CMT), autonomic dysfunction, 498 ChAT, see Choline acetyltransferase Cholecystokinin (CCK) bile secretion regulation, 205 pancreatic secretion regulation, 205 Choline acetyltransferase (ChAT), functional overview, 71 Chronic fatigue syndrome (CFS) autonomic dysfunction overview, 531 pathophysiology, 533 clinical presentation, 531 definition, 531 diagnosis, 531 epidemiology, 531 neurally mediated hypotension, 533 orthostatic intolerance, 531–532 postural tachycardia syndrome, 533 Chronic inflammatory demyelinating polyradiculopathy (CIDP), sympathetic microneurography, 393 Chronic kidney disease, muscle sympathetic nerve activity findings, 395–396 Chronic obstructive pulmonary disease (COPD), neural control of airways, 204 CIDP, see Chronic inflammatory demyelinating polyradiculopathy

695 Circadian rhythm cardiac events, 159 control of autonomic nervous system, 158–159 suprachiasmatic nuclei output and autonomic control, 157–158 CISS1, see Cold-induced sweating type 1 Clonidine adverse effects, 624 baroreflex failure management, 352 dosing, 624 mechanism of action, 624 CMT, see Charcot–Marie–Tooth disease CNES, see Cardiac norepinephrine spillover CO, see Carbon monoxide Cocaine autonomic effects on heart, 577–578 overdose management, 580 peripheral circulation effects, 577 pupil response, 240 thermoregulation effects, 579 Cold-induced sweating type 1 (CISS1), skin biopsy, 415 Colon, see Intestine Comparative biology, autonomic nervous system in vertebrates cardiovascular response to pressure alterations, exercise, and hypoxia, 670 cardiovascular system anatomy, 668–669 heart autonomic regulation, 669 overview, 667–668 pulmonary circulation autonomic regulation, 670–671 vasculature innervation, 669–670 Complex regional pain syndrome (CRPS) diagnosis, 381 edema, 584–585 inflammation, 585 initiating events, 586 skin sympathetic system cutaneous vasoconstrictor neurons and blood flow through skin, 583 sudomotor neurons and sweating, 584 sympathetically maintained pain, 586 trophic changes, 585 type I central changes, 584 peripheral changes, 584–585 somatomotor changes, 585–586 type II, 583 COMT, see Catechol-O-methyltransferase Congenital central hypoventilation syndrome (CCHS) gene mutation, see PHOX2B genotype–phenotype correlations cardiac asystole, 446 continuous ventilatory dependence, 446 facial dysmorphology, 447 frameshift mutations, 447 Hirschsprung disease, 446 later-onset disease, 447–448 tumors of neural crest origin, 446 management, 448–449 COPD, see Chronic obstructive pulmonary disease Coronary artery spasm, stress cardiomyopathy, 371–373

696

Index

Cotransmission cardiac neurons, 30 central nervous system, 27 enteric nervous system, 29–30 history of study, 27 parasympathetic nerves, 28–29 peripheral nervous system, 27 physiological significance different firing patterns, 30 excitation and inhibition, 32 false cotransmitters, 32 negative cross-talk, 32 neuromodulation, 30–31 neuropeptides, 32 postjunctional cell specificity, 30 synergism, 31 trophic factors, 32 plasticity, 32 sensory motor nerves, 29 sympathetic nerves, 27–28 CPA, see Caudal pressor area CRPS, see Complex regional pain syndrome CTS, see Carpal tunnel syndrome Cutaneous vasoconstriction (CVC), thermoregulation, 243–246 CVC, see Cutaneous vasoconstriction CVLM, see Caudal ventrolateral medulla CVS, see Cyclic vomiting syndrome Cyclic vomiting syndrome (CVS), 597–598 Cyclopentolate, pupil response, 239

D DA, see Dopamine Dantrolene, neuroleptic malignant syndrome management, 543 Darusentan, hypertension trials, 137 DBH, see Dopamine β-hydroxylase DCN, see Dorsal commissural nucleus Defecation, motor control, 209 Dementia with Lewy bodies (DLB) clinical features, 463 differential diagnosis, 463 management dementia, 465 dysautonomia, 465 hallucinations and psychosis, 465 overview, 463–464 Parkinsonism, 465 Detrusor overactivity (DO), incontinence, 231–232 DHPG, see Dihydroxyphenylglycol DI, see Diabetes insipidus Diabetes insipidus (DI), types and pathogenesis, 124 Diabetic autonomic dysfunction adrenal function, 479 bladder dysfunction, 478–479 cardiac autonomic neuropathy, 476, 479–480 colon dysfunction, 478 diabetic neuropathy and anesthesia precautions, 665 erectile dysfunction, 479 gallstones, 478 gastric emptying, 476–477 pupil findings, 476 sudomotor function, 479 vaginal function, 479

Dihydroxyphenylglycol (DHPG), biomarker of sympathetic innervation and function, 42 DLB, see Dementia with Lewy bodies DMV, see Dorsal motor nucleus of the vagus DO, see Detrusor overactivity Donepezil, 632 DOPA decarboxylase, see Aromatic amino acid decarboxylase Dopamine (DA) neuron classification and function, 63–65 synthesis, 38, 63 Dopamine β-hydroxylase (DBH) deficiency clinical features, 431–432 diagnosis, 432–433 differential dialysis, 433 genetics, 433 knockout mouse model, 433 management, 433, 619 Menkes disease, 435–437 expression regulation, 6 functional overview, 38, 431 metabolomics, 42 orthostatic hypotension mutations, 6–7 Dopamine receptor autonomic nervous system, 67 central nervous system, 67 classification, 67 functional selectivity, 69–70 gene structure, 67–68 ligand specificity, 69 oligomerization, 69 pharmacology, 69 signaling, 67–70 structure, 68–69 Dorsal commissural nucleus (DCN), sympathetic nervous system, 17 Dorsal motor nucleus of the vagus (DMV), central output, 12 Droxidopa (L-DOPS) autonomic failure indications, 619 dopamine β-hydroxylase deficiency management, 433 dopamine β-hydroxylase deficiency treatment, 619 history of study, 617 mechanism of action, 619 Menkes disease management, 437 norepinephrine synthesis, 617–618 pharmacokinetics, 617–618 structure, 617 Drug-induced autonomic dysfunction hypertension, 511–513 hypotension, 512, 514

E EAS, see External anal sphincter ECT, see Electroconvulsive therapy ED, see Erectile dysfunction Edrophonium, 631–632 EEG, see Electrencephalogram EGG, see Electrogastrogram Electrencephalogram (EEG), head-up tilt table testing monitoring, 384 Electroconvulsive therapy (ECT), neuroleptic malignant syndrome management, 544

Electrogastrogram (EGG), emotion studies, 296–297 Emotion, see Mind–body interactions Encephalitis, paraneoplastic autonomic dysfunction, 593 Endothelial function assessment nitric oxide activity, 320 pharmacologic testing, 320 physiologic testing, 320–321 autonomic interactions in cardiovascular pathophysiology, 322–323 biomarkers activation and dysfunction, 321 repair and regeneration, 321 consequences of dysfunction, 322–323 endothelial cell dysfunction, 319–320 normal function, 319 improvement strategies, 322 Endothelin (ET) aging effects, 273 animal models of pathophysiology, 136 endothelin-1 function cardiac effects, 136 essential hypertension role and therapeutic targeting, 136–137 renal effects, 136 expression regulation, 135 gene polymorphisms, 137 isoforms, 135 knockout mouse phenotypes, 135 nervous system distribution and pathology, 137–138 processing, 136 receptor polymorphisms and baroreflex function, 168 signaling, 135 salt-sensitivity of blood pressure role, 316 ENS, see Enteric nervous system Enteric nervous system (ENS), cotransmission, 29–30 Ephedrine adverse effects, 624 dosing, 624 mechanism of action, 623–624 Epilepsy central autonomic network infratentorial components, 549–550 supratentorial components, 549 ictal autonomic dysfunction cardiovascular, 550–551 cutaneous, 550 gastrointestinal, 550 pupil, 550 status epilepticus, 551 urogenital, 550 interictal autonomic dysfunction, 551–552 sudden unexpected death in epilepsy patients, 551 vagal nerve stimulation effects on cardiovascular function, 552 Epinephrine activation in stress, 42–43 panic disorder cotransmission in sympathetic nerves, 606 secretion, 605–607

697

Index

pupil response, 240 Erectile dysfunction (ED) associated conditions, 561 diabetic autonomic dysfunction, 479 epidemiology, 561 erection mechanism, 561–562 etiology, 562 hypogonadism, 562 neurogenic erectile dysfunction, 562 spinal cord injury, 508 treatment, 563 EROS-Clitoral Therapy Device, 238 Erythropoietin anemia in autonomic failure, 643 orthostatic hypotension management, 643–644 production modulation by autonomic nervous system, 643 ET, see Endothelin Evolution, see Comparative biology, autonomic nervous system in vertebrates Exercise benefits, 275, 277–278 blood pressure effects, 356–357 cardiovascular response acute exercise, 275–276 exercise training, 276–277 External anal sphincter (EAS), fecal continence, 601 External pressure, autonomic dysfunction management, 610

F

innervation, 236–237 sexual arousal afferent and sexual pathways, 235–236 neurotransmitters, 236 spinal cord injury, 508 FFT, see Fast Fourier transform FGIDs, see Functional gastrointestinal disorders Flow-mediated dilatation (FMD), endothelial function assessment, 320–321 Fludrocortisone history of study, 635 long-term effects, 636 mechanism of action, 635 neurally mediated syncope management, 342 pharmacology, 635–636 side effects, 636 structure, 636 Fluorescent probes, see Optogenetics FMD, see Flow-mediated dilatation fMRI, see Functional magnetic resonance imaging Functional gastrointestinal disorders (FGIDs) chronic abdominal pain, 598–599 classification, 597–598 cyclic vomiting syndrome, 597–598 overview, 597 treatment, 599 Functional magnetic resonance imaging (fMRI) brainstem sites in cardiovascular control, 13–16 neurovascular coupling, 197

Fabry's disease autonomic dysfunction, 496 clinical manifestations, 495–496 enzyme replacement therapy, 496 gene mutations, 495 Familial dysautonomia (FD) cardiovascular autonomic abnormalities, 500–501 clinical features, 499–500 genetics, 499–500 history of study, 499 pathology, 501 skin biopsy, 416–417 treatment, 502 Fast Fourier transform (FFT), power spectrum analysis, 405–407 FD, see Familial dysautonomia Fecal incontinence animal models, 602 physiology external anal sphincter, 601 internal anal sphincter, 601 puborectalis, 601 recto-anal reflexes, 601–602 prevalence, 601 sacral neuromodulation, 602 Female sexual function diabetic autonomic dysfunction and vaginal function, 479 dysfunction assessment, 237–238 classification, 235 etiologies, 237 management, 238 hormonal influences, 237

GABA, see γ-Aminobutyric acid Galantamine, 632 Gallstones, diabetic autonomic dysfunction, 478 Gastric emptying, see Stomach Gastric secretion, see Stomach GATA-3, autonomic nervous system development and specification role, 5 GBS, see Guillain–Barré syndrome GCHI, see GTP cyclohydrolase I GDNF, see Glial-derived neurotrophic factor Glial-derived neurotrophic factor (GDNF), autonomic neuron development role, 4 Glutamate autonomic function, 105–106 clearance, 105 excitotoxicity, 103 functional overview, 103 metabolism, 105 receptors, see specific receptors synthesis, 103 transporters, 103–104 GTP cyclohydrolase I (GCHI), gene therapy, 46–47 Guillain–Barré syndrome (GBS) clinical features, 493 course, 494 etiology, 493 investigations, 493 management, 494 prognosis, 494 sympathetic microneurography, 393

G

H Hair follicle, innervation, 413 Head-up tilt table testing (HUT) clinical applications, 384 indications, 384 monitoring, 383–385 neurally mediated syncope, 342, 384 orthostatic hypotension, 386–387 overview, 383 physiological basis, 383–384 postural tachycardia syndrome, 384, 386 types, 383 Heart, see also Cardiac vagal ganglia anatomy, 177–178 autonomic nervous control alterations, 179–180 overview, 178–179 beat control, 179 cocaine effects, 577–578 comparative biology of autonomic regulation, 669 conduction system, 178–179, 181 sympathetic imaging in Lewy body disease, 402 Heart failure central sleep apnea association, 569 individual variability, 367 muscle sympathetic nerve activity findings, 395 sympathetic activity, 367–370 Heart rate (HR) aging effects, 272 autonomic nervous system nitric oxide control, 101 baroreflex sensitivity control, 163–164 emotion studies, 297–298 power spectrum analysis, 405–407 variability in sleep apnea, 568–569 Heat stroke, management, 559 Hereditary sensory and autonomic neuropathy (HSAN) type III, see Familial dysautonomia types, 498 HfO, see High frequency oscillation HIF, see Hypoxia-inducible factor High altitude, see Altitude illness High frequency oscillation (HFO), blood pressure, 151–154 Hirschsprung disease, genotype–phenotype correlation, 446 Histamine, see Mastocytosis History taking autonomic disorder evaluation, 377 peripheral neuropathy with dysautonomia, 473 Hot flash, somatostatin analogs in management, 647 HR, see Heart rate HSAN, see Hereditary sensory and autonomic neuropathy HUT, see Head-up tilt table testing Hydrogen sulfide, carotid body hypoxic sensing, 332 Hydroxyamphetamine, pupil response, 240 11-β-Hydroxysteroid dehydrogenase type II, deficiency, 117 Hyperhidrosis causes, 553, 555

698

Index

Hyperhidrosis (continued) classification, 553–554 differential diagnosis, 555 treatment, 553–555, 649 Hypertension, see also Orthostatic hypertension; Preeclampsia aldosterone excess, 117–118 autonomic dysfunction complication by other disease, 346–347 evidence, 345 organ damage role, 345–346 therapeutic intervention, 347 baroreflex adaptation/resetting, 163 baroreflex stimulator therapy, 650 drug-induced, 511–513 drug-resistant hypertension, 650 endothelin-1 in essential hypertension and therapeutic targeting, 136–137 low-renin hypertension, 118–119 muscle sympathetic nerve activity, 395 obesity-associated hypertension and sympathetic activation, 360–361 renal dopamine role, 223 renal sympathetic nerve ablation for management, 650–652 sex differences, 263 spinal cord injury, 505 splanchic circulation, 212 sympathetic nervous system activation, 649–650 white coat hypertension, 356 Hyperthermia, thermoregulation, 288–289 Hypervolemia clinical manifestations, 123–124 etiology, 123 Hypoaldosteronism, 573 Hypohidrosis overview, 554–556 treatment of heat intolerance and heat stroke, 559 Hypotension, see also Neurally mediated hypotension; Orthostatic hypotension drug-induced, 512, 514 muscle sympathetic nerve activity testing, 393 postprandial hypotension, 356 Hypothalamus autonomic function, 9–10 thermoregulation, 245–245, 287 Hypothermia, thermoregulation, 288 Hypovolemia etiology, 123 postural orthostatic tachycardia syndrome, 523 Hypoxia, see also Altitude illness autonomic response acute hypoxia, 281–282 chronic hypoxia, 282 carotid body sensing, 331–332 comparative biology of response, 670 modeling sympathetic nerve activity following chronic intermittent hypoxia, 682–684 Hypoxia-inducible factor (HIF), carotid body hypoxic sensing, 332–333

I IAS, see Internal anal sphincter IBS, see Irritable bowel syndrome

IC, see Interstitial cystitis, Nucleus intercalatus spinalis ILF, see Intermediolateralis pars funicularis ILP, see Intermediolateralis pars principalis IML, see Intermediolateral cell column Immunoglobulin amyloidosis, see Amyloidosis Impedance threshold device (ITD), autonomic dysfunction management, 610 Incontinence, see Fecal incontinence; Lower urinary tract Inferior cervical ganglia, sympathetic nervous system, 17 Inflammation adrenomedullin attenuation, 128–129 autonomic nervous system effects on immune cells, 326–328 calcitonin gene-related peptide attenuation, 128–129 complex regional pain syndrome, 585 Innate immunity autonomic nervous system effects on cells, 326–328 overview, 325 Insular cortex, autonomic function, 9 Insulin, cardiovascular effects in nucleus of the solitary tract, 142 Insulin resistance (IR) complications, 307 sympathetic activity activation and resistance induction, 308–309 insulin resistance induction of activation, 307–308 overview, 307–308 therapeutic targeting, 309 Intermediolateral cell column (IML), sympathetic nervous system, 17 Intermediolateralis pars funicularis (ILF), sympathetic nervous system, 17 Intermediolateralis pars principalis (ILP), sympathetic nervous system, 17, 19, 21 Internal anal sphincter (IAS), fecal continence, 601 Interstitial cystitis (IC), stress effects, 25 Intestine autonomic control of secretion and absorption, 206 circulation, see Splanchic circulation diabetic autonomic dysfunction and colon function, 478 gut motility control, 206, 208 incontinence, see Fecal incontinence normal motor function, 208–209 spinal cord injury effects, 608 IR, see Insulin resistance Iris, see Pupil Irritable bowel syndrome (IBS), 599 ITD, see Impedance threshold device

J JH, see Joint hypermobility Joint hypermobility (JH) autonomic dysfunction, 535–537 clinical manifestations, 535 diagnosis, 535–536 epidemiology, 535

management, 537 pathophysiology, 535

K Kidney autonomic receptors, 215–217 blood volume and reflex regulation, 217–218 dopamine excretion, 221 hypertension role, 223 receptors expression, 221 sodium excretion regulation, 222 renin-angiotensin system interactions, 222–223–224 synthesis, 221 innervation, 215 pathophysiological states and autonomic control, 219–220 renal sympathetic nerve ablation for hypertension management, 650–652 renorenal reflex, 218–219

L LAAAD, see Aromatic amino acid decarboxylase Lambert–Eaton myasthenic syndrome (LEMS), 595 L-DOPS, see Droxidopa Leg crossing, autonomic dysfunction management, 609 LEMS, see Lambert–Eaton myasthenic syndrome Leptin functional overview, 131–132 interactions in hypothalamus melanocortin system, 133 neuropeptide Y, 133–134 receptor brain distribution, 132–133 isoforms, 131 signaling, 131–132 Lidocaine, see Anesthesia Liver circulation, see Splanchic circulation Lower urinary tract (LUT) central neural control, 227 clinical evaluation, 230 diabetic autonomic dysfunction and bladder dysfunction, 478–479 dysfunction bladder outlet obstruction, 232 incontinence, 230–232 neurology, 232 painful bladder syndrome, 232 treatment, 227–228 neuropathology, 227 neurotransmitter receptors, 227 normal sensory and motor properties, 229–230 parasympathetic pathways, 225–226 spinal cord injury effects, 508 structures, 226, 229 sympathetic pathways, 226–227 urethral sphincter somatic motor pathways, 227 Lumbodorsal splanchidectomy, 659 Lung, see Airway innervation LUT, see Lower urinary tract

Index

M Magnetic resonance imaging (MRI), see also Functional magnetic resonance imaging multiple system atrophy findings, 456 pure autonomic failure findings, 468 MAO, see Monoamine oxidase Mash1, autonomic nervous system development and specification role, 4–5 Mast cell activation disorder (MCAD), postural orthostatic tachycardia syndrome association, 522 Mastocytosis clinical features, 575–576 diagnosis, 576 mediators responsible for signs and symptoms, 575–576 MCAD, see Mast cell activation disorder MD, see Menkes disease Median preoptic nucleus (MnPO), thermoregulation, 243 Melanocortin receptor, types, 133 Menkes disease (MD) biochemical phenotype, 435–436 clinical features, 435 dysautonomia signs, 436–437 epidemiology, 435 molecular diagnosis, 437 neurochemical abnormalities, 437 Metabolic syndrome, muscle sympathetic nerve activity findings, 396 Metabolism regulation carbohydrate metabolism, 253 lipid metabolism, 253–254 protein metabolism, 254–255 resting metabolic rate and sympathetic stimulation contributions, 253 sypathoadrenal system dysregulation, 255 energy expenditure contribution, 253 Metabotropic glutamate receptor (mGluR), functional overview, 105 Methacholine, pupil response, 239–240 N-Methyl-D-aspartate (NMDA) receptor, functional overview, 104–105 mGluR, see Metabotropic glutamate receptor Microneurography, see Sympathetic microneurography Middle cervical ganglia, sympathetic nervous system, 17 Midrodine adverse effects, 622–623 dosing, 623 indications and efficacy, 621–622 mechanism of action, 621 pharmacology, 621 Migraine autonomic symptoms, 45–547 bio-behavioral model, 547 functional anatomy, 545 interictal autonomic dysfunction, 546 pain representation, 546–547 Mind–body interactions cardiovascular arousal studies of emotion, 297–298 electrogastrogram studies of emotion, 296–297 overview, 295

skin conductance response decision-making studies, 295 somatic marker hypothesis, 295 Mineralocorticoid receptor (MR), ligands, 117 MnPO, see Median preoptic nucleus Modeling, autonomic nervous system baroreceptor activation effects on respiratory pattern, 679, 681–682 brainstem sympatho-respiratory network, 679–680 sympathetic nerve activity following chronic intermittent hypoxia, 682–684 Virtual Physiological Human project/ Physiome project, 675–677 Monoamine oxidase (MAO) deficiency gene polymorphisms, 443–444 Norrie disease association, 443 functional overview, 41, 83 genes, 443 inhibitors and isoform specificity, 41 isoform function, 443 metabolomics, 42 Morvan's syndrome, 593–594 MR, see Mineralocorticoid receptor MRI, see Magnetic resonance imaging MSA, see Multiple system atrophy MSNA, see Muscle sympathetic nerve activity Multiple endocrine neoplasia type 2B, autonomic dysfunction, 497–498 Multiple myeloma, see Amyloidosis Multiple system atrophy (MSA) clinical features, 454 diagnosis, 454–456 differential diagnosis, 454–456 epidemiology, 453 history of study, 453 management, 456 muscle sympathetic nerve activity, 393 neuroprotective therapy, 456–457 pathophysiology, 303, 453–454 skin biopsy, 415 Muscarinic acetylcholine receptor, see Acetylcholine receptor Muscle sympathetic nerve activity (MSNA), see also Sympathetic microneurography applications bone loss, 396–397 chronic kidney disease, 395–396 heart failure, 367–369, 395 hypertension, 395 hypotensive attacks, 393 metabolic syndrome, 396 multiple system atrophy, 393 obstructive sleep apnea, 394–395 radiculoneuropathies, 393 insulin resistance studies, 307–310 power spectrum analysis, 406 sex differences, 261, 263

N NADPH oxidases, reactive oxygen species production, 336–337 ND, see Norrie disease NE, see Norepinephrine Nerve growth factor (NGF), autonomic neuron development role, 4

699 NET, see Norepinephrine transporter Neurally mediated hypotension, chronic fatigue syndrome, 533 Neurally mediated syncope (NMS) chronic fatigue syndrome, 533 diagnosis, 341–342 natural history, 342 pathophysiology, 341 syncope overview, 341 tilt table testing, 342, 384 treatment, 342–342 Neuroleptic malignant syndrome (NMS) clinical features, 541–542 differential diagnosis, 543 pathogenesis, 543 precipitants, 541–542 risk factors, 541, 543 treatment, 543–544 Neuromyotonia, paraneoplastic autonomic dysfunction, 593–594 Neuron, autonomic neural crest cell precursors, 3 neurotransmitters development acetylcholine, 7 noradrenaline, 6–7 phenotypes, 22–23 signaling in development, 3–4 transcription factors in autonomic nervous system development and specification AP2, 5–6 GATA-3, 5 Mash1, 4–5 Phox2 genes, 5 Neuropeptide Y (NPY) bone function, 258 cotransmission, 27–30, 32 leptin interactions in hypothalamus, 133–134 panic disorder and release, 607 vasomotor control, 187–190 Neurotrophin-3 (NT3), autonomic neuron development role, 4 NGF, see Nerve growth factor Nicotinic acetylcholine receptor, see Acetylcholine receptor Nitric oxide (NO) airway control, 203 autonomic nervous system nitric oxide heart rate control, 101 peripheral interactions, 100–101 cardiovascular effects in nucleus of the solitary tract, 141–142 central–autonomic nervous system interactions, 99–100 cotransmission, 27–31 endothelial function, 320–322 peroxynitrite formation, 335 salt-sensitivity of blood pressure role, 316–317 sympathetic function, 25 synthases, 99, 168 uncoupled nitric oxide synthase and reactive oxygen species production, 336 vasomotor control, 187, 190 NMDA receptor., see N-Methyl-D-aspartate receptor NMS, see Neurally mediated syncope; Neuroleptic malignant syndrome

700

Index

NO, see Nitric oxide Noradrenaline, see Norepinephrine Norepinephrine (NE) activation in stress, 42–43 autonomic function overview, 6 biomarkers of sympathetic innervation and function, 42 cardiovascular system neurons, 37–38 kidney function, 215–217 metabolism, 41–42 metabolomics, 42 panic disorder and reuptake, 606 release, 39 removal, 39–40 storage, 38–39 synthesis, 38, 617–618 transporter, see Norepinephrine transporter vasomotor control, 187–188 Norepinephrine transporter (NET) deficiency cardiovascular disease, 439 comorbidity of cardiovascular and neurobehavioral disorders, 442 gene polymorphisms, 439–442 orthostatic intolerance, 439–441 postural orthostatic tachycardia syndrome, 521–522 inhibition, see Atomoxetine regulation, 49–50 reuptake into nerve terminals, 39–40, 439 structure, 49 Norrie disease (ND), monoamine oxidase deficiency association, 443 NPY, see Neuropeptide Y NT3, see Neurotrophin-3 NTS, see Nucleus of the solitary tract Nucleus intercalatus spinalis (IC), sympathetic nervous system, 17 Nucleus of the solitary tract (NTS) anatomy, 141 autonomic function, 11, 13 cardiovascular effects adenosine, 142 angiotensin II, 142–143 insulin, 142 nitric oxide, 141–142 functional magnetic resonance imaging, 15–16

O Obesity, see also Hypertension; Insulin resistance epidemiology, 359 hypertension and sympathetic activation, 360–361 minority populations and sympathetic activation, 360 sympathetic activation, 359 Obstructive sleep apnea, see Sleep apnea Octreotide postprandial hypotension management, 640, 645–646 postural tachycardia syndrome management, 646–647 OI, see Orthostatic intolerance Optogenetics definition, 687 gene delivery, 688–689

reporters calcium, 687 effector proteins, 687–688 Organum vasculosum of the lateral terminalis (OVLT), osmoregulation, 121 Orthostatic hypertension associated conditions, 363–364 baroreflex failure, 350 overview, 363–364 Orthostatic hypotension aging, 271 definition, 529 delayed orthostatic hypotension, 529–530 dementia with Lewy bodies, 465 dopamine β-hydroxylase mutations, 6–7 erythropoietin management, 643–644 Guillain–Barré syndrome, 494 head-up tilt table testing, 386–387 midrodine efficacy, 621–622 Parkinson's disease, 460 pyridostigmine efficacy, 629 sex differences, 262–263 Orthostatic intolerance (OI), see also specific disorders chronic fatigue syndrome, 531–532 norepinephrine transporter defects, 439–441 space flight physiology, 284–285 Orthostatic stress arterial baroreceptor response, 193–194 capacitance vessels in orthostatic reflex adjustment, 194–195 humoral mechanisms, 195 local vasoconstrictor mechanisms, 194 skeletal muscle pump, 195 Orthostatic training, neurally mediated syncope management, 342–343 Osmoreceptors dysfunction, 124–125 Trpv4 role, 614 water drinking therapeutic utility, 614–615 water-induced pressor response evidence for sympathetic activation, 613–614 overview, 613 spinal sympathetic reflex, 614 Overactive bladder syndrome management, 227–228 overview, 231–232 OVLT, see Organum vasculosum of the lateral terminalis Oxidative stress adrenomedullin attenuation, 128 calcitonin gene-related peptide attenuation, 128 reactive oxygen species antioxidant defenses, 337 autonomic outflow, 337–338 biology, 335–336 sources mitochondrial respiration, 336 NADPH oxidases, 336–337 uncoupled nitric oxide synthase, 336 xanthine oxidase, 336 salt-sensitivity of blood pressure role, 316–317 Oxygen sensing carotid body gas messengers in hypoxic sensing, 332

hypoxic stimulus transduction, 331–332 molecular determinants of oxygen sensing, 332–333 morphology, 331 pathology, 333 unique aspects of oxygen sensing, 331 measures, 331 overview, 331

P PACAP, see Pituitary adenylate cyclaseactivating polypeptide PAD, see Paraneoplastic autonomic dysfunction PAF, see Pure autonomic failure PAG, see Periaqueductal gray Painful bladder syndrome, 232 Pancreas autonomic control of secretion, 205 circulation, see Splanchic circulation Panic disorder autonomic function, 292 cardiac risk, 607–608 neuropeptide Y release, 607 sympathetic nervous system function epinephrine cotransmission in sympathetic nerves, 606 secretion, 605–607 norepinephrine reuptake, 606 serotonin release, 605–606 Parabrachial complex (PBN) autonomic function, 11 thermoregulation, 243 Paraneoplastic autonomic dysfunction (PAD) classification, 593 clinical features, 593–594 diagnosis, 595–596 encephalitis, 593 enteric neuronopathy, 594 Lambert–Eaton myasthenic syndrome, 595 Morvan's syndrome, 593–594 neuromyotonia, 593–594 neuropathy, 491 treatment, 596 Parasympathetic nervous system (PNS) cotransmission, 28–29 functions, 24–25 neurotransmitter phenotypes, 22–23 overview, 18, 21–22 Parkinson's disease (PD) animal studies of autonomic function, 25 anticholinergic agent therapy, 461 denervation imaging, 402 diagnosis, 459 dopamine replacement therapy, 459–460 gene therapy targets, 46–47 L-DOPA therapy, 46 orthostatic hypotension, 460 skin biopsy, 415 α-synuclein in pathology, 303 PBN, see Parabrachial complex PD, see Parkinson's disease Periaqueductal gray (PAG), autonomic function, 10–11 Peripheral neuropathy, diagnostic workup with dysautonomia, 473–474 PET, see Positron emission tomography Phenylephrine, pupil response, 240

701

Index

Phenylethanolamine-N-methyltransferase (PNMT), functional overview, 38 Pheochromocytoma diagnosis, 421–424 differential diagnosis, 422 gene mutations and phenotypes, 421–422 malignant, 424 treatment, 424 Phox2 genes, autonomic nervous system development and specification role, 5 PHOX2B function, 445 genotype–phenotype correlations cardiac asystole, 446 continuous ventilatory dependence, 446 facial dysmorphology, 447 frameshift mutations, 447 Hirschsprung disease, 446 later-onset disease, 447–448 tumors of neural crest origin, 446 locus, 445 Physical activity, see Exercise Physical examination autonomic dysfunction, 379 peripheral neuropathy with dysautonomia, 473 Physiome project autonomic nervous system modeling, 675–677 overview, 673 physiome standards, 673–675 prospects, 677–678 Physostigmine, pupil response, 240 Pilocarpine, pupil response, 239–240 Pituitary adenylate cyclase-activating polypeptide (PACAP), migraine role, 545 PNMT, see Phenylethanolamine-Nmethyltransferase PNS, see Parasympathetic nervous system POA, see Preoptic area Porphyria autonomic dysfunction, 497 clinical manifestations, 497 gene mutations, 496–497 treatment, 497 Positron emission tomography (PET) dysautonomia sympathetic imaging, 401–402 Lewy body disease findings, 402 multiple system atrophy findings, 456 pure autonomic failure findings, 468 tracers for sympathetic imaging, 399–401 Post-traumatic stress disorder (PTSD), autonomic function, 292 Postprandial hypotension (PPH), 356, 639–641 Postural orthostatic tachycardia syndrome (POTS) chronic fatigue syndrome, 533 clinical features, 517 epidemiology, 517 exercise therapy, 278 follow-up, 518 head-up tilt table testing, 384, 386 joint hypermobility association, 536–537 management, 518 octreotide management, 646–647 pathophysiology hyperadrenergic POTS

central hyperadrenergic POTS, 521 mast cell activation disorder, 522 norepinephrine transporter deficiency, 521–522 hypovolemia and blood volume regulation, 523 neuropathic POTS, 522–523 stroke volume reduction, 523 phenotypes deconditioning-associated POTS, 518 hyperadrenergic POTS, 517 neuropathic POTS, 517 sex differences, 263, 517 symptoms, 378 tachycardia origins, 525–526 reflex tachycardia classification high flow POTS, 527 low flow POTS, 526–527 normal flow POTS, 528 water drinking therapy, 615 Posture, see Orthostatic hypertension; Orthostatic hypotension; Orthostatic stress POTS, see Postural orthostatic tachycardia syndrome PP2A, see Protein phosphatase 2A PPH, see Postprandial hypotension Preeclampsia, 267 Pregnancy baroreceptor reflex impairment, 265–267 preeclampsia, 267 sympathetic nervous system activation, 265 Preoptic area (POA), thermoregulation, 244– 246, 287 Protein phosphatase 2A (PP2A), norepinephrine transporter interactions, 49–50 Psychological stress, see Stress PTSD, see Post-traumatic stress disorder Puborectalis, fecal continence, 601 Pupil combined sympathetic and parasympathetic defects, 242 diabetic autonomic dysfunction findings, 476 epilepsy changes, 550 instilled drug response adrenergic blockers, 240 adrenergic drugs, 240 anticholinergic drugs, 239 cholinergic drugs, 239 iris pigment effects, 241 miscellaneous drugs, 240–241 parasympathetic defects, 242 sympathetic defects, 241–242 Pure autonomic failure (PAF) catecholamine studies, 467–468 clinical features, 467 differential diagnosis, 467 imaging studies, 468 management, 468 neuroendocrine studies, 468 neuropathology, 468 Purinergic receptors, see also Adenosine receptor classification, 89–91 P2X receptors, 90 P2Y receptors, 90, 92

Pyridostigmine efficacy, 629 mechanism of action, 629

Q QDIRT, see Quantitative direct and indirect test of pseudomotor function QSART, see Quantitative sudomotor axon reflex test QST, see Quantitative sensory testing Quantitative direct and indirect test of pseudomotor function (QDIRT), 410 Quantitative sensory testing (QST), female sexual dysfunction assessment, 237 Quantitative sudomotor axon reflex test (QSART), 410–411, 474, 519

R RAS, see Renin-angiotensin system Rasagiline, multiple system atrophy management, 456–457 Reactive amyloidosis, see Amyloidosis Reactive oxygen species, see Oxidative stress Recto-anal reflexes, fecal continence, 601–602 Renal norepinephrine spillover (RNES), heart failure studies, 367 Renin-angiotensin system (RAS) angiotensin II cardiovascular effects in nucleus of the solitary tract, 142–143 angiotensin receptors, 113–114, 168 angiotensin-converting enzyme, 113–116 brain system autonomic regulation, 115–116 components, 114–115 classical system autonomic regulation, 113–114 components and features, 113 low-renin hypertension, 118–119 orthostatic stress response, 195 renal dopaminergic system interactions, 222–223–224 salt-sensitivity of blood pressure, 314, 316 Renorenal reflex, see Kidney Respiratory-cardiovascular coupling cardiovascular modulation of respiratory activity, 154 high frequency oscillation cardiovascular disease response, 154 functions, 153 overview, 151 prospects for study, 154 respiratory pattern generator coupling to cardiovascular autonomic activity, 151–152 respiratory sinus arrhythmia cardiovascular disease response, 153–154 functions, 152–153 Respiratory sinus arrhythmia (RSA), respiratory-cardiovascular coupling, 151–154 Resting metabolic rate (RMR), sympathetic stimulation contributions, 253 Rifampicin, multiple system atrophy management, 457 Riley–Day syndrome, see Familial dysautonomia Rivastigmine, 632 RMR, see Resting metabolic rate

702

Index

RNES, see Renal norepinephrine spillover Rostral raphe pallidus area (rRPa), thermoregulation, 246 Rostral ventrolateral medulla (RVLM) adenosine receptors, 96 autonomic function, 11, 13 baroreceptor reflex impairment in pregnancy, 266–267 functional magnetic resonance imaging, 15 rRPa, see Rostral raphe pallidus area RSA, see Respiratory sinus arrhythmia RVLM, see Rostral ventrolateral medulla

S SA node, see Sinoatrial node SAH, see Subarachnoid hemorrhage Salivary gland, autonomic control of secretion, 205 Salt-sensitivity of blood pressure (SSBP) clinical significance, 317–318 defects arachidonic acid metabolites, 317 endothelin, 316 nitric oxide, 316 renin-angiotensin-aldosterone system, 314, 316 sympathetic nervous system neurotransmitters, 317 gene–environment interactions, 314 genetics, 313–316 SCG, see Superior cervical ganglia SCM, see Stress cardiomyopathy SCN, see Suprachiasmatic nuclei Scopolamine, pupil response, 239 SCR, see Skin conductance response Serotonin autonomic function, 85–86 false cotransmitter function, 28, 32 localization, 83 metabolism, 590 panic disorder and brain release, 605–606 sympathetic rhythms, 148 synthesis and metabolism, 83 transporters, 83–84 Serotonin receptor pharmacology, 85 signaling, 84–85 Sex differences, autonomic function hypertension, 263 normal autonomic function, 261 orthostatic hypotension, 262–263 overview, 261 Sexual function, see Female sexual function SIADH, see Syndrome of inappropriate antidiuretic hormone Sildenafil, erectile dysfunction management, 563 Single-photon emission computed tomography (SPECT) dysautonomia sympathetic imaging, 401–402 Lewy body disease findings, 402 multiple system atrophy findings, 456 tracers for sympathetic imaging, 399–401 Sinoatrial (SA) node, 178–179, 526 Skeleton, see Bone Skin biopsy applications, 414–416, 474 neuroanatomy arrector pilorum muscles, 414

blood vessels, 414 hair follicle, 413 sweat gland, 413 Skin conductance response (SCR) decision-making studies, 295 somatic marker hypothesis, 295 Skin sympathetic nerve activity (SSNA), see also Sympathetic microneurography applications radiculoneuropathies, 393 sweat disturbances, 394 cocaine effects, 580 Sleep apnea autonomic function during sleep, 565 central sleep apnea, 569 muscle sympathetic nerve activity, 394–395 obstructive sleep apnea acute changes during episodes, 565, 567 atrial fibrillation association, 567–568 chronic changes during episodes, 567 epidemiology, 565 heart rate variability, 568 sudden cardiac death association, 568 Small intestine, see Intestine SMP, see Sympathetically maintained pain SNS, see Sympathetic nervous system Somatostatin agonists, see also Octreotide adverse effects, 647–648 hot flash management, 647 postural tachycardia syndrome management, 646–647 autonomic neuropathy role, 645–646 receptors, 645 SP, see Substance P Space physiology management of disorders, 285 motion sickness, 284–285 orthostatic intolerance following flight, 284–285 overview, 284 SPECT, see Single-photon emission computed tomography Spinal cord injury bionic baroreflex system application, 662 cardiovascular system effects, 505–507 cutaneous circulation, 507 gastrointestinal effects, 608 reproductive system effects, 508 spinal shock, 505 sudomotor function, 508 thermoregulation, 507–508 urinary system effects, 508 Splanchic circulation lidocaine and hepatic blood flow, 665 local regulation, 211 overall circulatory function impact, 211 sympathetic control disease, 212–213 overview, 211, 212 special circumstances, 212 Spleen circulation, see Splanchic circulation Squatting, autonomic dysfunction management, 609–610 SSBP, see Salt-sensitivity of blood pressure SSNA, see Skin sympathetic nerve activity SSR, see Sympathetic skin response Stomach autonomic control of secretion, 205

circulation, see Splanchic circulation diabetic autonomic dysfunction and gastric emptying, 476–477 gut motility control, 206, 208 normal motor function, 208–209 Stress anxiety disorder, 292 autonomic dysfunction, 25 cardiac disease, 292 gastrointestinal effects, 292 noradrenergic versus adrenergic activation in stress, 42–43 panic disorder, 292 post-traumatic stress disorder, 292 psychosomatic disorders, 292 sympathetic nerve responses, 291–293 Stress cardiomyopathy (SCM) causes, 371–373 coronary artery spasm, 371–373 diagnosis, 371, 373 sympathetic activation, 373–374 Stress urinary incontinence (SUI), 232 Stroke volume, postural orthostatic tachycardia syndrome, 523 Subarachnoid hemorrhage (SAH), endothelin role, 138 Substance P (SP) airway control, 203 cotransmission, 29 Sudden cardiac death, obstructive sleep apnea association, 568 Sudden unexpected death in epilepsy patients (SUDEP), 551 SUDEP, see Sudden unexpected death in epilepsy patients Sudomotor function axon reflex testing, 409–411 diabetic autonomic dysfunction, 479 overview of tests, 409 skin potentials, 411 spinal cord injury, 508 sweating disorders and testing, 556, 558 thermoregulatory sweat test, 411 SUI, see Stress urinary incontinence Superior cervical ganglia (SCG), sympathetic nervous system, 17–20, 22–23 Suprachiasmatic nuclei (SCN) circadian and sleep control of autonomic nervous system, 158–159 output and autonomic control, 157–158 pacemaker cells, 157 Sweat glands denervation effects, 250–251 density and distribution, 249 factors affecting sweat response, 250 functions, 250, 289 innervation, 250 innervation, 413 normal factors affecting sweating, 553–554 physiology, 249–250 skin sympathetic nerve activity in disturbances, 394 thermoregulatory sweat test, 411 types, 249 Sweating disorders, see Anhidrosis; Hyperhidrosis; Hypohidrosis Sympathectomy, hyperhidrosis management, 649

703

Index

Sympathetic microneurography, see also Muscle sympathetic nerve activity; Skin sympathetic nerve activity analysis multi-unit activity, 391 single unit activity, 391 applications bone loss, 396–397 chronic kidney disease, 395–396 heart failure, 395 hypertension, 395 hypotensive attacks, 393 metabolic syndrome, 396 multiple system atrophy, 393 obstructive sleep apnea, 394–395 radiculoneuropathies, 393 sweat disturbances, 394 challenges, 392 electrode site, 392 equipment, 389 technique, 389–391 Sympathetic nervous system (SNS) cotransmission, 27–28 functions, 24–25 neurotransmitter phenotypes, 22–23 overview, 17–20 sympathoadrenal axis, 20–21 Sympathetic rhythm cardiac and respiratory rhythms entrainment, 147–148 mechanisms, 147 phasic inputs, 147 central oscillators, 148 definition, 147 functional significance, 148 spinal cord and sympathetic rhythms, 148 Sympathetic skin response (SSR) spinal cord injury, 508 sudomotor function testing, 411 Sympathetically maintained pain (SMP), complex regional pain syndrome, 586 Syncope, see Neurally mediated syncope Syndrome of inappropriate antidiuretic hormone (SIADH) autonomic dysfunction, 125 euvolemia, 123 Syntaxin 1A, norepinephrine transporter interactions, 49–50 α-Synuclein pathology Alzheimer's disease, 303 multiple system atrophy, 303 Parkinson's disease, 303 transgenic animal studies, 304 synucleinopathies, see Dementia with Lewy bodies; Multiple system atrophy; Parkinson's disease; Pure autonomic failure therapeutic targeting, 304 toxic species, 303–304

T Takotsubo syndrome, 371 TCD, see Transcranial Doppler TEF, see Thermogenic effect of feeding Tetrahydrobiopterin (BH4) deficiency clinical presentation, 427–428 diagnosis, 428–429 treatment, 429 functions, 427 TH, see Tyrosine hydroxylase Thermogenic effect of feeding (TEF), 254 Thermoregulation central control overview, 287 cocaine effects, 579 cutaneous thermal receptor afferent pathway, 243–244 dorsomedial hypothalamus, 245–246 hyperthermia response, 288–289 hypothermia response, 288 rostral raphe pallidus area, 246 sensorimotor integration in preoptic area, 244–245 spinal cord injury, 507–508 spinal sympathetic mechanisms, 246 sweat glands, 250, 289 thermoneutral environments, 287–288 Thermoregulatory sweat test, sudomotor function, 411 Tilt table testing, see Head-up tilt table testing Traditional Chinese medicine, see Acupuncture Transcranial Doppler (TCD), head-up tilt table testing monitoring, 384 Transient receptor potential channel (Trp), Trpv4 in osmoreception, 614 Transthyretin amyloidosis, see Amyloidosis Tropicamide, pupil response, 239 Trp, see Transient receptor potential channel Tyramine, pupil response, 240 Tyrosine hydroxylase (TH) activation, 38 deficiency clinical presentation, 428 diagnosis, 429 treatment, 429 functional overview, 38, 45, 427 gene mutations in deficiency, 46–47 splice variants, 45–46 structure, 45 gene therapy, 46–47

U Urethra, see Lower urinary tract Urination, see Lower urinary tract

V Vagal nerve stimulation (VNS), effects on cardiovascular function in epilepsy, 552

Valsalva maneuver, autonomic dysfunction evaluation, 380–381 Vasculature, see Blood vessel Vasoactive intestinal peptide (VIP) cotransmission, 27–31 vasomotor control, 187, 190 Vasomotor control, see also Endothelial function; Endothelin adrenergic vasoconstriction modulation, 191 aging effects, 273 differential control, 190 overview, 187–188 parasympathetic control, 189–190 sympathetic control neuroeffector junction, 188 neurotransmitters, 188–189 Vasopressin, see Arginine vasopressin Vesicular monoamine transporter (VMAT) functional overview, 38–39 serotonin transport, 83 VIP, see Vasoactive intestinal peptide Virtual Physiological Human (VPH) project autonomic nervous system modeling, 675–677 overview, 673 physiome standards, 673–675 prospects, 677–678 Visceral afferents anatomy, 171 autonomic reflex responses to activation, 173–174 fiber types, 171–172 functional overview, 23–24, 171 ischemia response, 171–173 pathological alterations, 174–175 stimuli, 171 VMAT, see Vesicular monoamine transporter VNS, see Vagal nerve stimulation VPH project, see Virtual Physiological Human project

W Water drinking, therapeutic utility, 614–615 WCH, see White coat hypertension White coat hypertension (WCH), 356 Wnt, autonomic neuron development role, 4

X Xanthine oxidase, reactive oxygen species production, 336

Y Yohimbine adverse effects, 624 dosing, 624 efficacy, 628 mechanism of action, 624, 628 sources, 627

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