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Calcium Antagonists: Mechanism of Action on Cardiac Muscle and Vascular Smooth Muscle [1st ed.]
 978-0-89838-655-4;978-1-4613-3810-9

Table of contents :
Front Matter ....Pages i-xiii
INTRODUCTION: Role of Ca++ in Contraction, and Wide Variety of Effects of Calcium Antagonists (Nick Sperelakis)....Pages 1-9
Calcium Channel Antagonists: Pharmacologic and Radioligand Binding Approaches to Mechanisms of Action (D. J. Triggle, R. A. Janis)....Pages 11-20
Effects of Calcium Slow Channel Blockers on the Slow Action Potentials of Cardiac Muscle and Vascular Smooth Muscle (Nick Sperelakis)....Pages 21-45
Voltage Clamp Studies Of Calcium Channel Blockage in the Heart (R. S. Kass, M. C. Sanguinetti)....Pages 47-62
Mechanisms of Selective CA Antagonist-Induced Vasodilation (C. Van Breemen, C. Cauvin, O. Hwang, P. Leyten, S. Lukeman, K. Meisheri et al.)....Pages 63-76
Action of Calcium Slow Channel Inhibitors on Cardiac and Vascular Smooth Muscle Membranes (Mohammed A. Matlib, Arnold Schwartz, A. DePover, G. Grupp, I. Grupp, S. W. Lee et al.)....Pages 77-93
Effects of Calcium Antagonists on Injured Cells (Philip D. Henry)....Pages 94-100
Antianginal Effects of Calcium Antagonists (John S. Schroeder)....Pages 101-110
Clinical Electrophysiology of the Calcium Antagonists (E. Rowland, D. M. Krikler)....Pages 111-117
Basic Features of The Frequency- and Voltage-Dependent Block By D600 of Calcium Channels in Ventricular Muscle (Terence F. McDonald, Dieter Pelzer, Wolfgang Trautwein)....Pages 118-136
Evidence for a Modulated Receptor Mechanism of Calcium Channel Blockade (C. W. Clarkson, M. Inazawa, S. Kanaya, L. M. Hondeghem, B. G. Katzung)....Pages 137-151
Effects of Calcium Antagonistic Drugs on Various Heart Tissues, Including Blockade of the Slow Channels and Depression of Postdrive Hyperpolarization. (P. A. Molyvdas, N. Sperelakis)....Pages 153-178
Effect of Nifedipine on Rat Papillary Muscle and Perfused Heart (P. Braveny, J. S. Juggi, G. Telahoun)....Pages 179-185
Excitation-Contraction Coupling in Rat Myocardium: Modulation with Verapamil and Calcium (Joseph M. Capasso)....Pages 187-202
The Potentially Direct Role of Intracellular Calcium Overload in the Electrophysiology of Cardiac Ischemia (William T. Clusin, Maurice Buchbinder, R. Hardwin Mead)....Pages 203-217
Mechanisms of the Beneficial Effects of Some Ca2+ Antagonists on the Ca2+-Paradox in Myocardium (N. S. Dhalla, P. K. Singal, S. Takeo, D. B. McNamara)....Pages 219-227
Comparative Effects of Ca Slow Channel Blockers on the Hamster Hereditary Cardiomyopathy (G. Jasmin, L. Proschek)....Pages 229-239
Calcium Antagonistic Agents: Uptake into Various Muscles and their Effects on Calcium Binding (David C. Pang, Nick Sperelakis)....Pages 241-256
The Effect of Verapamil on Lanthanum (La3+) Binding in the Isolated Rabbit Heart (S. A. Barman, J. T. Saari, M. D. Olson)....Pages 257-273
In Situ Small Blood Vessel Electrical Response to Verapamil in Spontaneously Hypertensive Rats (W. J. Stekiel, M. J. Burke, S. J. Contney, J. H. Lombard)....Pages 275-285
Comparison of Calcium Blocking Activities of Adenosine Potentiating Compounds With Calcium Blocking Drugs in Isolated Smooth Muscle and Atria (Y. Nakagawa, M. Gudenzi, S. Jamal Mustafa)....Pages 287-301
Effects of Inhibitors of Arachidonic Acid Metabolism and Calcium Entry on Hypoxic Contractions of the Isolated Canine Coronary Artery (Thomas J. Rimele, Paul M. Vanhoutte)....Pages 303-316
Effect of Diltia Zem on Calcium Cup Rents and Excitation-Contraction Coupling in Frog Twitch Muscle (H. Gonzalez-Serratos, R. Valle-Agilera, C. Phillips)....Pages 317-325
Inhibition of Human Platelet Aggregation and Calcium-Dependent, Phospholipase A2 Activity by Calcium Antagonists: Evidence for Intracellular Effects of Calcium Slow Channel Blockers (Richard C. Franson, Herbert Evans, Jayanti Thakkar, Nick Sperelakis)....Pages 327-338
Characterization of Cardiac Sarcoplasmic Reticulum: Dysfunction during primary myocardial ischemia: A potential source for intracellular calcium overload (Michael L. Hess, Stephen M. Krause)....Pages 339-352

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CALCIUM ANTAGONISTS

DEVELOPMENTS IN CARDIOVASCULAR MEDICINE Lancee, C.T., ed.: Echocardiology, 1979. ISBN 90-247-2209-8. Baan, J., Arntzenius, A.C., Yellin, E.L., eds.: Cardiac dynamics. 1980. ISBN 90-247-2212-8. Thalen, H.J.T., Meere, C.C., eds.: Funadmentals of cardiac pacing. 1970. ISBN 90-247-2245-4. Kulbertus, H.E., Wellens, H.J.J., eds.: Sudden death. 1980. ISBN 9O-247-2290-X. Dreifus, L.S., Brest, A.N., eds.: Clinical applications of cardiovascular drugs. 1980. ISBN 90-247-2295-0. Spencer, M.P., Reid, J.M., eds.: Cerebrovascular evaluation with Doppler ultrasound. 1981. ISBN 90-247-90-247-2348-1. Zipes, D.P., Bailey, J.C., Elharrar, V., eds.: The slow inward current and cardiac arrhythmias. 1980. ISBN 90-247-2380-9. Kesteloot, H., Joossens, J.V., eds.: Epidemiology of arterial blood pressure. 1980. ISBN 90-247-2386-8. Wackers, F.J.T., ed.: Thallium - 201 and technetium-99m-pyrophosphate myocardial imaging in the coronary care unit. 1980. ISBN 90-247-2396-5. Maseri, A., Marchesi, C., Chierchia, S., Trivelia, M.O., eds.: Coronary care units. 1981. ISBN 90-247-2456-2. Morganroth, J., Moore, E.N., Dreifus, L.S., Michelson, E.L., eds.: The evaluation of new antiarrhythmic drugs. 1981. ISBN 90-247-2474-0. Alboni, P.: Intraventricular conduction disturbances. 1981. ISBN 9O-247-2483-X. Rijsterborgh, H., ed.: Echocardiology. 1981. ISBN 90-247-2491-0. Wagner, O.S., ed.: Myocardial infarction. Measurement and intervention. 1982. ISBN 90-247-2513-5. Meltzer, R.S., Roelandt, J., eds.: Contrast echocardiography. 1982. ISBN 90-247-2531-3. Amery, A., Fagard, R., Lijnen, R., Staessen, J., eds.: Hypertensive cardiovascular disease; pathophysiology and treatment. 1982. ISBN 90-247-2534-8. Bouman, L.N., Jongsma, H.J., eds.: Cardiac rate and rhythm. 1982. ISBN 90-247-2626-3. Morganroth, J., Moore, E.M., eds.: The evaluation of beta blockers and calcium antagonist drugs. 1982. ISBN 90-247-2642-5. Rosenbaum, M.B., ed.: Frontiers of cardiac electrophysiology. 1982. ISBN 90-247-2663-8. Roelandt, J., Hugenholtz, P.O., eds.: Long-term ambulatory electrocardiography. 1982. ISBN 90-247-2664-8. Adgey, A.A.J., ed.: Acute phase of ischemic heart disease and myocardial infarction. 1982. ISBN 90-247-2675-1. Hanrath, P., Bleifeld, W., Souguet, eds.: Cardiovascular diagnosis by ultrasound, ansesophageal, computerized, contrast, Doppler echocardiography. 1982. ISBN 90-247-2692-1. Roelandt, J., ed.: The practice of M-mode and two-dimensional echocardiography. 1982. ISBN 90-247-2745-6. Meyer, J., Schweizer, P., Erbel, R., eds.: Advances in non-invasive cardiology. ISBN 0-89838-576-8. Perry, H.M., ed.: Life-long management of hypertension. ISBN 0-89838-572-2. Jaffe, E.A., ed.: Biology of endothelial cells. ISBN 0-89838-587-3. Surawicz, B., Reddy, C.P., Prystowsky, E.N., eds.: Tachycardiac. ISBN 0-89838-588-1. Simoons, M.L., Reiber, J.H.C., eds.: Nuclear imaging in clinical cardiology. ISBN 0-89838-599-7. Sperelakis, N., ed.: Physiology and pathophysiology of the heart. ISBN 0-89838-615-2. Messerli, F.H., ed.: Kidney in essential hypertension. ISBN 0-89838-616-0. Sambhi, M.P., ed.: Fundamental fault in hypertension. ISBN 0-89838-638-1. Marchesi, C., ed.: Ambulatory monitoring: Cardiovascular system and allied applications. ISBN 0-89838-642-X. Kupper, W., Macalpin, R.N., Bleifeld, W., eds.: Coronary tone in ischemic heart disease. ISBN 0-89838-646-2.

ii

CALCIUM ANTAGONISTS Mechanism of Action on Cardiac Muscle and Vascular Smooth Muscle From the Proceedings of the Meeting of the American Section of the International Society for Heart Research (ISHR) Hilton Head, South Carolina, September 21-24, 1983 Edited by Nicholas Sperelakis James B. Caulfield

Martinus Nijhoff Publishing

A member of the Kluwer Academic Publishers Group Boston/The Hague/Dordrecht/Lancaster

Distributors for North America: Kluwer Academic Publishers 190 Old Derby Street Hingham, MA 02043 Distributors Outside North America: Kluwer Academic Publishers Group Distribution Centre P.O. Box 322 3300 AH Dordrecht The Netherlands

Library of Congress Cataloging in Publication Data International Society for Heart Research. America American Section. Meeting (5th: 1983 : Hilton Head, S.C.) Calcium antagonists. (Developments in cardiovascular medicine) I. Calcium-Antagonists-Physiological effect-Congresses. 2. Heart-Muscle-Effect of drugs on-Congresses. 3. Vascular smooth muscle-Effect of drugs on-Congresses. I. Sperelakis, Nick, 1930. II. Caulfield, James B. III. Title. IV. Series. [DNLM: I. Calcium Channel Blockers-pharmacodynamics-congresses. 2. Muscle, Smooth, Vascular-drug effects-congresses. 3. Myocardium-drug effects-congresses. WI DE997VME I QV 15015903 1983cl QP535.C2I55 1983 615' .71 84-6139 e-ISBN-13: 978-1-4613-3810-9 ISBN-13: 978-1-4613-3812-3 DOl: 10.1007/978-1-4613-3810-9

Copyright 1984 © by Martinus Nijhoff Publishing, Boston Softcover reprint of the hardcover 1st edition 1984 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, mechanical, photocopying, recording, or otherwise, without written permission of the publisher, Martinus Nijhoff Publishing, 190 Old Derby Street, Hingham, Massachusetts 02043.

CONTENTS xiii

Acknowledgements

Introduction: Role of Ca + + in Contraction, and Wide Variety of Effects of Calcium Antagonists N. Sperelakis

Part I. Basic Science Aspects 2

Calcium Channel Antagonists: Pharmacologic and Radioligand Binding Approaches to Mechanisms of Action D.J. Triggle and R.A. Janis

11

3

Effects of Calcium Slow Channel Blockers on the Slow Action Potentials of Cardiac Muscle and Vascular Smooth Muscle. N. Sperelakis

21

4

Voltage Clamp Studies of Calcium Channel Blockage in the Heart R.S. Kass and M.C. Sanguinetti

47

5

Mechanisms of Selective Ca Antagonist-Induced Vasodilation C. VanBreemen, C. Cauvin, O. Hwang, P. Leyten, S. Lukeman, K. Meisheri, K. Saida, and H. Yamamoto

63

6

Action of Calcium Slow Channel Inhibitors on Cardiac and Vascular Smooth Muscle Membranes M.A. Matlib and A. Schwartz v

79

Part II: Clinical Science Aspects 7

Effects of Calcium Antagonists on Injured Cells P.D. Henry

93

8

Antianginal Effects of Calcium Antagonists J. S. Schroeder

101

9 Clinical Electrophysiology of the Calcium Antagonists E. Rowland and D.M. Krikler

111

Part III: Invited Poster Session

10 Basic Features of the Frequency and Voltage-Dependent Block by D-600 of Calcium Channels in Ventricular Muscle T.F. McDonald, D. Pelzer, W. Trautwein

118

11 Evidence for a Modulated Receptor Mechanism of Calcium Channel Blockade C.W. Clarkson, M. Inazawa, S. Kanaya, L.M. Hondeghem, and B.G. Katzung

137

12

Effects of Calcium Antagonistic Drugs on Various Heart Tissues, Including Blockade of the Slow Channels and Depression of Postdrive Hyperpolarization P.A. Molyvdas and N. Sperelakis

153

13 Effect of Nifedipine on Rat Papillary Muscle and Perfused Heart P. Braveny, 1.S. Juggi, and G. Telahoun

179

vi

14

Excitation-Contraction Coupling in Rat Myocardium: Modulation With Verapamil and Calcium J.M. Capasso

187

15 The Potentially Direct Role of Intracellular Calcium Overload in the Electrophysiology of Cardiac Ischemia W.T. Clusin, M. Buchbinder, and R.H. Mead

203

16 Mechanisms of the Beneficial Effects of Some Ca2 + Antagonists on the Ca2 +-Paradox in Myocardium N.S. Dhalla, P.K. Singal, S. Takeo, and D.B. McNamara

219

17 Comparative Effects of Ca Slow Channel Blockers on the Hamster Hereditary Cardiomyopathy G. Jasmin and L. Proschek

229

18 Calcium Antagonistic Agents: Uptake into Various Muscles and Their Effects on Calcium Binding D.C. Pang and N. Sperelakis

241

19 The Effect of Verapamil on Lanthanum (La3 +) Binding in the Isolated Rabbit Heart S.A. Barman, J.T. Saari, and M.D. Olson

257

20 In Situ Small Blood Vessel Electrical Response to Verapamil in Spontaneously Hypertensive Rats W.J. Stekiel, M.J. Burke, S.J. Contney, and J .H. Lombard

275

vii

21

Comparison of Calcium Blocking Activities of Adenosine Potentiating Compounds With Calcium Blocking Drugs in Isolated Smooth Muscle and Atria Y. Nakagawa, M. Gudenzi, and S.J. Mustafa

287

22

Effects of Inhibitors of Arachidonic Acid Metabolism and Calcium Entry on Hypoxic-Induced Responses in the Isolated Canine Coronary Artery T.J. Rimele and P.M. Vanhoutte

303

23 Effect of Ditiazem on Calcium Currents and ExcitationContraction Coupling in Frog Twitch Muscle H. Gonzalez-Serratos, R. Valle-Aguilera, and C. Phillips

317

24 Inhibition of Human Platelet Aggregation and CalciumDependent, Phospholipase A2 Activity by Calcium Antagonists: Evidence for Intracellular Effects of Calcium Slow Channel Blockers R.C. Franson, H. Evans, J. Thakkar, and N. Sperelakis

327

25 Characterization of Cardiac Sarcoplasmic Reticulum: Dysfunction During Primary Myocardial Ischemia: A Potential Source for Intracellular Calcium Overload M.L. Hess and S.M. Krause

339

viii

NAMES AND ADDRESSES OF AUTHORS Dr. S.A. Barman Department of Physiology School of Medicine University of North Dakota Grand Forks, ND 58202 Dr. Maurice Buchbinder Cardiology Division Stanford University School of Medicine Stanford, CA 94305 Dr. Joseph M. Capasso Division of Cardiology Department of Medicine Albert Einstein College of Medicine of Yeshiva University 1300 Morris Park Avenue Bronx, NY 10461 Dr. C. W. Clarkson Department of Pharmacology University of California San Francisco, CA 94143

Dr. P. Braveny Department of Physiology Faculty of Medicine Kuwait University Dr. M.J. Burke Department of Physiology Medical College of Wisconsin Milwaukee, WI 53226 Dr. C. Cauvin Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101 Dr. William T. Clusin Cardiology Division Stanford University School of Medicine Stanford, CA 94305

Dr. S.J. Contney Department of Physiology Medical College of Wisconsin Milwaukee, WI 53226

Dr. N.S. Dhalla Experimental Cardiology Section Department of Physiology Faculty of Medicine University of Manitoba Winnipeg, CANADA R3E OW3

Dr. Herbert Evans Department of Biochemistry Medical College of Virginia Richmond, VA 23298

Dr. Richard C. Franson Department of Biochemistry Medical College of Virginia Richmond, VA 23298

Dr. H. Gonzalez-Serratos Department of Biophysics University of Maryland School of Medicine Baltimore, MD 21201

Dr. M. Gudenzi Department of Pharmacology School of Medicine East Carolina University Greenville, NC 27834

ix

Dr. PhUip D. Henry Baylor College of Medicine Department of Medicine Cardiovascular Division Houston, TX 77030

Dr. Michael Hess Department of Medicine Division of Cardiology Medical College of Virginia Richmond, VA 23298

Dr. L.M. Hondeghem Department of Pharmacology University of California San Francisco, CA 94143

Dr. O. Hwang Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101

Dr. M. Inazawa Department of Pharmacology University of California San Francisco, CA 94143 Dr. R.A. Janis Miles Institute for Preclinical Pharmacology P.O. Box 1956 New Haven, CT 06509 Dr. S. Kanaya Department of Pharmacology University of California San Francisco, CA 94143 Dr. B.G. Katzung Department of Pharmacology University of California San Francisco, CA 94143 Dr. D.M. Krikler F.A.C.C. Consultant Cardiologist Hammersmith Hospital London, ENGLAND

Dr. G. Jasmin Department de Pathologie Faculte de Medecine University of Montreal C.P.6128 Montreal 101, P.Q. Dr. J.S. Juggi Department of Physiology Kuwait University P.O. Box 24923 Faculty of Medicine Alrazi, KUWAIT Dr. R.S. Kass Department of Physiology University of Rochester School of Medicine Rochester, NY 14642 Dr. Stephen M. Krause Department of Medicine Division of Cardiology Medical College of Virginia Richmond, VA 23298

Dr. J.H. Lombard Department of Physiology Medical College of Wisconsin Milwaukee, WI 53226 Dr. Mohammed A. Matlib Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine 231 Bethesda Avenue, M.L. 575 Cincinnati, OH 45267 x

Dr. P. Leyten Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101 Dr. S. Lukeman Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101

Dr. D.B. McNamara Experimental Cardiology Section Department of Physiology Faculty of Medicine University of Manitoba Winnipeg, CANADA R3EOW3 Dr. K. Melsheri Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101 Dr. S. Jamal Mustafa Department of Pharmacology School of Medicine East Carolina University Greenville, NC 27834 Dr. M.D. Olson Department of Anatomy School of Medicine University of North Dakota Grand Forks, ND 58202

Dr. Terrance F. McDonald Department of Physiology and Biophysics Dalhousie University Halifax NS B3H 4H7 CANADA Dr. R. Hardwin Mead Cardiology Division Stanford University School of Medicine Stanford, CA 94305 Dr. P.A. Molyvdas Department of Physiology Medical School University of Athens Athens 609, GREECE Dr. Y. Nakagawa Department of Pharmacology School of Medicine East Carolina University Greenville, NC 27834

Dr. Dieter Pelzer II. Physiologisches Institut Universitat des Saarlandes 6650 Homburg/Saar West Germany

Dr. David C. Pang Pharmacology Berlex Laboratories, Inc. 110 East Hanover Avenue Cedar Knolls, NJ 07927

Dr. L. Proschek Department de Pathologie Faculte de Medecine University of Montreal C.P.6128 Montreal 101, P.Q.

Dr. C. PhiUips Department of Biophysics University of Maryland School of Medicine Baltimore, MD 21201

Dr. E. Rowland F.A.C.C. Hammersmith Hospital London, ENGLAND

Dr. Thomas J. Rimele Department of Physiology and Biophysics Mayo Clinic Rochester, MN 55905

Dr. K. Saida Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101

Dr. J.T. Saari Department of Physiology School of Medicine University of North Dakota Grand Forks, ND 58202

xi

Dr. M.C. Sanguinetti Department of Physiology University of Rochester School of Medicine Rochester, NY 14642 Dr. Arnold Schwartz Department of Pharmacology and Cell Biophysics University of Cincinnati College of Medicine 231 Bethesda Avenue, M.L. 575 Cincinnati, OH 45267 Dr. Nicholas Sperelakis Department of Physiology University of Cincinnati College of Medicine 231 Bethesda Avenue, M.L. 576 Cincinnati, OH 45267 Dr. S. Takeo Department of Pharmacology School of Medicine University of Ryukyus Naha, JAPAN Dr. Jayanti Thakkar Department of Biochemistry Medical College of Virginia Richmond, VA 23298 Dr. D.J. Triggle Department of Biochemical Pharmacology School of Pharmacy State University of New York at Buffalo Buffalo, N.Y 14260 Dr. Casey VanBreemen Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101 Dr. H. Yamamoto Department of Pharmacology University of Miami P.O. Box 016189 Miami, FL 33101

xii

Dr. John S. Schroeder Department of Medicine Cardiology Divison School of Medicine Stanford University Palo Alto, CA 94304

Dr. P .K. Singal Experimental Cardiology Section Department of Physiology Faculty of Medicine University of Manitoba Winnipeg, CANADA R3E OW3 Dr. W.J. Stekiel Department of Physiology Medical College of Wisconsin Milwaukee, WI 53226

Dr. G. Telahoun Department of Physiology Faculty of Medicine Kuwait University

Dr. Wolfgang Trautwein II. Physiologisches Institut Universitat des Saarlandes 6650 Homburg/Saar West Germany Dr. R. Valle-Aguilera Department of Physiology University of Autonoma San Luis Potosi MEXICO Dr. Paul M. Vanhoutte Department of Physiology and Biophysics Mayo Clinic Rochester, MN 55905

ACKNOWLEDGEMENTS We gratefully acknowledge the support of the National Institutes of Health (Conference Grant Number HL-30581). The following companies and institutions also provided generous support for the meeting: Abbott Laboratories American Cyanamid Company Beckman Company Burroughs Wellcome Company Culpepper Foundation Drug Science Foundation. Medical University of South Carolina Fujisawa Smith-Kline Company International Society for Heart Research, American Section Merck and Company. Inc. Searle Pharmaceuticals, Inc. Sigma Xi, University of South Carolina Chapter Smith Kline-Beckman University of South Carolina University of South Carolina School of Medicine Upjohn Company

xiii

1 INTRODUCTION:

Role of

ca++ in Contraction, and Wide Variety of Effects of

Calcium Antagonists Nick SpereJakis

The calcium antagonistic drugs are a relatively new class of drugs that have in common the effect of reducing Ca++ ion entry into excitable nerve and muscle cells through the voltage-dependent and time-dependent slow channels.

Hence,

these drugs are also known as Ca ++ entry blockers, slow channel blockers, or organic calcium antagonistis (to contrast with the inorganic calcium antagonists, such as Mn++, Co++, Mg++, and La+++).

The organic structure of the calcium

antagonistic drugs varies widely, and the chemical formulae of some of the major sub-classes of these drugs are depicted in Figure 1.

VERAPAIlIL

.PlOIPIIiI

0(100)

g

IIlaUOIPINI

aCH3

I ~CODC2H5

",Ca DDC, .EPRIOIL

H,C

,

H

Pig. 1: The chemical structures of some calcium antagonists.

CH3

2

The process of excitation-contraction coupling in cardiac muscle cells and in vascular smooth muscle cells is depicted in Figures 2 and 3, respectively.

In

myocardial cells, the Ca++ influx from the interstitial fluid into the intracellular fluid controls the force of contraction of the cells (and hence of the entire heart).

Excitation-Contraction Coupling In Myocardial Cells Ca"" Influx Depolarization

--+

lSI

1

Slow Channels (V-DEP)

n.-.. ,. ... IIIL •• PItIOIL

l'

Ca""

SR Ca"

/ ' Release

SloW Channel Blocker.

/.-..

1

[call

Myofllamenl.

Contraction

Pig. 2: Summary diagram for excitation-contraction coupling in myocardial cells. Ca++ influx during excitation occurs through the voltage-dependent and timedependent gated slow channels. This entering Ca++ helps to raise the myoplasmic Ca++ concentration ([ Cal i) to the level necessary to activate the contractile proteins (e.g., 10-5 M), and acts to bring about the release of additional Ca++ from the SR. Taken from Sperelakis (1983).

The Ca++ ion that enters during cardiac excitation is primarily through the voltage- and time-dependent slow channels, and comprises the major portion of the inward slow current (lsi) (some of the lsi is also carried by Na + ions). The Ca++ that enters the myoplasm through the slow channels acts to help raise the intracellular concentration of free Ca++ ion ([ Cal i) to the level necessary to activate the contractile proteins, e.g., 10- 5 M brings about nearly maximal contraction (Fig. 4). In addition, the Ca ++ that enters across the cell membrane also acts on the sarcoplasmic reticulum (SR) to bring about the release of more

3

Ca++ stored in the SR (Fabiato & Fabiato, 1979). Thus, although the source of Ca++ for contraction is from the two pools, namely the extracellular fluid and the SR stores, the Ca++ entry across the cell membrane is the major regulatory factor of the force of contraction under a given set of conditions (e.g., constant drive rate).

Relaxation is produced by re-sequestration of the free myoplasmic Ca++

into the SR and pumping Ca ++ out of the cell, Ca-AT.Pase activity being utilized at both sites. Excitation Contr.ction Coupling in Vascillar Smooth Muscle

C:+INFLUX

Voltage-independent. Receptor-operated

1

Voltage-dependent

l

a-ADRENERGIC

AP

:tg~Ki~~:~~L ~ VERAPAMll)

.L Sarcolemmal

-

C.

DEPOLARIZATION

" Ca-Channels

i""/

Synaptic1 Ch.nnels

( RO'O~'

SLOW CHANNEL .LOCKER (e.g.

+- ~~~:~:I~;L

+

Slow Ca-Channels

~

~c++

1~ 8

SR

/'

ct+

SLOW CHANNEL

Release+-BLOCKER

(e.g. BEPAIDIL)

[Cali

1

Myofil.ments

CONTRACTION

Fig. 3: Diagram summarizing excitation-contraction coupling in vascular smooth muscle cells. Ca++ influx during excitation occurs through the voltage- and timedependent slow channels, which are Ca ++ -selective, and through several types of receptor-Qperated (voltage-independent) channels. Activation of the alphaadrenergic receptor (e.g., by norepinephrine) opens the gates of these channels. One type of receptor-Qperated channel is the postsynaptic channel; this channel is non-selective and allows most cations, e.g., Ca++, Na+, K+, to pass through (but screens out anions). The other major type of postulated receptor-operated channel is selective for Ca++ ion. See text for additional details. As depicted in Figure 2, the slow channel blocking drugs act, directly or indirectly, on the slow channels of the cell membrane to block a fraction (or all) of them, and thereby depress Ca++ entry during the action potential. In addition, those calcium antagonistic drugs that can readily enter the muscle cells, such as

4

bepridil and verapamil (Pang & Sperelakis, 1983), can exert their primary effect or a secondary effect on some intracellular site.

For example, since bepridil

depressed the contractions of cardiac muscle more than what could be accounted for on the basis of depression of lsi and the slow action potentials, it was suggested that bepridil enters the myocardial cells and acts on some intracellular site, such as the SR, to depress release of Ca++ (Vog.el, Crampton & Sperelakis, 1979). The effects of most of the calcium antagonistic drugs are dependent on frequency of stimulation, the effects being greater at higher driVe rates. The 1,4dihydropyridines, such as nifedipine and mesudipine, also have a frequencydependent component to their action, although this is less prominellt than for the other drugs, such as verapamil, bepridil, and diltiazem. In contrast, the inorganic calcium antagonists, such as Mn++ and La+++, are not frequency dependent. Some of the calcium antagonistic drugs affect Ca++ binding to sarcolemma, whereas others do not (Pang and Sperelakis, 1982). A derivative of nifedipine has recently been demonstrated to possess the property of opening slow channels in cardiac muscle and smooth muscle, and acting as a positive inotropic agent in cardiac muscle and as a vasoconstrictor in vascular muscle (Schramm et

!!.!.,

1983).

In vascular smooth muscle (VSM) cells, the process of excitation-contraction

coupling is quite similar to that in myocardial cells (Sperelakis, ·1982).

As

depicted in Figure 3, there is a Ca++ influx from the interstitial fluid into the myoplasm during excitation, and this Ca++ transport across the cell membrane is through the voltage- and time-

.~

-

m

NIF.

"0

c:::

iii

.... Q) :c

9

Me.

0

~

0

.

NIT



2CI

·2CN

NIM

• 3N 02

NIT NIF

••

B)

NIM. • 2CI 3N0 2 2Me

9

2CN • 2CF 3

8

H. 8

7

10

U H

c>

.2

6

7

/

I

/

/

/

/

/

/

/

.4Meo//

5 /

-log lC50. Gut Binding

Figure 5.A.

B.

/

/

-log

rc 50 •

Heart Response

Correlation between the activities of a series of 1,4-dihyropyridines as inhibitors of specific [3H]nitrendipine binding in microsomal fractions from guinea pig ileal longitudinal muscle and rabbit ventricle muscle. Correlation between the pyridines as inhibitors in rabbit ventricle and electrically stimulated Mannhold et al. (14).

activities of a series of 1,4-dihyroof specific [3H]nitrendipine binding as inhibitors of mechanical response in cat papillary muscle. Heart data from

For other tissues, including heart, rank order correlations occur, but there are large quantitative discrepancies between the high affinity binding and pharmacology. The data of Fi9ure 4 show for a series of 1,4-dihydropyridines their significantly higher activity as smooth muscle relaxants than as negative inotropic species. It is important to note, however, that this rank order correlation does indicate that a similar structure-activity relationship is being expressed at both sites. The correlation of Figure 4B reveals that a low affinity pharmacology state also exists in smooth muscle when the abilities of these antagonists to inhibit the

17

Verapamil

0600

Fi gure 6.

Schematic representation of organization of proposed binding sites at the Ca 2+ channel.

initial fast or phasic component of a depolarization-induced response is determined. The ability of 1,4-dihydropyridines to bind to cardiac and smooth muscle is very similar (Fig. 5), but there is a major quantitative discrepancy between binding and pharmacologic behavior in the heart (Fig. 5B). The origin of this discrepancy is not clear and in any event is not unique to cardiac tissue. Brain binding is of similarly high affinity to that found in cardiac and smooth muscle (13), but pharmacologic activity for inhibition of depolarization-induced neurotransmitter release or 45Ca 2+ uptake is generally very low (1,2,8,15,16). The origins of these discrepancies remain to be resolved (1,9,17,18) but may include loss of regulatory factors or processes or loss of pharmacologically relevant low affinity sites during cellular disruption. However, it is of interest that the density of the high affinity [3H]nitrendipine binding sites calculated for both smooth and cardiac muscle,

18

1-10 binding sites ~m-2, is very similar to the values estimated for functional Ca 2+ channel density in cardiac cells (19). Additional support for the thesis that the [3H]1,4-dihydropyridine binding site does indeed represent a pharmacologically relevant site derives from studies with other structural categories of Ca 2+ channel antagonists (1,8-11,20,21). With appropriate stereoselectivity, verapamil and 0600 behave as negative allosteric effectors, and diltiazem as a positive allosteric effector of [3H]1,4-dihydropyridine binding. This suggests the probable presence of three distinct sites, allosterically linked, at which the organic Ca 2+ channel antagonists interact to inhibit channel function (Figure 6). Recent determinations, by the technique of radiation inactivation, of molecular wei,ghts for the membrane associated [3H]nitrendipine binding site of 200,000-280,000 (22,23) provides further support for this view of the Ca 2+ channel as an oligomeric assembly. Two additional components of the model depicted in Figure 6 are worthy of attention. [3H]1,4-0ihydropyridine binding is divalent cation dependent (11,21,24,25), being significantly reduced or abolished in fully reversible fashion by treatment with chelating agents and restored by Ca 2+ and, to varying degrees, by other di- and trivalent cations in the sequence: Ca2+=Sr2+>Mg2+=Mn2+=Co2+>Ba2+=Ni2+>La3+=Sm3+ which parallels approximately the ability of these cations to serve as permeant or blocking species (24,25). The relationship of this site to the cation permeating machinery of the channel remains to be established, but one possibility is that the dihydropyridine antagonists function by enhancing the binding of a cation, presumably Ca 2+, to the channel and thus effectively blocking permeation. More plausibly, however, the site of cation interaction may be involved in the control of channel activation and inactivation (11). Additional evidence for a regulatory interaction of the 1,4-dihydropyridine site with the Ca 2+ channel derives from the recent report (26) that some dihydropyridines, including Bay K 8664 (methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)pyridine-5-carboxyl ate), closely related in structure to the antagonist species, actually serve as Ca 2+ channel activators. This clearly indicates that the 1,4-dihydropyridines do not function as "channel plugs" but serve as allosteric regulators, perhaps by stabilising the channel open state, increasing the probability of channel opening or by facilitating cation permeation. Because of the close structural similarity of these agonist and

19

antagonist molecules it is likely that, as in other receptor systems, there is a spectrum of activity from antagonist through partial agonist to agonist depending upon the ability of the ligand to stabilize or activate the appropriate channel conformation. It is thus possible that the apparent tissue selectivity exhibited by some 1,4-dihydropyridines has its origin, at least in part, in tissue-dependent differences in the expression of agonist-antagonist character of a ligand. It seems clear that the discovery of this new category of Ca 2+ channel ligands will activate new and exciting developments in the field of Ca 2+ channel regulation. ACKNOWLEDGEMENTS Preparation of this work was assisted by a grant from the National Institutes of Health (HL 16003). REFERENCES 1. Janis RA, Triggle OJ: New developments in Ca 2+ channel antagonists. J Med Chem (26): 776-785, 1983. 2. Triggle OJ, Swamy VC: Calcium antagonists. Some chemicalpharmacologic aspects. Circ Res (52: Suppl. I): 17-28, 1983. 3. Fleckenstein A: Calcium antagonism in heart and smooth muscle. Experimental facts and therapeutic prospects. Wiley and Sons, Inc., New York, N.Y., 1983. 4. Henry PO: Comparative cardiac pharmacology of calcium blockers. In: Flaim SF and Zelis R (Eds) Calcium blockers. Mechanisms of action and clinical applications. Urban and Schwarzenberg, Baltimore, 1982, pp. 135154. 5. Flaim SF: Comparative pharmacology of calcium blockers based on studies of vascular smooth muscle. In: Flaim SF and Zelis R (Eds) Calcium blockers. Mechanisms of action and clinical applications. Urban and Schwarzenberg, Baltimore, 1982, pp. 155-178. 6. Lee KS, Tsien RW: Mechanism of calcium channel blockade by verapamil, 0600, diltiazem and nitrendipine in single dialysed heart cells. Nature (302): 790-794, 1983. 7. Triggle OJ: Chemical pharmacology of calcium antagonists. In: Rahwan RG and Witiak 0 (Eds) Calcium regulation by calcium antagonists. American Chemical Society, Washington, D.C., 1982, pp. 17-37. 8. Triggle OJ, Janis RA: Ca 2+ channel antagonists. New perspectives from the radioligand binding assay. In: Back N and Spector S (Eds) Modern Methods in Pharmacology, Vol. II, Alan R. Liss, Inc., New York, N.Y., 1983, in press. 9. Bolger GT, Gengo P, Klockowski R, Luchowski E, Siegel H, Janis RA, Triggle AM, Triggle OJ: Characterization of binding of the Ca++ channel antagonist, [3H]nitrendipine. to guinea pig ileal smooth muscle. J Pharmacol Exptl Therap (22): 291-309, 1983.

20

10. Triggle DJ, Janis RA: Nitrendipine: binding sites and mechanisms of action. In: Scriabine A, Vanov S and Deck K (Eds) Nitrendipine. Urban and Schwarzenberg, Baltimore, 1983, in press. 11. Luchowski EM, Yousif F, Triggle DJ, Maurer SC, Sarmiento JG,Janis RA: The effects of metal cations and calmodulin antagonists on [3H]nitrendipine binding in smooth and cardiac muscle. Submitted for publication. 12. Fosset M, Jaimovich E, Delpont E, Lazdunski M: [3H]Nitrendipine receptors in skeletal muscle. Properties and preferential localization in transverse tubules. J Biol Chern (258): 6086-6092, 1983. 13. Bellemann P, Schade A, Towart R: Dihydropyridine receptor in rat brain labeled with [3H]nimodipine. Proc Nat Acad Sci USA (80): 2356-2360, 1983. 14. Mannhold R, Rodenkirchen R,Bayer R: Qualitative and quantitative structure-activity relationships of specific Ca antagonists. Prog Pharmacol (5): 25-52, 1982. 15. Nachshen DA, Blaustein MP: The efects of some organic "calcium antagonists" on calcium influx in presynaptic nerve terminals. Mol Pharmacol (16): 579-586, 1979. 16. Kaplita PV, Triggle DJ: Actions of Ca 2+ antagonists on the guinea pig ileal myenteric plexus preparation. Biochem Pharmacol (32): 65-68, 1983. 17. Triggle DJ: Ca 2+ channels revisited: problems and promises. Trends in Pharmacol Sci, in press, 1983. 18. Gengo PJ, Luchowski EM, Rampe DE, Rutledge A, Triggle AM, Triggle DJ, Janis RA: Chemical and pharmacological approaches to the definition and quantitation of Ca++ channels. In: Watson JD (Ed) Cold Spring Harbor Symposium on Molecular Neurobiology. Cold Spring Harbor, New York, in press. 19. Bean BP, Nowycky MC, Tsien RW: Electrical estimates of Ca channel density in heart cell membranes. Biophys J (41): 295a, 1983. 20. Murphy KMM, Gould RJ, Largent BL, Snyder SH:A unitary mechanism of calcium antagonist drug action. Proc Nat Acad Sci USA (80): 860-864, 1983. 21. Glossmann H, Ferry DR, LUbbecke F, Mewes R, Hofmann F: Identification of voltage operated calcium channels by binding studies: differentiation of calcium antagonist drugs with 3H-nimodipine radioligand binding. J Recept Res (3): 177-190, 1983. 22. Norman RI, Borsotto M, Fosset M, Lazdunski M, Ellory JC: Determination of the molecular size of the nitrendipine-sensitive Ca 2+ channel by radiation inactivation. Biochem Biophys Res Comm (111): 878-883, 1983. 23. Venter JC, Fraser CM, Schaber JS, Yung CY, Bolger G, Triggle DJ: Molecular properties of the slow inward calcium channel. J Biol Chem (258): 9344-9348, 1983. 24. Gould RJ, Murphy KMM, Snyder SH: [3H]Nitrendipine-labeled calcium channels discriminate inorganic calcium agonists and antagonists. Proc Nat Acad Sci USA (79): 3656-3660, 1982. 25. Hagiawara S, Byerly L: Calcium channel. Ann Rev Neuroscience (4): 69-125, 1981. 26. Schramm M, Thomas G, Towart R, Franckowiak G: Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels. Nature (303): 535-537, 1983.

3 EFFECTS OF CALCIUM SLOW CHANNEL BLOCKERS ON THE SLOW ACTION POTENTIALS OF CARDIAC MUSCLE AND VASCULAR SMOOTH MUSCLE NICK SPERELAKIS TABLE OF CONTENTS 1.

INTRODUCTION

2.

BLOCKADE OF THE SLOW APS

3.

ORDER OF POTENCY

4.

FREQUENCY DEPENDENCY

5. 6.

APPARENT REVERSAL BY ELEVATION OF EXTRACELLULAR Ca++ WASHOUT OF DRUGS

7.

SPECIFICITY OF CHANNEL BLOCKADE

8.

Ca++ BINDING

9.

INTRACELLULAR UPTAKE

10.

DRUG EFFECT ON MEMBRANE PROTEIN PHOSPHORYLATION

11.

SA NODE

12.

VASCULAR SMOOTH MUSCLE

13.

SUMMARY AND CONCLUSIONS

14.

REFERENCES

The work of the author and his colleagues reviewed and summaried in this article was supported in part by grants from the National Institutes of Health (HL-31942), Wallace Laboratories, Smith, Kline and French Laboratories, and Miles Institute for Medical Research.

22

L

INTRODUCTION

Ca++ influx into the myocardial cells during excitation occurs by means of the voltage-dependent slow channels. Some of the general and special properties of the voltage dependent slow channels in myocardial cells are listed in Table 1. The differences in the voltage inactivation of the slow channels and the fast Na+ channels are illustrated in Figure 1. Notice that the slow channels inactivate over a less negative (i.e., more depolarized) voltage range, and use will be made of this fact for induction of the slow action potentials (APs). TABLE 1 Some Properties of Myoeardial Slow Channels A.

B.

General Properties. 1.

The voltage-dependent slow channels allow Ca++ ions (and some Na+) to pass through.

2.

They are kinetically slow compared to the fast Na+ channels, i.e., their gates open, close, and recover more slowly.

3.

They operate over a less-negative voltage range than the fast Na+ channels.

4.

Tetrodotoxin (TTX), a blocker of fast Na+ channels, has no effect on slow channels.

Some Special Properties. 1.

CYClic AMP-dependence. (bet~-adrenergic agonists, histamine (H2), methyLxanthines, dibutyryl

cychc AMP, GPP (NH)P, cholera toxin, cyclic AMP injection). 2.

Metabolic (ATP)-dependence.

3.

pH-dependence.

(block by metabolic poisons, hypoxia, ischemia)

(selective blockade by acidosis) 4.

Drug blockade. (block by calcium-antagonistic drugs, such as verapamil, bepridil, nifedipine, diltiazem)

5.

Phosphorylation hypothesis.

6.

Protection hypothesis.

All calcium antagonistic drugs seem to have one property in common, namely an ability to block the voltage-dependent Ca++ slow channels in the cell membrane of cardiac muscle and vascular smooth muscle (VSM). (The slow channels in visceral smooth muscle, skeletal muscle, nodal cells of the heart, and cardiac Purkinje fibers are similarly affected.) The block of the slow channels thereby depresses the inward slow current (lsi) and Ca ++ influx during exci tation, and therefore the force of contraction. In myocardial cells, which also possess a

23

fast Na+ channel system, excitation-contraction uncoupling is produced by the calcium antagonistic drugs. 110

+Vmal (Ville)

140 120

100

80 60 40

,SLOW CHANNELS

20

Fig. 1: Graphic representation of differences in behavior, with respect to voltage inactivation, of the fast Na+ channels apd slow (Na+ and Ca++) channels. Maximal rate of rise of the action potential (+Ymax ) as a function of resting Em for the normal cardiac action potential (dependent on inward current through the fast Na+ channels) and for the slow action potential (dependent on inward current through the slow channels) elicited in cells whose.fast Na+ channels are blocked (by TTX or by depolarization to about -45 mY). +Ymax is a measure of the inward current intensity (everything else, such as membrane capacitance, held constant), which in turn is dependent on the number of channels available for activation. Taken from Sperelakis (1979). Since some of the drugs, such as verapamil, D600, and nifedipine, also block the slow Na+ channels found in young embryonic chick hearts (Table 2) and in cultured (monolayers) embryonic chick heart cells, these drugs are more accurately described

as

"slow-channel blockers" (McLean,

Shigenobu and

Sperelakis, 1974; Shigenobu, Schneider and Sperelakis, 1974). In contrast, bepridil, diltiazem, and mesudipine had no effect on the slow Na+ channels (Kojima and Sperelakis, 1983).

24

TABLE 2

1CSO values (M) for several types of calcium antagonistic drugs on depressing the slow action potentials Preparation

Verapamil

Bepridil

Nifedipine

Mesudipine

Guinea pig papillary muscle

1 x 10-6

1 x 10-5

1 x IO-7t

5 x 10-8

20, 40

Guinea pig Purkinje fibers

1 x IO-6t

1 x 10- 7t

5 x 10- 8

20

Cultured chick heart cells

1 x 10-6

S x 10-6

S x 10- 9

3-day-old embryonic chick heart

6 x 10- 6

No effect

7 x 10-6

No effect

Rabbit SA node

1 x IO- 6t

1 x 10- 6

1 x 10- 8

1 x 10- 7

Dog coronary artery

5 x 10-6t

S x 10- 6

K+ -contractures

References

3 x 10-7

18

No effect

14 7, 20 9, 10

1 x 1O- 7t

Cultured rat aortic cells

Diltiazem

2 x 10-8 1 x 10- 7

3 x 10-7

20, 22 1 x 10-6

rabbit aorta

38

t Concentration for complete block.

2.

BLOCKADE OF THE SLOW APs In order to study the effect of the drugs on the slow channels, the fast Na+

channels are voltage inactivated by elevation of [KJ 0 to about 25 mM to depolarize the cells to about -40 mY. This normally blocks excitability (Figs. 2 &: 3). The addition of a positive inotropic agent, such as isoproterenol or histamine, rapidly « 1 min) induces slowly-rising overshooting APs, the "slow APs", in both myocardial cells (Figs. 2 A-C &: 3 A-C) and Purkinje fibers (Fig. 2 E-G).

These

slow APs are blocked by verapamil (Fig. 2D) or by nifedipine (Fig. 2H) or other calcium antagonistic drugs. Figure 4 (A-C) illustrates the Ca++ dependency of the slow APs, namely elevated [Ca] AP, whereas lowered [Ca]

0

0

increases the overshoot and +Vmax of the slow The slow AP is also

lowers these two parameters.

dependent on [Na] 0 (Fig. 4 D-F). In addition, to the slow APs, voltage clamp experiments can be done to demonstrate that the drugs depress the inward slow Ca++ current, lsi (Fig. 5).

25

GUINEA PIG PAPILLARY MUSCLE NORMAL RINGER

25 I'IIM K+

1001 .. ISO

5Jl1O-e II VERAPA_

Orl ... r.t.: 05Hz

PUAKINJE FIBEP

Fig. 2: Induction of slow action potentials (APs) and block by calcium antagonistic drugs. AD: Papillary muscle (guinea pig). E-H: Purkinje fiber (guinea pig). A,E: Normal fast APs. B,F: Elevation of [KJ 0 to 25 mM (B) or 20 mM (F) depolarized to about -45 mV and blocked e~citability (shock artifacts only visible). C,G: Isoproterenol (10- 6 M) rapidly induced slowly-rising APs, the slow APs. D,H: Verapamil (5 x 10- 6 M) (D) or nifedipine (10-7 M) (H) rapidly depressed and blocked the slow APs. The driving rate for the slow APs was 0.5 Hz. The upper line in each panel is the zero potential level, and the lower trace is dV /dt, the peak excursion of which gives "max; the dV /dt calibration bars represent 500 V/sec for A and E, and 10 (B-D) or 20 V/sec (F-H). Modified from Molyvdas &: Sperelakis (1983 a,b).

GUINEA PIG PAPILLARY MUSCLE Slow Action potentials (In 25 mM K+ +105 M Histamine)

-5

10 M Histamine

I~

--v.'--"-C ]0 ]2J>,O 0/. C

,

20

-I

40mV ~

0.1

Mesudipine

·t

Drive rate: 0.5 Hz

3Xlrl M

Iii M

\0

-r\-~

-) L--I

sec

F

Fig. 3: Effect of mesudipine on the slow action potentials (APs) induced by histamine in guinea pig papillary muscle. A: Fast AP in normal Ringer solution. B: Partial depolarization and loss of excitability in 25 mM K+. C: Slow AP induced by 10-5 M histamine within 4 min. D-F: Mesudipine depressed (E, 3 x 10- 7 M) and abolished (F, 10- 6 M) the slow APs. Upper trace is dV /dt. Drive rate was 0.5 Hz. All records are from one impalement.

26

Fig. 4: Demonstration of the dependence of the slow APs of guinea pig ventricular myocardium on [ Cal o. Slow APs were induced by isoproterenol (10- 6 M). A-C: Variation in [Cal o. with [Na] 0 were held constant at 140 mM. Elevation of [Ca] 0 from 1 mM (A) to 2 mM (B) to 8 mM (C) increased the overshoot and maximal rate of rise (Vmax) of the slow APs. D-F: Variation in [Nal 0' with [Cal 0 held constant at 2 mM, lowering of [Nal 0 from 140 mM (F) to 110 mM (E) to 70 mM (0) decreased the amplitude and "max of the slow APs. Lower trace in each panel is dV Idt. Taken from Schneider &. Sperelakis (1975).

A

J,,,-__ o

Ca Z+ Conltant - vary Na+ E

.-- -' •• -2

~leontrol­ ~IY·r.Pllmh - --

Fig. 5: Effect of verapamil on membrane currents recorded from a reaggregate cell culture of chick embryonic heart (ventricular) using a two-microelectrode voltage clamp. Solid circles are peak lsi recorded with TTX (10- 6 M) and isoproterenol (10- 6 M) present; open circles represent outward currents (at 300 msec). Verapamil depressed lsi (filled triangle) and had almost no effect on the outward IK (unfilled triangles (10- 6 M, 3 min». Taken from Josephson &. Sperelakis (1982).

27

3.

ORDER OF POTENCY

The order of potency of the drugs on cultured myocardial cells is: mesudipine > diltiazem > verapamil > bepridil (Li and Sperelakis, 1983) (Fig. 6). The order of potency for guinea pig papillary muscle is: mesudipine > nifedipine > verapamil > bepridil; that for guinea pig Purkinje fibers is:

mesudipine >

nifedipine > verapamil.

The order of

These data are summarized in Table 2.

potency in SA-nodal and VSM cells is not much different from that in cardiac muscle.

The 1C50 values between cardiac muscle and VSM and SA node are not

greatly different also, and are summarized in Table 2. At 1 x 10- 5 M, bepridil produced a substantial inhibition of "'max of the slow AP of guinea pig papillary muscle, whereas the effect on "'max of the fast AP was considerably less. However, at this concentration, the contractions were completely suppressed. Therefore, bepridil depressed the contractions more than what could be accounted for by depression of the inward slow Ca++ current (lsi) (Fig. 7) (Vogel et a!., 1979). 100

\

----- ---- \

x

111

E

,

.,

r




·

• i ~

o

5.

12

6 12 Drive rete: 811m/min

4

APPARENT REVERSAL BY ELEVATION OF EXTRACELLULAR Elevation of [Ca]

0

ea++

tends to reverse or antagonize the effects of all of the

calcium antagonistic drugs (Fig. 7). The dose-response curves are shifted to the right in the presence of elevated (5.4 mM) [Ca]

0'

in both myocardial cells (Fig.

13) and Purkinje fibers (Fig. 14). This action could include competition with Ca++ ion for a binding site on or near the slow channel, as described above for verapamil and bepridil but not for nifedipine and diltiazem.

In all cases, the

increase in electrochemical driving force (Em - ECa) for an inward Ca ++ current in elevated [Ca]

0

would be a factor.

blocked by a drug, an increase in [Ca]

For example, if half of the channels were 0

would proportionally increase the Ca++

current (lCa) throllgh the unblocked channels (lCa

=gCa

[Em - ECa1 , where gCa

is the calcium conductance and ECa is the equilibrium potential for Ca++ as calculated from the Nernst equation (ECa = -61 mY log [Cal i/ [Cal

0»'

31

GUINEA PIG PURKINJE FIBERS

Jt:]:o

Slow ActiEln Potentials (in 20 mM K+ +106 M Iso)

CONTROL

':1\ 'j\ Tr\> A

B

.". . . ._

my

0.1 sec

,

-~MI-_

1.5 Hz

to

Hz

0.5 Hz

j

110

VIs

0.1 Hz

... ~,. __U.L.J. . . .I . .,-..: Fig. 11: Illustration of the frequency-dependency of the mesudipine effect on the slow action potentials (APs) in a guinea pig Purkinje fiber. Upper row; Control slow APs induced by 10-6 M isoproterenol in 20 mM K+ at 1.5 Hz (A), 1 Hz (B), 0.5 Hz (C), and 0.1 Hz (D). Lower row; Mesudipine (10- 7 M) rapidly (within 3 min) abolished the slow APs at a driving frequency of 1.5 Hz (E). Lowering of the driving frequency to 1 Hz (F), 0.5 Hz (G), and 0.1 Hz (H), partially restored the slow APs. All records are from the same impalement. The Three States of the Slow Channel

Fig. 12: Cartoon model for the three hypothetical states of a slow channel, after the Hodgkin-Huxley ~er:)' ~il:·':J.RAP' patterned states for the fast Na+ channel. In the resting state, the d(m) gate is closed d=O and the f(h) gate is open (d=O, f=l). 1=1 1= 1 Depolarization to the threshold 1 Reeov:::: In'cliv.,ion activates the slow channel to the rep;,~Y " " I (h) active state, the d gate opening rapidly and the f gate still being open ., (d=l; f=l). The activated channel spontaneously inactivates to the ~~A inactive state due to closure of the f Inactive st.te gate (d=l; f=O). The recovery process upon repolarization returns the channel from the inactive state back to the resting state, which is again available for reactivation. Ca ++ ion is bound to the outer mouth of the channel and poised for entry down its electrochemical gradient. Also depicted is the possible binding of nifedipine to the outer mouth of the channel in its active or inactive state, and thereby either blocking the activated channel or slowing the recovery process for converting from the inactive state back to the resting state. Modified from Sperelakis (1982). A.ctive state

Resting state

A~An~.";d=l

=I. •,. £. "J.RAP

32

f----

40

u0t:, Ca~1.8mM •• "

Ca+S,4 mM

+Vmax

~ 30

80

100

20

80

:

60

.§.

40

.9:

20

~~~~~-~~~~~-o-~h-~ [ MESUDIPINE

1 Ampl.

+vmax

(mV)

(VIs)

80

'0

A

"

~ Q

Fig. 13: Summary of the effects of me&udipine on the slow action potentials (APs) of guinea pig papillary muscles in the presence of normal (1.8 mM) [Ca] 0 (unfilled.symbols) and high (5.4 mM) [Ca] 0 (filled symbols). Slow APs were induced by 10- 6 M isoproterenol in 25 mM [K] o. The slow AP parameters plotted are +Vmax (circles), amplitude (squares), and duration at 50% repolarization (APD50) (triangles). Data plotted are the means + SE.

f--t H

~

60 50

iy

40 30 '0

1rl 4X1fl,07 APD 90

APD 50

(mSeC)

(msec)

100

8

C

fy i-;

106

.0 80

~

70

30

[ MESUDIPINE

1

0

5_10 6 M

Fig. 14: Summary of the effects of mesudipine on the slow action potentials (APs) of guinea pig Purkinje fibers in the presence of normal (1.8 mM) [Ca] 0 (unfilled symbols) and high (5.4 mM) [ Cal o (filled symbols). Slow APs were induced by 10-6 M isoproterenol in 20 mM [K] o. The slow AP parameters plotted are +Vrna (A), amplitude lB~, and duration at 50% and 90% repolarization (APD50, APD90) (C-D». Data plotted are the means + S.E.

33

6.

WASHOUT OF DRUGS

Speed of recovery upon washout of the drugs varies among the calcium antagonists.

Those drugs which permeate readily, i.e., bepridil and verapamil,

reverse more slowly (e.g., 30-60 min). In contrast, those drugs that permeate the least, i.e., diltiazem, niCedipine and mesudipine, wash out the most rapidly (e.g., 10-20 min). 7.

SPECIFICITY OF CHANNEL BLOCKADE

Some of the calcium antagonistic drugs have a weak effect on the fast Na + channels, in addition to their more marked effect on the slow channels. example, verapamil and bepridil, but not mesudipine, do depress

Vmax

For

of the

normal fast action potentials (APs) (Fig. 15) a small amount in concentrations that almost completely block the slow channels (Shigenobu et 1979; Labrid et

~.,

~.,

1974; Vogel et al.,

1979; Molyvdas & Sperelakis, 1983 a & b).

In this regard,

verapamil and bepridil have a slight local-anesthetic-like effect, i.e., some nonspecificity in block of the various types of membrane conductances. Nifedipine, diltiazem, and mesudipine appear to have little or no effect on the fast Na+ channels (Fleckenstein et ~., 1972; Saikawa et ~., 1977; Molyvdas and Sperelakis, 1983). The calcium antagonistic drugs, e.g., verapamil, seem to have little or no effect on the various K+ channels (Josephson and Sperelakis, 1982). However, it has been reported that some K+ can pass through the activated slow channels, and this K+ efflux is blocked by the Ca-antagonistic drugs (Lee and Tsien, 1982). 8.

ea++ BINDING Some of the calcium antagonistic drugs, namely verapamil and bepridil,

decrease the amount of Ca++ bound (to low-affinity sites) in isolated cardiac sarcolemmal vesicle Sperelakis, 1982).

preparations

in a dose-dependent

manner (Pang and

Verpamil was more potent than bepridil (about 5-fold);

nifedipine and diltiazem had no such effect (Table 3).

Thus, the calcium

antagonistic drugs are quite different with respect to this property.

Since the

first step in permeation of Ca++ ion through the slow channel would be binding to the mouth of the channel, bepridil and verapamil could act either by displacing Ca++ from this binding site, which is the presumed mechanism of action of Mn++, Co++, and La+++ ions in blocking lsi> or by permeating the membrane and displacing Ca++ from an internal obligatory binding site.

34

GUINEA PIG PAPILLARY MUSCLE

200

vis

1 ........

0.1sec

(MESUDIPINE)

A:

0

B: 10- 7 M C: 10- 6 M D:5X10 6 M

[BEPRIDIL)

E: 0

-6 F: 10 M

G: 10- 5 M

Fig. 15: Effect of mesudipine (A-D) and bepridil (E-G) on the normal fast action potentials (APs) of guinea pig myocardial cells. A: Control APs (no mesudipine present). B: Addition of mesudipine (10- 7 M) did not greatly affect the APs. C,D: Elevation of the mesudipine concentration to 10- 6 M (C), and 5 x 10-6 M (D) produced a marked shortening of the plateau. Upper trace given is the first derivative of the APs, whose peak excursion gives the maximal upstroke velocity (+ Vmax ), and is placed at zero potential level. E: Control AP (no bepridil present) and its first derivative (upper trace). F: Bepridil (10- 6 M) (60 min of exposure) did not greatly affect the AP. G: Elevation of the bepridil concentration to 10- 5 M produced a marked shortening of the plateau and +Vmax was reduced. Horizontal line gives zero potential level. +Vmax trace was arbitrarily shifted to the right.

35

TABLE 3 Effe.:'; of c~lcium-antagDnistic agents Dn the lDW-affinity Ca bmdmg s.tes Df sarcDlemma frDm guinea pig heart Percent Df control value

Agent Control

100

Bepridil

10-7 M 10-6 M 10-5 M

97 87' 75*

Verapamil

10-6 M 10-5 M

58* 41*

Nifedipine

10-6 M 10-5 M

97 94

Diltiazem

10-6 M 10-5 M

101 94

Mn++

10-3 M

La+++

10-3 M

6*

Acidosis

pH 6.4 pH 5.6 '

71' 49*

34*

*Statistically significant difference from the controls. The ea++ binding under control conditions averaged 15.3 ~~~l~s/mg protein. Data taken from Pang '" Sperelakis, 1981,

9.

INTRACELLULAR UPTAKE

Some of the drugs, particularly bepridil, verapamil, and nitrendipine, readily enter into the muscle cells (Mras and Sperelakis, 1982; Pang and Sperelakis, 1983a,b), and so have the option of (a) exerting a second effect on some intracellular organelle, e.g., to depress release of Ca++ from the sarcoplasmic reticulum (SR) during excitation-contraction coupling, or (b) exerting their effect on the slow channels from the inner surface of the cell membrane. The order of uptakes is: bepridil > verapamil 2 nitrendipine »nifedipine > diltiazem (Fig. 16). This order is similar to that for the lipid solubilities of these compounds, as measured from their oil:water partition coefficients.

36

Chick Embryonic Ventricles

-... c

200

II)

o

Q.

en

150

~ o E

100

Q.

50 NIFEOIPINE

o

OILTI

o

20

40

60

80

100

Z M

120

Time I minutesl Fig. 16: Time course of uptakes of nifedipine, diltiazem, bepridil and verapamil into chick embryonic ventricular muscle (9-day-old). Uptakes of calcium antagonists were measured in the presence of 2 mM CaCI2, 10-6 M calcium antagonists, 1 J.lCi/mI 3 H-calcium antagonists at 37o C. Data expressed as mean ~ S.E. Taken from Pang and Sperelakis (1983a).

We showed that bepridil depressed the contractions of cardiac muscle more than what could be accounted for by the depression of maximal rate of rise

(Vmax )

of the slow (slow-current-dependent) action potential and lsi, and so

suggested that bepridil enters the cells and depresses Ca++ release from the SR (Vogel et al., 1979).

It was recently shown that a quaternary ammonium

derivative of D600 (D890), which is less permeant because of its charge, has no effect when added to the outside of single cardiac myocytes, but blocks the slow channels when injected intracellularly (Hescheler et

~.,

1982).

These results

support the hypothesis that verapamil and its methoxy derivative (D600) enter the myocardial cell in the uncharged lipid-soluble form and block the slow channel by binding perhaps to the inner surface of the cell membrane. action might also be true for bepridil.

A similar site of

37

10.

DRUG EFFECT ON MEMBRANE PROTEIN PHOSPHORYLATION In

preliminary experiments, we have found that verapamil, bepridil,

diltiazem (all at 10-6, 10-5 M) and nifedipine (10- 7 , 10-6 M) inhibited the cyclic AMP-dependent phosphorylation of three membrane proteins (Carty, Sperelakis and Villar-Palasi, 1983).

Rinaldi and colleagues (1982) have reported that the

voltage-dependent Ca++ uptake into isolated cardiac sarcolemmal vesicles is proportional to the degree of membrane phosphorylation.

Since we have

previously shown that the functioning of the myocardial slow channels is dependent on cyclic AMP and on metabolism, presumably by a cyclic AMPdependent phosphorylation of the slow channel protein (or an associated regulatory protein) (reviewed by Sperelakis, 1980), the possibility that the slow channel blocking drugs act on the phosphorylation process must be considered (Figs. 17, 18). Phosphorylation Hypothesis for Slow Channel

A

B

Dephosphorylated

linoperatlve)

Phosphorylated

(operative)

Fig. 17: Cartoon model for a slow channel in myocardial cell membrane in two hypothetical forms: dephosphorylated (or electrically silent) form (left diagrams) and phosphorylated form (right diagrams). A protein constituent of the slow channel itself (part A) or a regulatory protein associated with the slow channel (part B) must be phosphorylated in order for the channel to be in a functional state available for voltage activation. Phosphorylation occurs by a cyclic AMPdependent protein kinase in the presence of ATP. Presumably, a serine or threonine residue in the protein becomes phosphorylated. Phosphorylation may produce a conformation change that effectively allows the channel gates to operate or increases the diameter of the water-filled pore. Modified from Sperelakis &: Schneider (1976) and Sperelakis (1983).

38

PROPERTIES OF MYOCARDIAL MEMBRANE ION CHANNELS HIS'.

CAFFEINE

t

THEOPHYLLINE

I tP

C\MP PhosPhodiesterase. 5-AMP

Ca++-Calmodulin act.

Protein

• (+)

Kin •••

A

Prot.ln rne••

(PDE)

~(_) Metabolic ell.ct. by Ilchemia

Hypoxia Metabolic pollonl Acidosis

Metabolic

Phosphorylation of Varloue Prolelna

EII.ct. SR

Fig. 18: Diagrammatic summary of some of the properties of the ion channels in myocardial cell membrane. The mechanism of action of some positive inotropic agents, such as beta-adrenergic agonists, histaminic H2 agonists, and methylxanthines (phosphodiesterase inhibitors) are depicted. The beta-agonists and H2-agonists act on the regulatory component (guanine nucleotide binding protein) of the adenylate cyclase complex to stimulate cyclic AMP production. The myocardial slow channels are dependent on cyclic AMP and on metabolism, presumably because a protein constituent (or regulatory component) of the slow channel must be phosphorylated in order for it to be in a form that is available for voltage activation. The sites of action of GPP(NH)P and cholera toxin on the regulatory component of adenylate cyclase are shown. Also depicted are the facts that the slow channels are selectively blocked by acidosis and by calcium antagonistic drugs. Also schematized are two types of ion channels that are activated by internal Ca++ ion: a K+-selective channel and a mixed Na-K channel. Taken from Sperelakis (1983). 11.

SA NODE

In SA nodal cells, which do not possess functional fast Na+ channels, the drugs depressed automaticity and +Vmax of the action potentials (APs), and produced depolarization to about -45 mY. The order of potency was: nifedipine> mesudipine > verapamil > bepridil (Molyvdas and Sperelakis, 1983; Goto and Sperelakis, 1983). As shown in Figures 19 and 20, bepridil and mesudipine blocked the naturally-occurring slow APs of SA node in a dose-dependent manner. It is possible that the depolarization was produced by a lowered K+

conductance (gK)'

The effects of the drugs on the automaticity and the APs of

the SA node may be explained by direct action on the slow inward current, which

39

has been proposed to be a major participating current in the pacemaker potential (Yanagihara and Irisawa, 1980).

An alternative view that the pacemaker

depolarization of nodal cells is due to a mixed (Na-K) current (similar to that of Purkinje

fibers)

has

been presented

by several invesigators

DiFrancesco, 1980; Brown, 1982; Osterrieder et

!!!.,

(Brown

and

1982; Grand and Strauss,

1982). CONTROL

RABBIT SA NODE BEPRIOIL 10· o M

30 MIN

~

)I)UUl

--r-r-r-r )~~0

I 5V18DC

200msec BEPRIDIL 5x10"M

10 MIN

15MI"

20 .. 110

Fig. 19: Effect of bepridil on the naturally-occurring slow action potentials (APs) of isolated rabbit SA node. Bepridil (10- 6 M) depressed the slow APs, +Ymax and rate of spontaneous firing. Elevation of the bepridil to 5 x 10- 6 M further depressed (10 min) and blocked (l.S min) the slow APs. Taken from Goto and Sperelakis (1983).

Mesudipine

)WWl ~iXL WW I.. k. Normal Tyrode

10- 8 M

10- 7M

v,

~ .-JII......J. ---'. ---'. - ..Gi$ ~ 1II---'._..b~j_d"IIIII_"t %

~aa-V

E

. . . . . . . . . . . ._..

6 min

&sta

atu ...

12 min F

__

t] 10 V I s Washout 10 min

)VVJ

..

.i

Of sab.L ~ ~•

Fig. 20: Effect of the calcium antagonistic drug, mesudipine, on the naturallyoccurring slow action potentials (APs) of a spontaneously-firing isolated rabbit SA node. A: Control APs in normal Ringer solution. B: 10-8 M mesudipine had little or no effect on the AP amplitude, Ymax , and slope of the diastolic depolarization. C-F: Elevation of mesudipine to 10- 7 M (C) and 3 x 10- 7 (D-F) depressed (D-E) and abolished (F) the APs within 12 min. G: Washout of the drug restored the spontaneous APs within 10 mi.n. Lower trace gives dV /dt. Time calibration bar represents 40 msec for the superimposed faster sweeps. All records are from the same impalement.

40 12.

VASCULAR SMOOTH MUSCLE The naturally-occurring slow APs in vascular smooth muscle (YSM) are also

depressed and blocked by the calcium antagonistic drugs (Fig. 21). The slow APs in YSM are dependent on a pure Ca++ inward current through voltage-dependent slow Ca++ channels (Sperelakis, 1982). Thus, the drug depresses the Ca++ influx into the YSM cell during excitation, and so can account for its vasodilating action. Consistent with this, the K+-induced contractures of YSM, which are dependent on a Ca++ influx from the interstitial fluid space, are markedly depressed by verapamil and bepridil in a dose-dependent manner (Fig. 22).

A Normal RInger

r--

-1= =r=-Lmv

B +lOmM

-=L o

CONTROl l..

1

TEA

C +VERAPAMIL.

---~---- -r

E lPRIDll 00'"SM) If IWASI-OJr C20 mh)

ffA-~ -~

Il0¥

-LL -t-----

0 mV

1---4

0.55«

Fig. 21: Effect of verapamil and bepridil on the TEA +-induced spontaneous action potentials (APs) of dog coronary artery. A: Control in normal Ringer's solution showing a lack of spontaneous APs or responses to electrical stimulation (one shock artifact visible). B: Record from the same cell taken 10 min after addition of 10 mM TEA, illustrating a large overshooting AP in response to stimulation. C: Addition of verapamil (5 x 10-6 M) abolished the AP in 5 min. D: Spontaneous AP recorded in the presence of TEA (10 mM). E: Complete inhibition of the AP by 10-5 M bepridil. F: Restoration of the AP following washout of bepridil for 20 min. Taken from Harder and Sperelakis (1979, 1981).

41

100

lfI

,;

75

OJ

i

.~

ii

i

-o-controt

-00-- '10" Mbtlprldil ..... 1O·I Mbepridll •• 0(10 •• 'IC)"'M ....r.,.rnil

..... 'to'IM Q,.,.mil

50

25 .... 20 25 30

5

45

..•.. 60

100

[KJ O > mM

Fig. 22: Effect of bepridil and verapamil on the contractions of rabbit aortic rings induced by elevated K+. Cumulative K+ dose-response curves were obtained in the presence and absence of bepridil (10- 5 and 10-6 M) and verapamil (10- 5 and 10- 6 M). Tissues were equilibrated with the blocking agents for 20 min. Taken from Sperelakis and Mras (1983). 13.

SUMMARY AND CONCLUSIONS

The calcium antagonistic drugs have the common property of blocking the slow channels in cardiac muscle and vascular smooth muscle, as well as in other tissues.

The effect on the slow channels is relatively specific, i.e., some of the

drugs (e.g., nifedipine and diItiazem) have almost no effect on the other types of ion channels, such as the fast Na+ channels and various types of K+ channels. Some of the drugs (e.g., verapamil and bepridil) have a relatively small effect on depressing the fast Na+ channels as well.

The order of potency of the drugs in

blocking the slow channels is generally: mesudipine ~ nifedipine > diltiazem > verapamil > bepridil. The blocking effect of these drugs on the slow channels is very frequency dependent (use-dependent), although nifedipine has a lesser frequency dependency than the other drugs.

Elevation of [Cal

0

appears to

antagonize or reverse the inhibition of the slow channels produced by the drugs, although at least part of this effect of elevated [Cal 0 is probably mediated by a larger electrochemical driving force for Ca++ influx through any unblocked channels. The effect of the drugs can also be reversed by washout of the drugs,

42

nifedipine and diltiazem being the fastest to reverse, and verapamil and bepridil being slower. As might be expected from their quite diverse chemical structures, there are a number of differences among the calcium antagonistic drugs. For example, verapamil,D600, and nifedipine block the slow Na+ channels found in early embryonic chick hearts, whereas bepridil, diltiazem, and mesudipine do not. Verapamil and bepridil inhibited Ca++ binding to isolated sarcolemmal vesicles in a dose-dependent manner, whereas nifedipine and diltiazem did not. The order of uptake into cardiac muscle and vascular muscle was:

bepridil > verapamil 2:

nitrendipine » nifedipine > diltiazem; this order followed their order of lipid solubilities. Those drugs that readily permeate into the cell interior thus have the option of having their primary site of action on some intracellular location. For example, verapamil and D600 may block the slow channels by acting on the inner surface of the cell membrane. In addition, those drugs that readily enter the cells could have a second site of action intracellularly, e.g., bepridil might also act to depress Ca++ release from the SR.

Finally, because of the requirement of the

myocardial slow channels to be phosphorylated by a cyclic AMP-dependent protein kinase in order for them to be available for voltage activation (i.e., functional), it is possible that some drugs might act to inhibit the rate of phosphorylation (or accelerate the rate of dephosphorylation). 14. 1.

REFERENCES

Brown, HF: Electrophysiology of the sinoatrial node. Physiological Reviews (62): 505-530, 1982.

2.

Brown HF, DiFrancesco D:

Voltage clamp investigations of membrane

currents underlying pacemaker activity in rabbit sinoatrial node. J Physiol Lond (308): 331-335, 1980. 3.

Carty D, Sperelakis N, Villar-Palasi C:

Ca++-antagonistic drugs reverse

cyclic A MP-dependent phosphorylation of heart sarcolemmal proteins. (unpublished observations). 4.

Fabiato A, Fabiato F: Calcium and cardiac excitation-contraction coupling.

5.

Ferrier GR, Moe GK:

Ann Rev Physiol (41): 473-484, 1979. Effect of calcium in acetyl strophanthidin-induced

transient depolarizations in canine Purkinje tissue. Circ Res (33): 508-515, 1973.

43 6.

Fleckenstein A, Tritthart H, Doring HJ, Byon KY:

Bay a 1040-ein

hochactiver inhibitor der electro-mechanischen Koppelungspzozesse in warmbluter-Myokard Arzwein-Forsch (Drug Res) (22): 22-33, 1972. 7.

Goto J, Sperelakis N: Depression of automaticity of the rabbit SA node by nifedipine and bepridil. Europ J Pharmacol (in press), 1984.

8.

Grant AO, Strauss HC: Intracellular potassium activity in rabbit sinoatrial node. Circ Res (51): 271-279, 1982.

9.

Harder DR, Sperelakis N:

Action potentials induced in guinea pig arterial

smooth muscle by tetraethylammonium. Am J Physiol/Cell Physiol (237): 10.

C75-C80, 1979. Harder DR, Sperelakis N:

Bepridil blockade of

Ca++~ependent

action

potentials in vascular smooth muscle of dog coronary artery. J Cardiovasc Pharmacol (3): 906-914, 198!. 11.

Hescheler J, Pelzer D, Trube G, Trautwein W:

Does the organic calcium

channel blocker D600 act from inside or outside on the cardiac cell membrane? Pflugers Arch (393): 287-291, 1982. 12.

Hondeghem S, Kanaya P, Arlock, Katzung B: Verapamil and diltiazem block inactivated calcium channels. Circulation (66): 11293, 1982.

13.

Josephson I, Sperelakis N: On the ionic mechanism underlying adrenergiccholinergic antagonism in ventricular muscle.

J Gen Physiol (79): 69-86,

1982. 14.

Kojima M, Sperelakis N:

Calcium antagonistic drugs differ in ability to

block the slow Na+ channels of young embryonic chick hearts.

Eur J

Pharmacol (94): 9-18, 1983. 15.

Kohlhardt M, Krause H, Kubler M, Herdey A: Kinetics of inactivation and recovery of the slow inward current in mammalian ventricular myocardium. Pflugers Arch (355): 1-17, 1975.

16.

Kohlhardt M, Fleckenstein A:

Inhibition of slow inward current by

nifedipine in mammalian ventricular myocardium.

Naunyn Schmiedbergs

Arch Pharmacol (298): 267-272, 1977. 17.

18.

Labrid C, Grosset A, Dureng G, Mironneau J, Duchenne-Marullaz P: Some membrane interactions with bepridil, a new antianginal agent. J Pharmacol Exp Ther (211): 546-554, 1979. Li T, Sperelakis N: Calcium antagonist blockade of slow action potentials in cultured chick heart cells. Can J Physiol Pharmacol (61): 957-966, 1983.

44

19.

McLean MJ, Shigenobu K, Sperelakis N: Two pharmacological types of slow Na+ channels as distinguished by verapamil blockade.

Europ J Pharmacol

(26): 379-382, 1974. 20.

Molyvdas P-A, Sperelakis N: Comparison of the effects of several calcium antagonistic drugs (slow-channel blockers) on the electrical and mechanical activities of guinea pig papillary muscle. J cardiovasc Pharmacol (5), 162169,1983a.

21.

Molyvdas P-A, Sperelakis N: Comparison of the effects of several calcium antagonistic drugs on the electrical activity of guinea pig Purkinje fibers. Eur J Pharmacol (88): 205-214, 1983b.

22.

Mras S, Sperelakis N: Bepridil (CERM-1978) blockade of action potentials in cultured rat aortic smooth muscle cells.

Europ J Pharmacol (71): 13-19,

1981. 23.

Mras S, Sperelakis N:

Comparison of [3 H] bepridil and [3 H] verapamil

uptake into rabbit aortic rings. J Cardiovasc Pharmacol (4): 777-783, 1982. 24.

Osterrieder W, Yang QF, Trautwein W: Effects of barium on the membrane

25.

currents of the rabbit SA node. Pflugers Arch (394): 78-84, 1982. Pang D, Sperelakis N: Inhibitory action of bepridil (CERM-1978) on calcium binding to cardiac sarcolemma of guinea pig.

Biochem Pharmacol (30):

2356-2358, 1981. 26.

Pang DC, Sperelakis N:

Veratridine stimulation of calcium uptake by

embryonic heart cells in culture. J Molec Cell Cardiol (14): 703-710, 1982. 27.

Pang DC, Sperelakis N:

Nifedipine, diltiazem, bepridil and verapamil

uptakes into cardiac and smooth muscles. Europ J Pharmacol (87): 199-297, 1983a. 28.

Pang DC, Sperelakis N: Uptake of 3H-nitrendipine into cardiac and smooth muscles. Biochem Pharmacol (32): 1660-1663, 1983b.

29.

Pelzer D, Trautwein W, McDonald TF: Calcium channel block and recovery from block in mammalian ventricular muscle treated with organic channel inhibitors. Pflugers Arch (394): 97-105, 1982.

30.

Rinaldi ML, Capony J-P, Demaille JG: The cyclic A MP-dependent modulation of cardiac sarcolemmal slow calcium channels. J Mol Cell

31.

Saikawa T, Nagamoto Y, and Arita M:

Cardiol (14): 279-289, 1982. Electrophysiologic effects of

diltiazem, a new slow channel inhibitor, on canine cardial fibers. Jap Heart J. (18): 235-245, 1977.

4S

32.

Schneider JA, Sperelakis N:

Slow Ca++ and Na+ responses induced by

isoproterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K+. J Molec Cell Cardiol (7): 249-273, 1975. 33.

Shigenobu K, Schneider JA, Sperelakis N: Verapamil blockade of slow Na+ and Ca++ responses in myocardial cells.

J Pharmacol Exp Therap

(190):

280-288, 1974. 34.

Sperelakis N:

Origin of the cardiac resting potential.

IN: Berne RM,

Sperelakis N (eds) Handbook of Physiology, The Cardiovascular System, Vol. 1: The Heart. Am Physiol Soc, Bethesda, 1979, pp 187-267. 35.

Sperelakis

N:

Changes

development of the heart.

in In:

membrane

electrical

properties

during

Zipes OP, Bailey JC, Elharrar V (eds) The

Slow Inward Current and Cardiac Arrhythmias.

Martinus Nijhoff, The

Hague, 1980, pp 221-262. 36.

Sperelakis N:

Electrophysiology of vascular smooth muscle of coronary

artery. In: Kalsner S (ed) The Coronary Artery. Croom Helm, Ltd, London, 1982, pp 118-167. 37.

Sperelakis N: Properties of calcium-dependent slow action potentials, and their possible role in arrhythmias.

In:

Antagonists and Cardiovascular Disease.

Opie LH, Krebs R (eds) CalciumRaven Press, New York, 1983, (in

press). 38.

Sperelakis N, Mras S: Depression of contractions of rabbit aorta and guinea pig vena cava by mesudipine and other slow channel blockers. Blood Vessels (20): 172-183, 1983.

39.

Sperelakis N, Schneider JA: A metabolic control mechanism for calcium ion influx that may protect the ventricular myocardial cell. Am J Cardiol (37): 1079-1085, 1976.

40.

Vogel S, Crampton R, Sperelakis N: Blockade of myocardial slow channels by bepridil (CERM-1978). J Pharmacol Exp Ther (210): 378-385, 1979.

41.

Yanagihara K, Irisawa H:

Potassium current during the pacemaker

depolarization in rabbit sinoatrial node cell. Pflugers Arch (388): 255-260, 1980.

4

VOLTAGE CLAMP STUDIES OF CALCIUM CHANNEL BLOCKAGE IN THE HEART R.S. KASS AND M.C. SANGUINETTI

1.

INTRODUCTION

It is now recognized that calcium channels exist in a wide variety of cells and participate in many aspects of cellular function (for review, see refs. 1 and 2). In the heart, voltage-dependent calcium ion influx underlies spontaneous acti vi ty and impulse conduction in the sinoatrial and atrioventricular nodes, maintains the plateau phase of ventricular action potentials, and is closely associated with activation of contraction (3-6). Thus, it is not surprising that compounds which block calcium channels are of considerable interest to cellular electrophysiologists and, in particular, to those interested in the electrophysiology of the heart. Organic compounds that block calcium channel current in cardiac and other cells have been, and continue to be, extensively investigated using electrophysiological and biochemical techniques. The compounds are referred to as calcium channel antagonists, calcium entry blockers, or calcium channel blockers (7). In addition to their usefulness as electrophysiological tools, these drugs have important therapeutic applications, particularly in the treatment of angina, hypertension, and certain cardiac arrhythmias (8,9). Important differences between the modes of action of these drugs have been found and have resulted in the indentification of at least three classes of these compounds (see ref. 10 for review). One important distinction between the drugs is the manner in which

48

3.

RESULTS

3.1.

Action Potential Experiments: use-dependent effects When iso11'1.ted cardiac ventricular cells (either ventricular muscle ce11s or Purkinje fibers) are exposed to compounds that block calcium channel current, the plateau phase of the action potential is altered. The manner in which organic calcium channel blockers affect the action potential plateau depends on drug concentration and the rate at which the preparation is being driven. Under conditions of constant stimulation rate, action potentials of ventricular muscle or Purkinje fibers respond similarly to exposure to D600 (a methoxy derivative of verapamil) or to dihydropyridines such as niso1dipine. At moderate to high concentrations (500 nM 10 11M), these drugs lower and shorten the plateau (14). Differences in the specificity of these compounds for the calcium channel probably account for contrasts in lowconcentration effects of the drugs that can be observed during initial exposure to the compounds (Fig. 1).

A

B

o ~

mV

~,

control \~~~

~J~ _

~::S=~

d

i3'~~ 500 ms

-100 500ms Figure 1. Effect of Ca channel blockers on the Purkinje fiber action potential. A. Before, and 6,7, and 12 min after exposure to 1 11M D600 (Data from Kass and Tsien (1975)). B. Before, and 6 and 13 min after exposure to 10 11M niso1dipine. (Data from Kass (1982)). Preparations driven at 0.5 Hz.

49

blockage of the calcium channel by them is modulated by membrane potential. Voltage clamp studies are the most direct means to investigate voltage-dependent calcium channel inhibition. The purpose of this chapter is to review the evidence for voltage-dependent block of cardiac calcium channels and to show differences that have been observed between calcium channel block by various calcium channel antagonists. The chapter will focus on results obtained for verapami 1, D600, and the dihydropyr i dine der iva ti ves nisoldipine and nitrendipine. METHODS AND NOMENCLAUTURE Many of the results discussed in this chapter have been reported in the literature and thus methods used in obtaining these results have been previously described. However, some results have not been previously discussed. These experiments were carried out in calf cardiac Purkinje fibers injected with tetrabutylammonium (TBA) to block outward currrents (11). Membrane current was recorded with a two microelectrode technique arranged to optimize the measurement of inward current (12). Dihydropyridine compounds were applied to preparations under subdued illumination as described in another paper 2.

(13) •

The calcium channel is permeable to calcium and other divalent cations such as strontium and barium (1). Thus in this paper, current through this pathway is referred to as calcium channel current. In some figure legends, this current is also called lsi (slow inward current), a designation that has been used in cardiac electrophysiology to distinguish calcium current from the regenerative sodium current INa (4).

50

When the frequency of electrical stimulation is changed, clear contrasts in actions of these drugs are apparent. Verapamil (and D600) becomes a more potent inhibitor of contractile activity of ventricular muscle when drive rate is increased. Inorganic calcium channel blockers also produce negative inotropic responses, but are not sensitive to drive rate (15). Other experiments have since shown similar contrasts between the influence of drive rate on the effects of verapamil (and D600) and dihydropyridines on the cardiac action potential (16; Fig. 2 this paper).

A

(-) - VEAAPAMll 2X

a

eM

B

2. Influence of stimulation rate on action potential configuration before and after exposure to verapamil (A) or nifedipine (B). Preparatiops (cat papillary muscles) stimulated at a rate of 6 min- and 60 min-. Data from Bayer and Ehara (1978). Figure

Modulation of drug action caused by changes in tissue stimulation rate have been referred to as frequency-or use-dependent effects. Local anesthetic drugs such as lidocaine block sodium channels in nerve and heart ce11s in a use-dependent manner consistent with a model (modulated receptor hypothesis) that relates this action both to the state of the sodium channel and to the degree of ionization of the blocking compound (17-19). According

51

to this model, permanently charged drugs should be characterized by marked use-dependence. Block by drugs that are permanently uncharged will not be use-dependent, but may be modulated by membrane potential (17). Since verapamil and D600 are virtually entirely in the charged form and dihydropyridines such as nifedipine are uncharged at physiological pH (20), it is attractive to speculate that similar drug-channel interactions underlie differences in voltage-sensitivity of calcium channel block by these compounds. Since membrane potential modulates channel blockage in the model, the key to testing it is to control potential. Thus voltage clamp experiments are essential to determining the modes of action of these drugs. 3.2.

Voltage clamp studies: steady-state block of calcium channels The simplest experiments designed to test whether a compound blocks calcium channel current are carried out by applying depolarizing voltage steps from a fixed potential (holding potential) at a constant pulsing rate. In order to determine that calcium channel current is affected by the drug of interest, other membrane currents must first be reduced, or eliminated. This can be done in Purkinje fibers and ventricular cells by injection of quaternary ammonium ions (11) or replacement of intracellular K+ by Cs+ (21) to block outward currents and by the use of tetrodotoxin (TTX) or choice of holding potential to block sodium current (22). An experiment showing that nisoldipine blocks calcium channel current under these conditions is shown in Figure 3. The inset shows that injection of the quaternary ammonium compound tetrabutylammonium (TBA) blocks the transient outward current and reveals time-dependent inward calcium current. Subsequent exposure to nisoldipine (10 ~M) completely blocks this time-dependent inward current. In this experiment current records were

52

_

o

CONTROL



100 p.M NISOLDIPINE



I8A

100

10llM NISOLDIPINE

--~

~

-100

o

0 0--..---0

~.

-50

25

mV

Figure 3. Block of calcium current by nisoldipine: constant positive (-50 mV) holding potential. Inset: Calcium current revealed by TBA injection during voltage step to -5 mV blocked by nisoldipine (10 ~M). Currentvoltage curve indicates block of calcium current over more complete voltage range by 10 ~M (.) and 100 ~M (.) nisoldipine. obtained by applying 500 ms steps to a series of voltages from a-50 mV holding potential. Pulses were applied once every 5 sec. Under these condi tions, nisoldipine completely blocks calcium channel current over all voltages tested (see current-voltage relation in Fig. 3). When D600 is applied to cardiac cells under similar conditions, this drug also completely blocks all available calcium channels (23,24). According to the results of experiments like these, it would appear that D600 and the dihydropyridines block calcium channels in the same manner. However, these drugs show marked di fferences in their actions when voltage protocols are altered.

53

3.3.

Calcium channel blockage: voltage-dependent inhibition Initial voltage-clamp experiments suggested that verapamil, but not nifedipine blocks calcium channel current in a use-dependent manner (25). Trautwein and McDonald (24) and co-workers have carefully investigated voltage-dependent modulation of D600-induced calcium Comparison of their results with the current inhibition. results of similar experiments with dihydropyridines shows that calcium channel blockage by the latter group of drugs may be more complicated than the earlier experiments suggested. A

-IO[ Jl nTl:JL 55

(1

30 MIN RES'!

m\l

-50

(40

_

2SOHSfI

30MIN REST



flTlM

_xN

DRuG

-r-r -r ~:~ 20HIN-1

B

01

-r;, 'pA[' SS

(4'

ss

C

=

0.6

~

~A

0.4

0600

,........-'-!-~~IO~-'-'-:';-'S PULSE NUMBER INI

..

3O;;;in

AeA

0600

Figure 4. Influence of repeti ti ve depolarization on Ca current block by D600 and AQA 39. A: Voltage clamp pulse protocol. AQA 39 (2 x 10- 5 M) or D600 (2 x 10-6 M) was present during the second 30 min rest period (no stimulation). Pulsing was then resumed at 20 min-I. B: Curre~t records. lsi was not blocked during first pulse after 30 min rest in presence of drug. C: Usedependent block of lsi (stimulation rate 20 min-I) by AQA 39 and D600 after 30 min rest. Data from Trautwein et al. (1983).

Some of the key findings of McDonald et al. (26) and Pelzer et al. (27) (also see ref. 24) are summarized in Figure 4 which compares the influence of membrane potential on two drugs: D600 and a related compound AQA 39. Voltage clamp protocols (Panel A) and the resulting currents (Panel B) show the effects of imposing stimulusfree periods of rest between trains of depolarizing voltage pulses in the absence and presence of these drugs. It is very important to notice that the

54

depolarizing pulses are applied from a-50 mV holding potential at a rate of 20 min-I. Small changes in the measured inward current are apparent in the absence eC l ) and presence (ss) of steady trains of depolarizing pulses even when no drug is present. These small changes are expected because of slow inactivation of cardiac calcium channels (3). However, what is striking in this figure is that, although D600 is aplied for 30 min, because the fiber is not being depolarized during this period, little or no inhibi tion of calcium channel current is measured until the train of voltage depolarizations is resumed. Instead, there is a pulse-by-pulse decrease in calcium channel current after the train of pulses is reimposed (Panel C), with near maximal block being attained by the 15th pulse of the train. As shown in the figure, under these condi tions, AQA-39 causes very similar effects. Similar observations of use-dependent calcium channel blockage by D600 (or verapamil) have been observed in Purkinje fibers (13) and in isolated ventricular muscle cells (28). The results of these, and other experiments with D600, have suggested that calcium channels must first be opened (upon depolarization) and then inactivated (during the depolarizing pulses) before D600 and verapamil block calcium channels (29). In other words, block of the channel depends on the state of the channel which is modulated by membrane potential. In experiments designed to study the actions of dihydropyridines (nitrendipine, nisoldipine, nifedipine) and compare them to D600 and verapamil, li ttle evidence for use-dependent effects has been found (28,13). Figure 5 is an example that shows marked differences between the development of calcium current blockage by nisoldipine and D600 when a train of voltage pulses is applied from an elevated holding potential following a 2 min stimulationfree period at a negative holding potential. However, in this experiment a brief (4 sec), but crucial, pause was

55

FRACTION OF I" BLOCKED PER PULSE

456

9

10

PULSE NUMBER NISOlOIPINE: 10~M (6.)

.05j.lM (0) .02,.'-" (0)

P,

P,!)

~J"L VR

Influence of membrane potential on block of Ca current by D600 and nisoldipine. Membrane potential held at VR (-75 mY) without stimulation for 2 min. Holding potential was then changed to VH (-44 mY) for 4 sec before applying a train of pulses (to -5 mY) at 0.67 Hz. Usedependent block of lsi at VH is expressed as fraction of lsi blocked during each pulse of the train. Data from Kass (1982).

Figure 5.

imposed at the elevated holding potential before starting the train of depolarizing voltage pulses. Thus, from this experiment, although D600 and nisoldipine show marked differences, it is not clear if voltage modulated the block of calcium channels by nisoldipine. This question is addressed more completely in the experiment shown in Figure 6 in which voltage protocols (insets) were imposed to test for specific effects of membrane potential on calcium channel blockage by nisoldipine. As in the previous experiments, the preparation was exposed to the drug (200 nM) and calcium channel blockage was assessed by measuring drug-induced changes in current during repetitive application of voltage pulses from a depolarized holding potential. After attaining steady-state block of the current, the pulses were interrupted and the membrane was held at -70 mV for 2 min.

56

Three experiments were carried out. In the first experiment, the holding potential was changed from -70 mV to -45 mV and a train of depolarizing pulses was applied. In order to compare this wi th other similar experiments, the amount of ca1ci urn channel current blocked was determined during each depolarizing pulse (13) and plotted against the time after change in holding potential. Initially, almost none of the current is blocked, but after applying a train of brief (20 ms) pulses from the depolarized holding potential for 30 sec, approximately 60% of calcium channel current is blocked (!). If the duration of each voltage pulses is increased tenfold but the interval between pulses is held constant, the fraction of current blocked during each pulse increases by about 25% (.). In either case, a clear change occurs in the amount of current blocked by nisoldipine after the holding potential is changed. These changes can be resolved here because, unlike the experiment shown in Figure 5, only 10 msec elapsed at the -45 mV holding potential before the first test pulse of the train was imposed. Is the resumption of repetitive electrical activity necessary to enhance calcium channel blockage by nisoldipine as it is for block of the channel by D600, or can channel blockage be modulated by the change in holding potential without repetitive stimulation? In order to test for the latter possibility, the third experimental protocol was carried out as illustrated in Figure 6 (D). In this experiment the holding potential was again stepped from -70 mV to -45 mY. Then after an interval (t) at the more depolarized holding potential only one test pulse was applied to assay inhibition of current. The holding potential was reset to -70 mV for 2 min, and then returned to -45 mV where another test pulse was imposed after a different delay (t). This cycle was repeated several times. The fraction of current blocked during each test pulse was again determined and plotted (against time at the -45 mV holding potential, 0 ). The change in calcium

57

-~.Jl

-70

;]WLll .

-45 -70

0.8

0.6

..

0.4

0.2

.

.

q

0

t

.,--

20 rnsec pulse • 200 msec pulse

.. . . •

..

Figure 6. Vol tagedependent block of calcium channel current by nisoldipine (200 nM): influence of holding potential. Membrane potential was held at -70 mV for 2 min and then changed to -45 mV . Fraction of inward current blocked measured during 20 msec ( ... ) and 200 ms (.) pulses applied at .5 Hz plotted against time at -45 mV holding potential (inset shows pulse protocol). Fraction of inward current blocked measured during single test pulses after variable periods (t) at -45 mV holding potential (0).

channel blockage by nisoldipine measured using this protocol was almost the same as that observed with the repetitive pulse procedure. Thus, changing the resting potential from -70 mV to -45 mV in the absence of stimulation markedly increases the effectiveness of this dihydropyridine compound as a calcium channel blocker. This result clearly contrasts with observations of voltage-dependent modulation of D600 block of calcium channels which show that inhibition of current is increased only if trains of depolarizing pulses accompany changes in holding potential (see Fig. 4). 3.4

The contrast between actions of D600 and nisoldipine: significance Calcium channel blockage by both D600 and nisoldipine is affected by changes in membrane potential, but the response of each drug depends critically on the values of the imposed voltage change and thus, probably on the state of the calcium channel. In the case of D600, voltage pulses must be applied to potentials positive enough to

58

open calcium channels before block develops (24). This accounts for the observation of use-dependent changes in block by this compound, and is consistent with the interpretation that calcium channels must first be opened before D600 can block them (24). On the other hand, considerable modulation of calcium current inhibition by nisoldipine / is observed when membrane potential is changed from -70 mY to -45 mY even in the absence of trains of depolarizing pulses. This voltage range is too negative to open the calcium channels (4), and so membrane potential must be affecting the nisoldipine drug-channel interaction in a different manner. One interesting possibili ty that is consistent with the modulated receptor hypothesis (17) is that nisoldipine shifts the apparent steady-state calcium channel inactivation curve (30) in the hyperpolarizing direction. Such an effect would strongly suggest that nisoldipine preferentially blocks channels that are in the inactivated state. This possibility remains to be tested. What are the important implications of these findings? First of all, because membrane potential markedly influences the fraction of calcium channel current blocked by a given nisoldipine concentration, concentration-response relationships for this drug must be expressed relative to membrane potential. This is particularly important when comparisons are made between binding data that may be obtained in chronically electrophysiological preparations and depolarized information obtained from preparations partially or fully polarized. This effect may underlie the present discrepancy between binding and pharmacological studies of dihydropyridines in heart cells (10). suggest experimental results very basic These predictions about the clinical usefulness of these drugs. For example drugs such as verapamil, D600 and dil tiazem that are characterized by pronounced use-dependent effects ought to be effective as antiarrhythmic agents against

59

disturbances due to repetitive firing of calcium dependent action potentials. There same disturbances should be relatively insensitive to compounds like nisoldipine that display little use-dependence. The clinical data support thi s view because verapamil and dil t iazem (31), but not nifedipine (32), have marked antiarrhythmic actions. Compounds like nisoldipine, very sensitive to changes in holding potential, but showing little use-dependence should be more effective than D600 (and related drugs) at blocking calcium channels in cells that are chronically depolarized. Because vascular smooth muscle is characterized by rather positive resting potentials (-60 mV), it is not surprising that the dihydropydidines are more potent vasodilators than verapamil and are thus the drugs of choice in the treatment of angina (33) and hypertension (34). In conclusion, it now appears that membrane potential plays a very important role in the blockage of calcium channels by the organic compounds known as the calcium channel antagonists. Perhaps future investigations will eventually show that all of these drugs interact with a common voltage-sensitive structure such as the inactivation gating particle. If so, the drug-channel interactions may yield important information about the structure and regulation of the calcium channel itself.

ACKNOWLEDGEMENT This work was supported by USPHS grants #HL 21922 and HL 00556. We thank Dr. Alexander Scriabine (Miles Laboratories) for supplying us with nisoldipine and for supporting this work. We thank Ms. Karen Vogt for her excellent help in preparing this manuscript.

60

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11.

12.

13. 14.

15.

Hagiwara S, Byerly L: Calcium channel. Ann Rev Neurosci (4): 69-125, 1981. Tsien RW: 1983. Calcium channels in excitable cell membranes. Ann Rev Physiol (45): 341-358, 1983. Kass RS, Scheuer T: Slow inactivation of calcium channels in the cardiac Purkinj e fiber. J Molec Cell Cardiol (14):1615-618,1982. Reuter H: 1979. Properties of two inward membrane currents in the heart. Ann Rev Physiol (41): 413424, 1979. Reuter H: Divalent cations as charge carriers in excitable membranes. Prog Biophys Mol BioI (26): 143, 1973. Fozzard HA: Heart: Excitation-contraction coupling. Ann Rev Physiol (39): 201-220, 1977. Flaim SF, Zelis R: Calcium Blockers, Mechanisms of Action and Clinial Implications. Urban and Schwarzenburg, Baltimore, Munich, 1982, p. 303. Singh BN, Nademanee K, Baky SH: Calcium antagonists. Clinical use in the treatment of arrhythmias. Drugs (25): 125-153, 1983. Schwartz A, Taira N: Symposium on calcium channel blocking drugs: A novel intervention for the treatment of cardiac disease. Circ Res (52) (Part II, Suppl.): 1983, 181 pp. Janis RA, Triggle DJ: New Developments in Ca 2 + channel antagonists. J Med Chern (26): 775-785, 1983. Kass RS, Scheuer T, Malloy KJ: Block of outward current in cardiac Purkinje fibers by injection of quaternary ammonium ions. J Gen Physiol (79): 10411063, 1982. Kass RS, Siegelbaum SA, Tsien RW: Three-microelectrode voltage clamp experiments in calf cardiac Purkinje fibers: Is slow inward current adequately measured? J Physiol (290): 201-225, 1979. Kass RS: Niso1dipine: a new, more selective calcium current blocker in cardiac Purkinj e fibers. J Pharm Exp Ther (223): 446-456, 1982. Kass RS: Measurement and block of voltage-dependent calcium current in the heart. In: Merrill, GF, Weiss HR (eds) Ca 2 + entry blockers, adenosine, and neurohumors. Urban and Schwarzenberg, Baltimore, 1983. Bayer R, Hennekes R, Kaufman R, Mannhold R: Inotropic and e1ectrophysiological actions of verapami1 and D600 in mammalian myocardium. I. Pattern of inotropic effects of the racemic compounds. Naunyn-Schmiedeberg's Arch Pharmaco1 (290): 49-68, 1975.

61

16.

17. 18.

19. 20.

21.

22. 23.

24.

25. 26. 27.

28.

29.

30.

Bayer R, Kaufmann R, Mannhold R, Rodenkirchen R: The action of specific Ca antagonists on cardiac electrical activity. Prog Pharmacol (5): 53-85, 1982. Hille B: Local anesthetics: Hydrophilic and hydrophobic pathways for the drug-receptor reaction. J Gen Physiol (69): 497-515, 1977. Hondeghem LM, Katzung BG: Timeand voltagedependent interactions of antiarrhythmic drugs with cardiac sodium channels. Biochim Biophys Acta (472): 373-398, 1977. Bean BP, Cohen CJ, Tsien RW: Lidocaine block of cardiac sodium channels. J Gen Physiol (81): 613642, 1983. Rodenkirchen R, Bayer R, Mannhold R: Specific and nonspecific Ca antagonists. A structure-activity analysis of cardiodepressi ve drugs. Prog. Pharmacol (5): 9-23, 1982. Marban E, Tsien RW: Effects of nystatin-mediated intracellular ion substitution on membrane curents in calf Purkinje fibres. J Physiol (329): 569-587, 1982. Colatsky TJ: Voltage clamp measurements of sodium channel properties in rabbit cardiac Purkinje fibres. J Physiol (305): 215-234, 1980. Kass RS, Wiegers SE: Ionic basis of concentrationrelated effects of noradrenline on the action potential of cardiac Purkinje fibres. J Physiol (322): 541-558, 1982. Trautwein W, Pelzer D, McDonald TF: Interval-and voltage-dependent effects of the calcium channelblocking agents D600 and AQA 39 on mammalian ventricular muscle. Circ Res (52)(Part II, Suppl.): 160-168, 1983. Bayer R, Ehara T: Comparative studies on calcium antagonists. Prog. Pharmacol. (2): 31-37, 1978. McDonald TF, Pelzer D, Trautwein W: On the mechanism of slow calcium channel block in heart. Pflugers Arch (385): 175-179, 1980. Pelzer D, Trautwein W, McDonald TF: Calcium channel block and recovery from block in mammalian ventricular muscle treated with organic channel inhibitors. Pflugers Arch (394): 97-105, 1982. Lee KS, Tsien RW: Mechanism of calcium channel block by verapamil, D600, diltiazem, and nitrendipine in single dialyzed heart cells. Nature (302): 790-794, 1983. Kanaya S, Arlock P, Katzung B, Hondeghem LM: Diltiazem and verapamil preferentially block inactivated cardiac calcium channels. J Molec Cell Cardiol (15): 145-148, 1983. Kass RS, Sanguinetti MC: Calcium channel inactivation in the cardiac Purkinje fibre: evidence for voltage- and calcium-mediated mechanisms. 1983. In Preparation.

62

31.

32. 33.

34.

Rowland E, McKenna WJ, Gulker H, Krikler DM: The comparative effects of diltiazem and verapamil on atrioventricular conduction and atrioventricular reentry tachycardia. Circ Res (52) (Part II, Suppl.): 1163-1168, 1983. Rowland E, Curry P, Fox K, Krikler DM: Relation between atrioventricular pathways and ventricular response during atrial fibrillation and flutter. Br Heart J (45): 83-87, 1981. DePonti C, Mauri F, Ciliberto GR, Caru B: Comparative effects of nifedipine, verapamil, isosorbide dinitrate and propranolol on exerCIse induced angina pectoris. Eur J Cardiol (10): 47-58, 1979. Klein W, Brandt D, Vrecko K, Harringer M: Role of calcium antagonists in the treatment of essential hypertension. Circ Res 52 (Part II, Suppl.): Il74Il8l, 1983.

5 MECHANISMS OF SELECTIVE

CA ANTAGONIST-INDUCED VASODILATION

Co VAN BREEMEN, Co CAUVIN, 00 HWANG, Po LEYTEN, So LUKEMAN, Ko l1EISHERI, Ko SAIDA AND Ho YAHAMOTO

Vascular smooth muscle is the primary target organ for Ca antagonist (CAt) therapy

0

It is now well established that CAts bind to

and block excitable channels in the cell membrane (1)0

The question

which remains to be answered is why are these agents selective for certain blood vessels and certain modes of activation.

Our presentat.ion

addresses this question and is divided into the following three topics: 1)

A brief discussion of the evidence for the basic mechanisms of CAt

action; nanely blockade of Ca 2)

2+

.

entry through excltable Ca

2+

Evidence supporting the hypothesis that the vascular smooth muscle

plasma membranes possess at least two different types of Ca potential 3)

channels.

2+

channels,

dependent channels (PDC) and receptor operated channels (ROC ) 2'

s

The influence of receptor mediated release of intracellular Ca -'on

arterial sensitivity to CAtso The data presented here

or~ginates

from work done in our laboratory

on rabbit aorta and mesenteric arterieso employed are discussed in detail elsewhere

The experimental techniques (2,3) and will not be covered

here. i'lechanisms of Ac tion The two experimental models of in vitro arterial smooth muscle excitation used most frequently are membrane depolarization by raising the external K+ concentration with a corresponding reduction in [ Na ]

e

(high K) and application of norepinephrine to the bathing solution (NE). These procedures are thought to represent some approximation of physiological excitation by depolarization and neurotransmitters o In the rabbit aorta the most striking difference between high K and 2-'-

1lE

is that the

former stimulates only Ca ' entry·while the latter stimulates both Ca 2-'-

2+

entry and intracellular Ca ' release. Thus an agent which interferes with smooth reuscle excitation

contraction coupling solely by blocking

64 stimulated Ca 2+ entry should cause closely related inhibitions of high K stimulated 45 Ca influx and contraction while being reletively less effective

in blocking NE,induced contraction. Figure 1 illustrates that 2+ this pattern holds true for the Ca antagonists D600 and diltiazem and the same hasbeen found for the dihydropyridine nisoldipine.

45Ca INFLUX CONTRACTION

;,/f; l

+ LOW HIGH

80 K

A

z

o IIn

/,

50

o I i l.''

:r: Z

~

I

~~

/./

NE

80 K+

NE

I

*-/

-7

-6

-5

j*

*

/"

:to

-4

HIGH NE

Ii'"

*'

0

......

.....

~~W

./ )1 I Ii./ I

0

/'



/,0' /

I

B

I If *1,/ ........... -8

.--.

OILTIAZEM

0600

100

*-*

-8

-7

-6

*

*

,

'

/ .' * _0" '"

-5

-4

-3

LOG [CATl, M

Figure 1. Ca 2+ antagonist m!f:diated inhibition of 45Ca influx and contraction stimulated by SOw! K , 10- 7 norepinephrine, and 10- 5 norepinephrine in isolated rings o£ rahbit aorta A. D600 B. diltiazem. CAts also inhibit spontaneous smooth muscle action potentials in veins (4) and those induced in arterial smooth muscle by the K+ channel blocker tetraethyl ammonium (5). Since these action potentials rca 2j' their hlockade by e 2+ CAts is further evidence for the CAt induced blockade of Ca channels 2 which are opened by depolarization. The Ca + ions which enter the are tetrodotxin insensitive and dependent on

cells under conditions when there is neither depolarization nor receptor activation is totally insensi,tive to CAts although it is inhihited by

65 This indicates that the inherent Ca 2+ leak in the smooth plasmalemma does not occur through excitable ca 2+ channels. At thera-

Lanthanum.

peutically relevant concentrations dihydropyridines, verapamil and its analogues and diltiazem have been shown to have no direct effects on 2+. 2+ myofilament activation by Ca nor on lutracellular Ca release (6). POTENTIAL DEPENDEN'l CA CHANNELS AND RECEPTOR OPERATED CA CHANNELS Edman and Schild originally demonstrated that acetylcholine could cause a sustained contraction in a completely depolarized (high K) smooth muscle (7).

In 1964 Su and Bevan (8) reported an arterial

contraction initiated through nerve stimul

0.5

~

« .-J

w

0:::

0.25

0

0.25 0.5 0.75 10 FRACTION OF CHANNELS OPENED (d oo )

FIGURE 6. Evidence suggesting that the degree of Ca channel block by 0600 is related to the fraction of Ca channels opened on the conditioning pulses. The graph is a plot of relative channel block versus the fraction of channels opened (d oo ) . In the experimental procedure, unblock (90 sec at -90 mY) was followed by fifteen 30 msec depolarizations from -50 mY, and a 300 msec test step to 0 mY. The fractional number of channels opened varied with the amplitude (5 to 100 mY) of the 30 msec depolarizations (see ref. 17) and the relative block was estimated by measuring I . amplitudes evoked by the test pulse. The filled circles are v~iues obtained from 4 muscles while the straight line is a reference line indicating a strict correlation between block and d 00.

We next investigated whether block was related directly to the duration of a conditioning pulse, or indirectly in a way consistent with the availability of open Ca channels. exposed to 2

~M

A muscle

0600 for 110 min was blocked with regular

stimulation from -50 to 0 mY, and then unblocked by a 90 sec hyperpolarization to -90 mY.

Ten seconds later, a conditioning

pulse was applied from -50 to 0 mY.

The conditioning pulse had

a duration ranging from 30 to 2400 msec and, as indicated by the schematic of Fig. 7, it was followed by a standard test pulse to 0 mY.

130 Block

o[ j l

mV

-50

300 msec 0.33 Hz

- nx1-n Conditioning

Test

-t-

1.0

:.:::

u 0

-l

co L L

0.75

::>

X

otent; nifedipine and mesudipine had about the same potency. The data with mesudipine are summarized in Table lB.

TABLE 1. Summary of effects of mesudipine on the fast and slow action

potentials of guinea pig papillary muscles

Mesudipine concentration (M)

AP [Cal 0 amplitude (mM) (mY)

0

Relative contraction

Vm x +(Vis

APD50 (ms)

+ + + + :£

122 + 5.1 108 + 5.9 100 + 5.1 94 +7.9 78:£ 5.6

100 68 52 30 0

100 92 58

(%)

A. Fast APs O· x 10-7 x 10-6 x 10- 6 x 10-5

1.8 1.8 1.8 1.8 1.8

121 + 1.0 117+3.1 120 + 1.8 117 + 3.4 121:£ 2.1

0 1 x 10-8 4 x 10- 8 1 x 10- 7

1.8 1.8 1.8 1.8

75 + 1.9 74 + 4.0 69:; 2.6 0

22 + 2.3 18 + 2.5 12 :£2.1 0

95 + 2.4

0 1 x 10-7 1 x 10-6 4 x 10-6

5.4 5.4 5.4 5.4

86 + 1.6 83:; 3.2 67:£ 4.4 0

43 + 8.6 32:; 5.9 7 £'1.1 0

88 + 3.6 69:; 2.4 42:; 4.9 0

1 1 5 1

250 191 238 223 225

18.0 21.6 26.6 41.0 25.4

B. Slow APs

~~ :£ 1.~ 10 T

2.u

69

Data given as means .:t. SE. N was 9-15. J?rive rate was 0.5 Hz (30/min) throughout. Abbreviations: AP, action potential; +Vmax , maximum rate of rise of action potential; APD, action potential duration.

158

GUINEA PIG PAPILLARY MUSCLE Slow ",Uon PotenllMe (In 25 .......·.10... " IN ) CONTROL

VERAPAIilIL

,illil

til III

•• . , .

iT=~tt_~~ 0.'_

-L- .J_ . ...,-- -"-- ~_.II""

__

••

ItOw.

H

--"'---_

_~_--' -

",-I

----

Drive rate ; 0.5 Hz

Fig. 3: Verapamil, nifedipine, and mesudipine blockade of the slow action potentials (APs) of guinea pig papillary muscle. A: Control slow AP induced by 10-6 M isoproterenol in 25mM [Kl o' B-D: Addition of verapamill x 10- 7 M (B), 10-6 M (C), and 5 x 10- 6 M (D) depressed and blocked the slow APs within 4 min. E: Control slow AP. F-H: Addition of nifedipine 1 x 10-8 M (F) and 1O-7M (G & H) depressed and blocked the slow APs within 8 min. I: Control slow AP. J-L: Addition of mesudipine 1 x 10-8 M (J), 4 x 10-8 (K), and 1 x 10- 7 (L) depressed and blocked the slow APs within 10 min. M: Washout of mesudipine for 15 min restored the slow APs. The drive rate was 0.5Hz. The upper solid line gives the z~ro potential level. The lower trace is dV Idt, the peak excursion of which gives +Vmax ' Each row presents records taken from the same impalement. (b) Antagonism by elevated [Cal o' After the blockade of the slow APs by a calcium antagonistic drug, elevation of [Cal IB). That is, high [Cal

0

0

reverses the effects (Table

shifts the dose-response curve to the right (Table IB).

This antagonism of the drug effect by Ca++ may be explained by two possibilities: (1) the increase in electrochemical driving force (Em-ECa) for an inward Ca++

current through unblocked slow channels would increase lsi and +Vmax of the APi

159

(2) competition between drug and Ca++ ions for the same binding site on or near the slow channel.

Consistent with this possibility, Pang and Sperelakis (1982)

\"eported that verapamil and bepridil (but not nifedipine and diltiazem) depress the binding of Ca++ ions to isolated, sarcolemmal membranes in a dose-oependent manner, verapamil being the more potent. (c) Frequency dependency. The effect of mesudipine on the slow APs was partly dependent on the stimulation frequency. In Figure 4, the amplitude +Vmax of the slow APs in the presence of 10- 7 M mesudipine, normalized as

and

percent of the control values, are plotted against the stimulation frequency, to illustrate the frequency (use-) dependency. At a drive rate of 1/min, 10-7 M mesudipine reduces Vmax to only 57%, whereas complete block occurs at a drive rate of 24/min. Thus, mesudipine-blocked slow APs recovered when the stimulation

GUINEA PIG PAPILLARY MUSCLE 100

....

'0 c

-8

Mesudipine 3x 10M

-... o o o

..

C GI

C)

GI

0.

11/

III

11/

GI

::J

iii

>

GI

>

:;:;

III

'i p:

4

Drive rate: stlm/mln

Fig. 4: Dependence on the stimulation frequency of the mesudipine (10- 7 M) effect on the slow action potentials (APs) of guinea pig papillary muscle. Slow APs were induced by 10-6 M isopro-terenol in 25 mM K+. Parameters of the slow APs plotteq are amplitude (circles) and maximum rate of rise (+Vmax) (squares). The ordinate gives the relative values of these two parameters as a percent of the control value before drug addition. Lowering of the stimulation frequency from the control driving rate of 24/min to l/min restored tt}e AP amplitude to 88% and +Vmax to 57%. Data plotted are the mean values (+ SE) from 8 experiments. -

160

frequency was lowered.

With longer exposure periods, the blocking effect of

mesudipine was completely independent of frequency. This frequency-dependency.(or use-dependency) of the drug action, which has been described also for verapamil and bepridil (Vogel et

!!!.,

1979; Sperelakis,

1981), but not for nifedipine (Kohlhardt and Fleckenstein, 1977), seems to be a common property of Ca++ slow channel blockers, but the degree of frequency dependency varies for the different drugs.

This phenomenon might reflect

increase in the recovery time required for the slow channels in the presence of the drug (Kohlhardt et al., 1975), and the degree of inhibition of the recovery process may be different among the various Ca antagonists (Kohlhardt and Fleckenstein, 1977). One possibility is that the drugs may bind to the inactivated form (j.e.,

A~ate

open and

I~ate

closed) of the slow channels to slow recovery

(Kanaya et al., 1982; Pelzer et al., 1982). 3.

Contractions. The contractions accompanying the fast or the slow APs are also

blocked by the Ca antagonistic drugs. Figure 5 illustrates the contractions in the presence of Normal Tyrode solution (upper row) and in high [Kl 0 plus 10-6 M isoproterenol (lower row).

The contractions accompanying the slow APs (lower

row of Fig. 5) are depressed by mesudipine in a parallel course with the depression of the slow APs (Fig. 3). However, in the case of the fast APs (upper row of Fig. 5), the concentration of mesudipine required to block the contractions was much higher (10- 5 M). Even at these high concentrations, the amplitude and +Vmax of the fast APs were not significantly affected. Thus, excitation-contraction uncoupling is produced by the drug. The depression of contractions was reversed when [Cal 5).

0

was elevated (Fig.

The recovery of contractions in the case of the slow APs was almost

complete, but was only partial in the case of the fast APs. This difference may be due to the different concentrations of the calcium antagonists required to block the contractions: 10-7 M for the slow APs, and 10-5 M for the fast APs. The requirement of a higher mesudipine concentration (10- 5 M) to block the contractions in normal Ringer solution (fast APs) compared to that (10- 7 M) in high [K]

0

solution (slow APs), is in agreement with the findings of Nabata (1977)

for verapamil, nifedipine, and diltiazem. A possible explanation of the different concentrations needed to block contractions in normal [K]

0

and in K+-

depolarized hearts could be the following. In a partially depolarized membrane, a critical mass of slow channels is needed to propagate the slow AP (which allows

161 the necessary Ca++ entry and initiates contraction), whereas in a preparation perfused with normal Ringer solution, the fast AP is propagated utilizing fast Na+ channels (which are unaffected by mesudipine).

As a result of this, a lower

concentration of the Ca++ antagonist is sufficient to block the contractions accompanying the slow APs.

That is, failure of the slow APs, and hence the

contractions, will occur at lower'drug concentrations, even though the fraction of the slow channels blocked should be the same in both cases.

GUINEA PIG PAPILLARY MUSCLE Contractions In Normal Ringer Solution Control

Mesudipine

Ca" 5.4

I ... - .....

Ca" 8.0 mM

A,

B. 107 M C. 10.6 M D. 5X106M E.10S M F.10 fi M G. H;5 M

I! I

.111111111111111111111111111111.

.

..

.

II1II

,

Contractions In 25mM it-Ringer Solutlon+lsoproterenol (106 M) Control Mesudipine Ca" 5.4 mM H. I. 168 M J. 41110 8 M K.10· 7 M L. 10'7 M

' . llllillWllIl\11 Ullluilll\U'UI

t

--24 sec

Fig. 5: Effect of mesudipine on the force of contraction of guinea pig papillary muscle. Upper row: Experiment in normal (4.7 mM K+) Ringer solution. A: Control contractions recorded on a pen writer after the preparation was equilibrated in normal Ringer solution for 20 min. B-E: In the presence of 10-7 (B), 10- 6 (C), S x 10-6 (D) and 10- SM (E) mesudipine. Complete block of contractions occurred in 10-5M mesudipine. F-G: Elevation of [Ca] 0 from 1.8 mM to 5.4 mM (F) and 8.0 mM (G) in the presence of 10-SM mesudipine partially restored contractions. Lower row: Experiment in high [K] 0 (25 mM) solution containing 10- 6 M isoproterenol. H: Control contractions. I-K: In the presence of 10-8 (I), 4 x 10-8 (J), and 10-7M (K) mesudipine. L: Elevation of [Ca] 0 from 1.8 mM to 5.4 mM restored contractions in 10- 7 M mesudipine. Drive rate was O.S Hz (30/min). All records illustrated are after 10 min equilibration in each drug concentration; steady-state contractions occurred within 3-7 min.

162 In cardiac cells, net Ca++ influx acts to raise the internal Ca++ concentra-

tion ([ Cal i) and to trigger Ca++ release from the sarcoplasmic reticulum (SR) to contraction.

~nitiate

The AP depolarization of the surface membrane and T-

tubules may also lead to depolarization of the SR, and thereby trigger Ca++ release from the SR (Endo, 1977).

Ca antagonistic drugs should not block this

mechanism for the contraction aecompanying the fast APs, unless we assume that the drug enters the cell and also blocks the release of Ca++ from the SR. This actually has been suggested for one site of action of bepridil (Vogel et

!!l.,

1979).

Bepridil and verapamil readily enter myocardial cells, whereas nifedipine and diltiazem do not (Pang & Sperelakis, 1983). B.

Purkinje Fibers

1.

Fast APs.

The action of mesudipine on the normal fast action

potentials (APs) of isolated guinea pig Purkinje fibers, perfused with normal Ringer solution, is summarized in Table 2.

As shown in Fig. 6, mesudipine, in

concentrations between 10- 7 M and 10- 6 M, had no significant effect on the amplitude and maximum upstroke velocity (+Vmax ) of the fast APsj the AP duration at 90% of repolarization (APD90) was not affected, but the APD50 was slightly depressed (Fig. 6C compared to A). However, elevation of the mesudipine concentration to 5 x 10-6 M abolished excitability within 4 min (D). The cell stopped firing and remained depolarized at about -45 mV; the cells were inexcitable despite intense electrical stimulation.

Elevation of the Ca++

concentration to 5.4 mM, and/or addition of 10-6M isoproterenol, did not restore excitability. ~econd

It is possible, that this depolarization, to what appeared to be a

stable state, i.e., a second resting potential (Wiggins and Crane field, 1976;

Sperelakis, 1978), is due to a lowered K+ conductance (gK) produced by the drug (Kass & Tsien, 1975; Lee & Tsien, 1982, 1983). Washout of the drug restored large APs within 3 min (E), but the APD50 remained somewhat depressed.

The block

and subsequent recovery upon washout are illustrated at slower speed in Fig. 6 FH. Thus, the recovery was very fast but partial; full recovery occurred after 1-2

hr. 2.

Slow Action Potentials. (a) Blockade of slow channels. The actions of verapamil, nifedipine,

and mesudipine on slow APs were studied in Purkinje preparations, depolarized with 20 mM [Kl

0

to -45 mV to inactivate the fast Na+ channels (Fig. 7B). These

cells were inexcitable despite intense electric field stimulation.

However,

addition of 10- 6 M isoproterenol to the perfusate allowed slow-rising overshooting APs to be elicited by electrical stimUlation (Fig. 7C).

163

GUINEA PIG PURKINJE FIBERS Normal Fast Action Potentials

CONTROL

MESUDIPINE

1C? M

-6

WASHOUT

-6

5x10 M

10 M

-"~

c

I -6

MESUDIPINE 5'10 M

WASHOUT

4 min

......-----.

0.2 sec

3 min F

G

t

H

[0 mV I------
-------