Calcium Regulation by Calcium Antagonists 9780841207448, 9780841209688, 0-8412-0744-5

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

Calcium Regulation by Calcium Antagonists

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

Calcium Regulation by Calcium Antagonists Ralf G. Rahwan, EDITOR Donald T. Witiak, EDITOR

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

The Ohio State University

Based on a symposium sponsored by the A C S Division of Medicinal Chemistry at the 182nd Meeting of the American Chemical Society, New York, New York, August 23-28, 1981.

ACS SYMPOSIUM SERIES

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1982

201

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

Library of Congress Cataloging in Publication Data Calcium regulation by calcium antagonists. (ACS symposium series, ISSN 0097-6156; 201) "Based on a symposium sponsored by the ACS Division of Medicinal Chemistry at the 182nd Meet­ ing of the American Chemical Society, New York, New York, August 23-28, 1981." Includes bibliographies and index. 1. Calcium—Physiological effect—Congresses. 2. Calcium—Antagonists—Congresses. I. Rahwan, Ralf G., 1941- . II. Witiak, Don­ ald T. III. American Chemical Society. Division of Medicinal Chemistry. IV. American Chemical Society. National Meeting (182nd: 1981: New York, N.Y.) V. Series. [DNLM: 1. Calcium—Metabolism—Con­ gresses. 2. Calcium—Antagonists and inhibitors— Congresses. 3. Ion channels—Metabolism—Congresses. 4. Calcium antagonists, Exogenous—Metabolism— Congresses. QV 276 C1443] QP535.C2C265 1982 612'.01524 82-16451 ISBN 0-8412-0744-5 ACSMC8 201 1-208 1982 Copyright © 1982 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of thefirstpage of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of thefirstpage of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, repro­ duce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATESOFAMERICA

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

ACS Symposium Series M.

Joan Comstock,

Series Editor

Advisory Board David L. Allara

Marvin Margoshes

Robert Baker

Robert Ory

Donald D . Dollberg

Leon Petrakis

Robert E. Feeney

Theodore Provder

Brian M . Harney

Charles N . Satterfield

W . Jeffrey Howe

Dennis Schuetzle

James D . Idol, Jr.

Davis L. Temple, Jr.

Herbert D . Kaesz

Gunter Zweig

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.fw001

FOREWORD The ACS S Y M P O S I U M SERIES was founded in 1974 to provide a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing A D V A N C E S I N C H E M I S T R Y SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.pr001

PREFACE ^ L L T H O U G H T H I S B O O K W A S C O N C E I V E D as a result of a Symposium entitled "Calcium Regulation and Drug Design," the topics presented at the Symposium have undergone revision, expansion, and updating, and additional chapters from invited authors have been incorporated into the present volume. This book, therefore, represents a collage of various topics involving the roles of calcium and calcium antagonists in health and disease. Unlike other excellent recent books on calcium antagonists, the present volume does not focus exclusively on the cardiovascular system, but covers other systems as well. The emphasis is on basic science, although clinical correlates are included wherever applicable. Numerous authorities in the field have contributed their expertise in their respective areas to the compilation of this book.

The opening chapter by Dr. Nayler attempts to create order from chaos by presenting a rational classification of membrane calcium channel blockers based upon their pharmacological effects on the cardiovascular system. Dr. Triggle then provides an overview of structure-activity studies with the calcium channel blockers, and emphasizes the chemical and pharmacological heterogeneity of this class of compounds. The latter fact provides a strong impetus for the adoption of the pharmacological classification of calcium channel blockers proposed by Dr. Nayler. A comprehensive review of the cardiovascular electrophysiological and hemodynamic effects of the calcium channel blockers is presented by Dr. Muir. Dr. Daniel and his colleagues provide insight into the role of calcium in the regulation of vascular and nonvascular smooth muscle contractility, and outline the methodological approaches for studying calcium fluxes, sequestration, and mobilization in smooth muscle. While the first four chapters focus on the membrane calcium channels, the next two explore the intracellular compartment. Dr. Brostrom and his colleagues review present-day knowledge about the intracellular calcium receptor, calmodulin, while the Editors contribute an update on the basic and applied pharmacology of a novel class of intracellular calcium antagonists, the methylenedioxyindenes. The roles of calcium and calcium antagonists in the central nervous system are dealt with in the next two chapters. From Dr. Leong Way's laboratory comes an overview of the mechanism of interaction between calcium and opioid alkaloids and peptides, while Dr. Ferrendelli reviews ix

the evidence for an inhibitory effect of certain antiepileptic drugs on neuronal calcium conductance. Dr. Le Breton and his colleagues review the relationship of cyclic nucleotides and calcium in platelet function, while novel information on the salutary effects of the calcium antagonist nifedipine in atherosclerosis is presented from Dr. Henry's laboratory. Dr. Borowitz and coworkers review the role of calcium and the modulating influence of calcium antagonists on secretory systems. We wish to acknowledge with gratitude the cooperation of the participants in the Symposium and the contributors to this volume with whom we had the distinct privilege of collaborating. RALF

G.

RAHWAN

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.pr001

D O N A L D T. W I T I A K

The Ohio State University College of Pharmacy Columbus, OH June

1982

χ

1 Calcium

Antagonists:

C l a s s i f i c a t i o n and P r o p e r t i e s

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

WINIFRED G. NAYLER University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Victoria, Australia Calcium antagonists (slow channel blockers, slow Ca antagonists) are a heterogeneous group of sub­ stances with widely differing tissue specificities, potency and properties. Some of them exhibit other properties in addition to that of Ca antagonism. In general these drugs can be considered as a sub­ group of a much larger group of compounds which impede the entry of Ca irrespective of the route of entry. The 'slow channel blockers' can be sub­ divided on the basis of their differing tissue spec­ ificities. The purpose of this article is to explore the possibility that a classification which is based on differing tissue specificities may reflect differing modes of action. 2+

2+

2+

Verapamil, nifedipine and diltiazem (Figure 1) belong to a relatively newly recognized group of drugs known collectively as "calcium antagonists", (1) "slow channel inhibitors", (2) or "calcium entry blockers" (3,4). Other substances which are now thought to belong to this group include niludipine, nimodipine, prenylamine, fendiline, caroverine, cinnarizine and perhexeline. The possibility of using these and other closely related sub­ stances in the management of a variety of cardiovascular dis­ orders - including infarction, (5.,^, 7) arrhythmias, (8,9) angina, (JL0,_11) hypertension (12) and hypertrophic obstructive cardiomyopathies (13) is now being considered. However the "calcium antagonists" that are currently available for such use differ from one another not only in terms of their chemistry, bio-availability and stability, (1,14) but also in potency, (1,14) tissue specificity and possibly in their precise mode of action (15,16,17). Because no attempt has yet been made to subclassify these drugs, the purpose of this article is to explore the problem of providing a suitable classification. To do this i t is necess­ ary to briefly discuss the physiological significance of the 0097-615 6/82/0201 -0001 $06.00/0 © 1982 American Chemical Society

2

C A L C I U M R E G U L A T I O N B Y C A L C I U M ANTAGONISTS

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

Verapamil (mol.wt. 454.59) Compound D-600 (gallopamil, mol.wt. 485.59)

M,C CM,

CM' CM, CH,Ο-Α-C CM, CM, CM, Ν CH, CH,-tfS-OCH, 3°-V O N V-OCM, CH

CH

M,C CM, CM CM, 3 ° 1 ^ Ç CM, CM, CM, Ν CM, CM,

Ç3-NO, JLM

Nifedipine (mol.wt., 346.34)

M,C O O C -/S- C O O C M, HjC-y-CM, 1

M Niludipine (mol.wt. 490.55)

Μ^,Ο-H,C

- M,C - ooc -/as-COO - CM,-CM, - OC/ty MjC-V^CM,

M

Nimodipine (mol.wt. 418.45)

H

J

C

Y H

\ M C O O C

v

J T ^ C O O C M , - C M ,- ο - C M ,

^.jC MjC^iT^CM,

M,C ^ Diltiazem (mol.wt. 414.52)

i

CH.

C M , C H I , N < > HCI Ch

Figure 1.

Structural formulas of some Ca

2+

antagonists and their derivatives.

1.

NAYLER

"slow C a els.

Classification

2 +

and

3

Properties

c u r r e n t " (18) and the a s s o c i a t e d i o n - s e l e c t i v e chann2+

The

Slow Ca

Current

2+ The slow Ca c u r r e n t i n heart muscle. In normal heart muscle e x c i t a t i o n i n v o l v e s the a c t i v a t i o n of two d i s t i n c t inward currents (_19^20) . The f i r s t of these currents i s c a r r i e d by Na+ and i s recorded as the f a s t upstroke of the a c t i o n p o t e n t i a l . The Na ions move across the c e l l membrane through v o l t a g e - a c t i v a t e d "channels" that are h i g h l y , but not t o t a l l y , s e l e c t i v e f o r N a . The second inward current i s c a r r i e d mainly (21,22), but not e x c l u s i v e l y (23), by C a . It i s activated slowly, c o n t r i b u t e s to the p l a t e a u phase of the a c t i o n p o t e n t i a l and i s known as "the slow C a c u r r e n t " . The Ca ions i n v o l v e d pass across the c e l l membrane through channels that are h i g h l y s e l e c t i v e f o r C a . L i k e t h e i r Na"" counterparts, the Ca2+s e l e c t i v e channels are v o l t a g e a c t i v a t e d but t h e i r t h r e s h o l d of a c t i v a t i o n (about-55mV) i s higher than that of the N a channels (-35mV). I t i s p o s s i b l e to envisage these "channels" as being p o r e - l i k e s t r u c t u r e s i n the plasmalemma, each pore having i t s own set of " a c t i v a t i o n " and " i n a c t i v a t i o n " gates. In t h i s analogy v o l t a g e a c t i v a t i o n can be l i k e n e d to the opening of "gates" which are c l o s e d during the r e s t i n g s t a t e (24). Taking t h i s hypothesis one step f u r t h e r i t can be argued that the normal opening and c l o s i n g of these "gates" i n v o l v e s voltage-dependent changes i n the c o n f i g u r a t i o n a l s t a t e of the membrane. T h i s same argument a p p l i e s i r r e s p e c t i v e of whether the i o n - s e l e c t i v e "channels" are "porel i k e " s t r u c t u r e s or a p a r t i c u l a r combination or o r g a n i z a t i o n of the membrane p r o t e o l i p i d s that f a c i l i t a t e s the inward movement of c e r t a i n i o n s . Within t h i s framework drugs which i n t e r a c t with the c e l l membrane may a l t e r the configurâtional s t a t e of the membrane, thereby a l t e r i n g the i o n s e l e c t i v i t y or responsiveness of the "gated" channels and p r o t e o l i p i d complexes. +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2 +

2 +

2 +

1

+

2+ The

slow Ca

current and e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g .

2+ The slow Ca current accounts f o r the entry i n t o the c y t o s o l of between 5 and 10 ymoles C a / k g heart weight/beat (25). This i s approximately one tenth of the C a needed to a c t i v a t e cont r a c t i o n (26). Probably the Ca ions that enter as the main charge c a r r i e r s f o r the slow current serve as a t r i g g e r f o r the m o b i l i z a t i o n of C a from the i n t r a c e l l u l a r s t o r e s (27,_28). However, s i n c e the magnitude of the mechanical response v a r i e s according to the e x t r a c e l l u l a r c o n c e n t r a t i o n of C a (29) i t i s probably the c u r r e n t - c a r r y i n g Ca ions of the slow inward c u r r e n t which d e t e r mine the amount of C a m o b i l i z e d f o r i n t e r a c t i o n with the myofilaments. In other words, the C a - i n d u c e d " t r i g g e r " r e l e a s e of Ca from the i n t r a c e l l u l a r storage s i t e s simply provides an 2+

2 +

2 +

2 +

2 +

2+

2 +

C A L C I U M R E G U L A T I O N BY

4

CALCIUM

ANTAGONISTS

i n t e r n a l a m p l i f i c a t i o n f a c t o r . S k e l e t a l muscle d i f f e r s from c a r d i a c muscle i n that i t m o b i l i z e s a l l the C a i t requires for e x c i t a t i o n - c o n t r a c t i o n coupling d i r e c t l y from i t s own i n t r a c e l l u l a r stores. 2 +

2+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

The slow Ca tissues.

current i n smooth muscle, nodal and

conducting

2+ The occurrence of "Ca - s e l e c t i v e " , voltage a c t i v a t e d "channels" i s not l i m i t e d to the myocardium. They occur i n most smooth muscle c e l l s - i n c l u d i n g those i n the coronary, c e r e b r a l and p e r i p h e r a l v a s c u l a t u r e (30). The normal a c t i v i t y of pacemaker, nodal and conducting t i s s u e s (31) i s a l s o l a r g e l y dependent upon them. Because of t h e i r widespread d i s t r i b u t i o n i t follows that substances which a f f e c t the f u n c t i o n i n g of these channels w i l l have a profound e f f e c t on the c i r c u l a t i o n . By c o n t r a s t , s k e l e t a l muscle i s r e l a t i v e l y unaffected (32). 2+ S p e c i f i c i t y of slow channels f o r Ca 2+ Although Ca i s the main current c a r r y i n g i o n f o r the slow inward current part of the current i s c a r r i e d by N a (23). Certain divalent cations, including B a and S r , can s u b s t i t u t e for C a as the main charge c a r r i e r (33). Others, i n c l u d i n g N i , C o , and M n are i n h i b i t o r y (34). 2+ The I d e n t i f i c a t i o n of Ca -antagonists Techniques which are c u r r e n t l y being used to i d e n t i f y substances that a l t e r slow channel transport i n v o l v e measurements of the height and d u r a t i o n of the a c t i o n p o t e n t i a l , monitoring the r a t e of uptake of r a d i o a c t i v e l y l a b e l l e d C a , and e l e c t r o p h y s i o l o g i c a l techniques that i n v o l v e suppression of the f a s t N a current. Use of these techniques i s based on the t a c i t assumpt i o n that the drugs we are d e a l i n g with act only on the slow channels. Later i n t h i s chapter we w i l l summarize the data which suggests that such an assumption may no longer be j u s t i f i e d . For the moment, however, i t i s p e r t i n e n t to work w i t h i n t h i s framework. +

2 +

2 +

2 +

2 +

2 +

2 +

+

(a) Height and

d u r a t i o n of the a c t i o n p o t e n t i a l .

To i l l u s t r a t e the use of changes i n the c o n f i g u r a t i o n of the a c t i o n p o t e n t i a l to e s t a b l i s h the presence or absence of "Ca antagonism" we w i l l concentrate on the c a r d i a c a c t i o n potential. The currents which c o n t r i b u t e to the height and d u r a t i o n of the c a r d i a c a c t i o n p o t e n t i a l are complex (20). In a d d i t i o n to the inward c u r r e n t s already r e f e r r e d to there are at l e a s t two and p o s s i b l y three outward (35) K"*" c u r r e n t s . A c c o r d i n g l y , unless a substance i s s p e c i f i c f o r only the inward Ca"*" current s t u d i e s 2 +

2

1.

NAYLER

Classification

and

5

Properties

which depend upon the monitoring of changes i n the height and duration of the a c t i o n p o t e n t i a l are not p a r t i c u l a r l y h e l p f u l i n i d e n t i f y i n g e i t h e r i n h i b i t o r s or a c t i v a t o r s of slow channel t r a n s port. T h i s d i f f i c u l t y i s f u r t h e r compounded by the f a c t that the subsarcolemmal concentration of C a i n f l u e n c e s the outward K c u r r e n t s , (35) and hence the shape and d u r a t i o n of the a c t i o n potential. 2 +

+

(b) I s o t o p i c Techniques 2+ The use of r a d i o a c t i v e l y l a b e l l e d Ca to monitor changes i n the magnitude and duration of the slow C a current i s a l s o d i f f i c u l t , p a r t l y because of the small amounts of C a that are i n v o l v e d . When considered on a beat to beat b a s i s the C a that enters heart muscle c e l l s by way of the slow C a current represents l e s s than 2 percent of the t o t a l t i s s u e C a and i f , as seems probable, t h i s small component i s r a p i d l y r e c y c l e d to the e x t e r i o r i t s accurate d e t e c t i o n during the time course of the a c t i o n p o t e n t i a l presents s u b s t a n t i a l t e c h n i c a l d i f f i c u l t i e s . There i s , however, another and more s e r i o u s o b j e c t i o n to the use of l a b e l l e d C a f o r t h i s purpose. T h i s d i f f i c u l t y centres around the f a c t that Ca2+ can enter the myocardium and other e x c i t a b l e c e l l s through s e v e r a l d i f f e r e n t routes - i n c l u d i n g i n exchange f o r Na , by passive d i f f u s i o n , and (Figure 2) i n exchange f o r K . Consequently even i f the uptake of r a d i o a c t i v e l y l a b e l l e d Ca + i s reduced there can be no c e r t a i n t y that the p a r t i c u l a r route of entry that i s being a f f e c t e d i s "the slow channels". 2 +

2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2 +

2 +

2 +

2 +

+

+

2

(c) E l e c t r o p h y s i o l o g i c a l techniques These i n v o l v e the use of techniques to suppress the f a s t inward N a current. E i t h e r a voltage can be a p p l i e d so that the transmembrane p o t e n t i a l d i f f e r e n c e i s clamped above the l e v e l a t which the N a current i s a c t i v a t e d . (36) or t e t r o d o t o x i n (37) which s p e c i f i c a l l y i n h i b i t s the Na current, can be added, or the membrane can be d e p o l a r i z e d by r a i s i n g the e x t e r n a l K (38,39). Under each of these c o n d i t i o n s the slow inward current can be a c t i v a t e d by adding aminophylline or i s o p r o t e r e n o l , (39) and by e l e c t r i c a l stimulation. +

+

+

Substances That A l t e r Slow Channel

Transport

T h e o r e t i c a l l y , substances or i n t e r v e n t i o n s that a l t e r slow channel transport may do so i n a v a r i e t y of ways:(a) they may a l t e r the amount of C a which i s a v a i l a b l e to a c t as the charge c a r r i e r ; (b) they may, by i n t e r a c t i n g with the c e l l s u r f a c e , evoke a configurât i o n a l change which e i t h e r f a c i l i t a t e s or impedes the approach of C a to the channels; a l t e r n a t i v e l y (c) the configurâtional change i n the membrane may induce a 2 +

2 +

6

C A L C I U M REGULATION

BY

CALCIUM

ANTAGONISTS

change i n the Ca^ - c a r r y i n g c a p a c i t y of each channel; or (d) the number of channels that are o p e r a t i v e at any given time may be a f f e c t e d . T h i s could be achieved by a l t e r i n g the t h r e s h o l d of a c t i v a t i o n , changing the k i n e t i c s of channel a c t i v a t i o n and/or recovery, by f a c i l i t a t i n g the formation of "de novo" channels or by a c t i v a t i n g " s l e e p i n g " channels.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

A c t i v a t o r s of slow channel t r a n s p o r t . The catecholamines, i n c l u d i n g norepinephrine, epinephrine and i s o p r o t e r e n o l augment the slow Ca current (39,40). They do t h i s by i n c r e a s i n g the number of channels that are a c t i v a t e d at a given v o l t a g e , without a f f e c t i n g the r a t e of channel a c t i v a t i o n or d e a c t i v a t i o n (40). C y c l i c AMP has the same e f f e c t (41). T h i s a b i l i t y of the catecholamines to r e c r u i t new channels may i n d i c a t e the e x i s t e n c e of a heterogenous p o p u l a t i o n of v o l t a g e - a c t i v a t e d channels, one p o p u l a t i o n being under c y c l i c AMP c o n t r o l . Altern a t i v e l y there may be s e v e r a l d i f f e r e n t s t a t e s of a c t i v a t i o n f o r each channel. I r r e s p e c t i v e of which, i f e i t h e r , of these a l t e r n a t i v e s i s c o r r e c t , i t f o l l o w s that the amount of slow channel a c t i v i t y that i s a v a i l a b l e at any one moment i s i n f l u e n c e d by the c i r c u l a t i n g l e v e l of catecholamine. The recent d i s c o v e r y of c a l c i d u c t i n , a p r o t e i n that appears to be a s s o c i a t e d with the slow channels and which can be phosphorylated by a c y c l i c AMPdependent pathway points towards the p o s s i b i l i t y of heterogeneity w i t h i n the channels. There are other reasons f o r b e l i e v i n g that the channels themselves must be heterogenous. Thus, f o r example, the drugs we are d i s c u s s i n g have no i n h i b i t o r y e f f e c t on the Ca -dependent e x c i t a t i o n - i n d u c e d r e l e a s e of norepinephrine from the sympathetic nerve t e r m i n a l s . T

1

2+

I n h i b i t o r s of slow channel transport. Many substances i n h i b i t slow channel t r a n s p o r t . In a d d i t i o n to the d i v a l e n t c a t i o n s already c i t e d ( M n , Co + and N i + ) , protons, La^+, the metabolic i n h i b i t o r s cyanide and d i n i t r o p h e n o l , are e f f e c t i v e i n h i b i t o r s (24). Other i n h i b i t o r y agents i n c l u d e a c e t y l c h o l i n e , (42) papaverine, (43) p e n t o b a r b i t a l , l i d o f l a z i n e , (44) and adenosine (45) as w e l l as verapamil, n i f e d i p i n e and d i l t i a z e m ( 1 ) . P r e c i s e l y how many of these substances i n t e r f e r e with slow channel transport i s unknown, although i n the case of the metabolic i n h i b i t o r s we can probably account f o r t h e i r e f f e c t i n terms of the energy requirements (24) needed f o r maintaining the configurâtional s t a t e of the c e l l membrane compatible with the maintenance of normal slow channel u l t r a s t r u c t u r e . 2+

2

2

C l a s s i f i c a t i o n of Slow Channel B l o c k e r s (or Antagonists) F l e c k e n s t e i n (1) o r i g i n a l l y c l a s s e d some of the substances l i s t e d i n Table I as "calcium a n t a g o n i s t s " on the b a s i s of two requirements :

NAYLER

Classification

passive transport

and

Properties

Ca /Na exchange 2 +

+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

Ca2+ Figure 2.

voltage activated transport

K+

Ca'2+

Ca2+

Schematic representation of possible routes of Ca * myocardial cell. 2

+

T a b l e I : S u b s t a n c e s C l a s s e d a s Ca

entry into a

Antagonists

References Verapamil

1

methoxyverapami1 (D-600)

1

prenylamine

1

nifedipine

1

lidoflazine

44

nimodipine

52,53

diltiazem

1

bepridil

68

caroverine

70

niludipine

53

fendiline

1

8

CALCIUM

R E G U L A T I O N BY C A L C I U M

ANTAGONISTS

(a) the predominant c h a r a c t e r i s t i c of these substances i s t h e i r a b i l i t y to i n h i b i t the slow C a current; and 2+ (b) t h i s i n h i b i t i o n could be overcome by adding Ca 2 +

These c r i t e r i a s t i l l apply. However, the continued u n q u a l i f i e d use of the term "calcium antagonist" r e q u i r e s r e a p p r a i s a l because of i t s l a c k of s p e c i f i c i t y with respect to the s i t e and p r e c i s e mode of drug a c t i o n . Thus "calcium antagonism" can be expressed at a v a r i e t y of s i t e s , i n c l u d i n g the c e l l membrane, the m y o f i b r i l s , the sarcoplasmic r e t i c u l u m and the mitochondria. When used i n t h e r a p e u t i c concentrations, however, the drugs we are d i s c u s s i n g express t h e i r "calcium a n t a g o n i s t i c " p r o p e r t i e s a t only one s i t e - the c e l l membrane. Even a t the c e l l membrane there are other ways i n which substances can i n t e r f e r e with transmembrane C a movements - apart from the entry of C a through the v o l t a g e - a c t i v a t e d "channels". P o s s i b l y , t h e r e f o r e , there i s some merit i n c o n s i d e r i n g drugs of the type shown i n Table I as being a subgroup of a much l a r g e r group of drugs which, f o r want of a b e t t e r term, may be c a l l e d " C a - e n t r y b l o c k e r s " or " C a entry a n t a g o n i s t s " (34). T h i s group of drugs - "the Ca + entry b l o c k e r s " or " C a - e n t r y a n t a g o n i s t s " (Figure 3) would i n c l u d e any drug which impedes the inward movement of C a , i r r e s p e c t i v e of the route of entry. The known routes of C a entry i n t o c a r d i a c and smooth muscle c e l l s i n c l u d e by passive d i f f u s i o n , i n exchange f o r Na , i n exchange f o r K+, and through the v o l t a g e a c t i v a t e d , i o n s e l e c t i v e channels we have been d i s c u s s i n g . As f a r as c a r d i a c and smooth muscle c e l l s are concerned, t h e r e f o r e i t i s p o s s i b l e that four d i f f e r e n t sub groups of Ca -entry b l o c k e r s (or antagonists) w i l l u l t i m a t e l y become a v a i l a b l e . However, the drugs which are c u r r e n t l y a v a i l a b l e are s p e c i f i c only f o r the subgroup that i n v o l v e s the i n f l u x of Ca2+ through the voltage a c t i v a t e d , i o n s e l e c t i v e channels. Since these channels are slowly a c t i v a t e d r e l a t i v e to the channels that s e l e c t i v e l y f a c i l i t a t e the r a p i d i n f l u x of N a during the f a s t upstroke phase of the a c t i o n p o t e n t i a l i t may be more appropriate to r e f e r to these substances as " i n h i b i t o r s of slow channel t r a n s p o r t " . An a l t e r n a t i v e term - " C a channel b l o c k e r " has already appeared i n the l i t e r a t u r e but may be i n a p p r o p r i a t e because the slow channels are not t o t a l l y s e l e c t i v e f o r C a . They a l s o admit some N a and the r e s u l t a n t "slow" Na -dependent current i s blocked by some of the c u r r e n t l y a v a i l a b l e antagonists, i n c l u d i n g verapamil and methoxy verapamil (23). Presumably as new C a entry b l o c k i n g drugs are developed substances that s p e c i f i c a l l y i n h i b i t the i n f l u x of C a through routes of entry other than the slow channels w i l l become a v a i l a b l e . For example, substances that s p e c i f i c a l l y i n h i b i t the entry of C a i n exchange f o r N a or K may be developed. Such substances could be of c l i n i c a l importance because these other routes of C a entry may be i n v o l v e d (43) i n the massive i n f l u x of C a that occurs (46) when flow i s re-introduced to a p r e v i o u s l y ischaemic zone - as may occur, f o r

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2 +

2 +

2+

2 +

2

2+

2 +

2 +

+

+

2 +

2

+

+

2 +

2 +

2 +

+

2 +

2 +

+

1.

NAYLER

Classification

and

Properties

9

example, when coronary vasospasm i s r e l i e v e d , a thrombus i s d i s s olved or removed, or r e v a s c u l a r i z a t i o n takes p l a c e . Subclassif ication Substances which i n h i b i t slow channel transport can be conv e n i e n t l y subdivided i n t o two major subgroups, on the b a s i s of t h e i r chemistry. Thus there are the i n o r g a n i c (Co +, Mn +, La3+) and the organic i n h i b i t o r s . The organic i n h i b i t o r s can be f u r t h e r subdivided i n t o three main c l a s s e s , on the b a s i s of t h e i r t i s s u e s p e c i f i c i t y . According to t h i s scheme drugs which predominately a f f e c t slow channel a c t i v i t y i n the myocardium - example v e r a pamil, can be c l a s s e d as having strong C l a s s I a c t i v i t y (47). C l a s s I I could i n c l u d e those drugs which are most e f f e c t i v e i n b l o c k i n g slow channel transport i n v a s c u l a r smooth muscle. N i f e d i p i n e would provide the prototype f o r t h i s subgroup (47). C l a s s I I I would i n c l u d e those substances which are most potent i n b l o c k i n g slow channel transport i n pacemaker, nodal and conducting t i s s u e s . Verapamil, t h e r e f o r e would have strong C l a s s I I I a c t i v i t y , (8) whereas n i f e d i p i n e would have only weak C l a s s I I I a c t i v i t y (48,49). D i l t i a z e m (11) e x h i b i t s strong C l a s s I I a c t i v i t y (50,51). These r e l a t i v e a c t i v i t i e s are summarized i n Table I I . Working from such a c l a s s i f i c a t i o n i t i s r e l a t i v e l y easy to d e t e r mine which p a r t i c u l a r slow channel b l o c k e r should be s e l e c t e d f o r use i n a p a r t i c u l a r s i t u a t i o n . For example, because of i t s strong C l a s s I I I a c t i v i t y verapamil i s the drug of choice i f i t i s the Ca + currents i n nodal, pacemaker or conducting t i s s u e s that are to be suppressed. On the other hand n i f e d i p i n e may be the drug of choice f o r r e l i e v i n g coronary a r t e r y spasm - an a c t i v i t y which would r e f l e c t i t s strong C l a s s II a c t i v i t y . C l a s s I I drugs can be f u r t h e r subdivided i n t o at l e a s t three subgroups (Figure 3). For example the e f f e c t of d i l t i a z e m on the slow channels i s more marked i n the smooth muscle c e l l s of the coronary (51) than the p e r i p h e r a l v a s c u l a t u r e . Nimodipine (52) a c t s p r e f e r e n t i a l l y on slow channel transport i n the c e r e b r a l v e s s e l s , where i t blocks thromboxane-induced c o n t r a c t i o n s (53). By c o n t r a s t , l i d o f l a z i n e i s more potent i n the periphery. Even w i t h i n the coronary c i r c u l a t i o n there i s room f o r f u r t h e r subc l a s s i f i c a t i o n according to whether i t i s the small or l a r g e v e s s e l s (Figure 3) that are being a f f e c t e d (45). For example, adenosine, which i s a r e l a t i v e l y weak C a entry b l o c k e r , a c t s on smooth muscle c e l l s i n the small coronary a r t e r i e s , whereas d i l t iazem and n i f e d i p i n e act p r e f e r e n t i a l l y on the l a r g e v e s s e l s . Why the v a r i o u s organic i n h i b i t o r s d i f f e r with respect to t h e i r p r e f e r r e d s i t e of a c t i o n i s unknown. Is i t because of t h e i r d i f f e r e n t chemical s t r u c t u r e ? Or are the slow C a channe l s themselves t i s s u e s p e c i f i c ? Some substances - eg. pentobarbitone and adenosine, which are r e l a t i v e l y weak slow channel b l o c k e r s do not d i s p l a y t i s s u e s p e c i f i c i t y with respect to t h e i r C a 2 antagonism. Indeed i n these substances i t i s questionable

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2

2

2

2 +

2 +

+

C A L C I U M REGULATION B Y C A L C I U M ANTAGONISTS

Ca

Entry Blockers /Antagonists

2 +

Ca /Na Exchange 2 +

Passive Transport

+

Slow Ca : K Channel Exchange Transport 2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

Inorganic

Class I myocardium

Organic

/ κ II

/ /

S

Table I I :

S

conducting + nodal tissue

ii iii coronary cerebral

small vessels Figure 3.

III

vasculature ^ / J \ ^ /

i peripheral

+

large vessels

Proposed subdivision of Ca *-entry blocking drugs. 2

S u b d i v i s i o n of Slow Channel I n h i b i t o r s

Class

Slow Channel I n h i b i t o r Verapamil

Nifedipine

Diltiazem

Lidoflazine

I

strong

weak

weak

absent

II

weak

strong

strong

strong

III

strong

absent

weak

absent

1.

NAYLER

Classification

and

11

Properties

whether, because of t h e i r n o n - s p e c i f i c i t y , these substances be c l a s s i f i e d as slow channel b l o c k e r s .

should

S i t e and Mode of A c t i o n . In the c l a s s i f i c a t i o n shown i n F i g u r e 3 the organic and i n o r g a n i c i n h i b i t o r s of slow channel transport have been grouped together as subgroups of a common type of C a entry b l o c k e r . T h i s does not mean that they have a common s i t e of a c t i o n . For i n s t a n c e , the i n o r g a n i c i n h i b i t o r s - M n , N i a n d Co2+ - act at the outer surface of the c e l l membrane, where they compete with Ca f o r b i n d i n g s i t e s (34,54). By c o n t r a s t the organic i n h i b i t o r s - eg. verapamil, D-600 appear to act at the c y t o s o l i c surface (55,56). Even the organic i n h i b i t o r s d i f f e r i n t h e i r p r e c i s e mode of a c t i o n . For example, n i f e d i p i n e (46) reduces the number of channels that are o p e r a t i o n a l at a given time without a l t e r i n g the k i n e t i c s of channel a c t i v a t i o n or i n a c t i v a t i o n , whereas verapamil (17) a l t e r s the k i n e t i c s of channel r e a c t i v a t i o n so that recovery i s delayed a f t e r a p r i o r p e r i o d of a c t i v i t y . P o s s i b l y t h i s e f f e c t of verapamil i s a s s o c i a t e d with i t s a b i l i t y to d i s p l a c e Ca + from s u p e r f i c i a l l y l o c a t e d b i n d i n g s i t e s (47). T h e o r e t i c a l l y i t should be p o s s i b l e to c l a s s i f y the slow channel b l o c k e r s according to whether or not they a f f e c t the k i n e t i c s of slow channel t r a n s p o r t . In t h i s way we could r e a d i l y separate the n i f e d i p i n e type of drugs from those that are more l i k e verapamil. A l t e r n a t i v e l y can these drugs be subgrouped according to t h e i r chemistry? T h i s p o s s i b i l i t y seems to be remote. Thus d i l t i a z e m i s a benzothiazepine d e r i v a t i v e (Figure 1\ n i f e d i p i n e i s derived from d i h y d r o p y r i d i n e w h i l s t verapamil has some s t r u c t u r a l features i n common with papaverine. S t r u c t u r e a c t i v i t y r e l a t i o n s h i p s are a v a i l a b l e f o r verapamil and n i f e d i p i n e but not, as y e t , f o r d i l t i a z e m . The a c t i v i t y of verapamil as a slow channel i n h i b i t o r depends upon the presence of the two benzene r i n g s and the t e r t i a r y amino n i t r o g e n . Loss of the t e r t i a r y amino n i t r o g e n - as occurs, f o r example, upon q u a r t e r n i z a t i o n , r e s u l t s i n a t o t a l l o s s of a c t i v i t y , w h i l s t s u b s t i t u t i o n s w i t h i n the benzene r i n g s r e s u l t i n a decreased potency. There are s e v e r a l ways of i n t e r p r e t i n g t h i s data. The t e r t i a r y amino n i t r o g e n may be a c t i n g as an e s s e n t i a l "spacer u n i t " , ensuring that the d i s t a n c e between the two aromatic r i n g s i s optimal f o r " r e c e p t o r " occupancy. A l t e r n a t i v e l y permanent i o n i z a t i o n of the n i t r o g e n , as occurs upon q u a r t e r n i z a t i o n , may r e s u l t i n the molecule being unable to penetrate i n t o the membrane. Now i f , as seems l i k e l y , the binding s i t e f o r verapamil i s l o c a t e d at or near the c y t o s o l i c surface of the membrane permanent i o n i z a t i o n could prevent the molecule from reaching i t s b i n d i n g s i t e , thus rendering i t i n a c t i v e . L i k e verapamil, n i f e d i p i n e contains two aromatic r i n g s (Figure 1), but t h i s seems to be the only property shared by these 2 +

2+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2 +

2

2 +

12

C A L C I U M REGULATION

BY

CALCIUM

ANTAGONISTS

two molecules. In the case of n i f e d i p i n e s u b s t i t u t i o n s at the e s t e r p o s i t i o n of the N - h e t e r o c y c l i c r i n g that i n c r e a s e i t s l i p i d s o l u b i l i t y decrease i t s potency as a slow channel i n h i b i t o r . A low l i p i d s o l u b i l i t y , t h e r e f o r e , must be of some importance. In a d d i t i o n , and although the presence of the NO2 group at the ortho p o s i t i o n i s not e s s e n t i a l , s u b s t i t u t i o n s at the ortho, meta or para p o s i t i o n s of the benzene r i n g r e s u l t i n a decreased a c t i v i t y , p a r t i c u l a r l y i f e l e c t r o n donating or e l e c t r o n withdrawing r a d i c l e s are added, or i f long s i d e arms are introduced. I t seems l i k e l y , t h e r e f o r e , that so f a r as n i f e d i p i n e i s concerned low l i p i d s o l u b i l i t y and i t s s t e r i c c o n f i g u r a t i o n are both important. 2+

The heterogeneity of the Ca

antagonists.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2+

We have a l r e a d y mentioned the p o s s i b i l i t y of the Ca channels being heterogeneous. There i s i n c r e a s i n g evidence that the heterogeneity of the slow channel b l o c k e r s may extend beyond t h e i r chemistry to t h e i r p r e c i s e mode of a c t i o n . Thus w h i l s t F l e c k e n s t e i n ' s o r i g i n a l r e c o g n i t i o n of these compounds was based on t h e i r a b i l i t y to i n h i b i t slow channel transport i n the myocardium the potency of the v a s o d i l a t o r a c t i v i t y which some of these substances exert may i n v o l v e more than slow channel i n h i bition. Indeed i t i s always p e r t i n e n t to remember that the i n h i b i t o r y e f f e c t of these substances on membrane a c t i v i t y i n smooth muscle c e l l s has always r e l i e d upon the presence of an abnormal e x t r a c e l l u l a r i o n i c environment - u s u a l l y a r a i s e d K . Data i s beginning to accumulate now which i n d i c a t e s that some of these substances - i n c l u d i n g c i n n a r i z i n e , f l u n a r i z i n e , f e n d i l i n e (57), and f e l o d i p i n e (58) may i n t e r a c t with calmodulin to reduce i t s Ca binding a c t i v i t y . Since the i n t e r a c t i o n of the calmodulin -Ca complex with myosin l i g h t chain kinase p l a y s an important r o l e i n determining the c o n t r a c t i l e s t a t e of smooth muscle c e l l s , t h i s i n t r a c e l l u l a r a c t i o n may account, i n part at l e a s t , f o r the greater s e n s i t i v i t y which some of these drugs e x h i b i t f o r smooth muscle c e l l s - that i s these h i g h l y potent v a s o d i l a t o r s may i n t e r act with the C a - c a l m o d u l i n complex as w e l l as i n h i b i t i n g slow channel t r a n s p o r t . T h i s i s an i n t r i g u i n g p o s s i b i l i t y and i t may provide the b a s i s f o r the d i v i s i o n of these substances as shown i n F i g u r e 3. Thus C l a s s I I compounds could be those which act predominately w i t h i n the c e l l , whereas C l a s s I compounds could i n c l u d e those which act predominately on the v o l t a g e a c t i v a t e d slow channels. An i n t r a c e l l u l a r s i t e of a c t i o n need not be l i m i ted to an e f f e c t on the C a - b i n d i n g a c t i v i t y of calmodulin. Thus there i s some evidence to support the p o s s i b i l i t y that d i l t i a z e m which, because of i t s powerful coronary v a s o d i l a t o r a c t i v i t y (59) would be i n c l u d e d i n the C l a s s I I s u b d i v i s i o n of the slow channel b l o c k e r s , i n h i b i t s the r e l e a s e of Ca + from i n t r a c e l l u l a r storage s i t e s i n smooth muscle c e l l s (60). How then can we account f o r the C l a s s I I I (Figure 3) compounds? To do t h i s we may have to take i n t o account yet another +

2 +

2 +

2+

2 +

2

1.

NAYLER

Classification

and

13

Properties

property of these compounds - that i s the a b i l i t y of some of them, i n c l u d i n g verapamil and d i l t i a z e m (59) to i n t e r f e r e with the f a s t N a channels. This i s an a c t i v i t y which i s expressed at r e l a t i ­ v e l y high dose l e v e l s but which i s absent from those compounds ( n i f e d i p i n e , n i l u d i p i n e and nimodipine) which e x h i b i t strong C l a s s I I but no C l a s s I I I (Figure 3) a c t i v i t y . +

Other P r o p e r t i e s of Slow Channel Blockers Although suppression of the v o l t a g e - a c t i v a t e d inward d i s ­ placement of C a i s undoubtedly the most widely discussed pro­ perty of the substances l i s t e d i n Table I, some of these drugs e x h i b i t other p r o p e r t i e s that may be of c l i n i c a l relevance. For example verapamil (61,62) and n i f e d i p i n e bind to α receptors i n b r a i n , neuroblastomaglioma h y b r i d (63) and c a r d i a c muscle c e l l s (64). Verapamil and methoxyverapamil a l s o bind f a i r l y t i g h t l y to muscarinic receptors (65). Verapamil and D-600 have an i n h i ­ b i t o r y e f f e c t on N a and conductance, (23,63) an e f f e c t which apparently i s not shared by n i f e d i p i n e . D i l t i a z e m slows the r e l e a s e of C a from smooth muscle sarcoplasmic r e t i c u l u m (66). As f a r as the c i r c u l a t i o n i s concerned, however, these other p r o p e r t i e s of the slow channel b l o c k e r s are probably i n s i g n i f i c a n t when compared with t h e i r i n h i b i t o r y e f f e c t on slow channel and p o s s i b l y on calmodulin- Ca2+ b i n d i n g a c t i v i t y .

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2 +

+

2 +

2+ The I d e n t i f i c a t i o n of Ca

-Antagonist Binding S i t e s

P o s s i b l y , and i n the very near f u t u r e , we may be able to subdivide the C a - e n t r y b l o c k e r s i n terms of t h e i r i n t e r a c t i o n with s p e c i f i c membranes-located b i n d i n g s i t e s . Thus, i n a recent study (67) h i g h l y s p e c i f i c b i n d i n g s i t e s have been i d e n t i f i e d i n r a b b i t c a r d i a c homogenates f o r H ^ - l a b e l l e d n i f e d i p i n e . Other C a entry b l o c k e r s have been found to compete with ^ H - n i f e d i p i n e f o r these b i n d i n g s i t e s , the order of potency being n i f e d i p i n e >> D-600 = verapamil > cinnarizine. Interestingly, diltiazem and perhexeline were found not to i n h i b i t % n i f e d i p i n e b i n d i n g . Thus there i s now good reason f o r b e l i e v i n g that i t i s not only the slow C a channels themselves which are heterogeneous: the drugs which we now b e l i e v e to i n t e r a c t with them are a l s o h e t e r ­ ogeneous not only i n t h e i r chemistry, but a l s o i n t h e i r mode of a c t i o n . As might have been expected substances with C a anta­ gonist p r o p e r t i e s are now being i s o l a t e d from some of the t r a d i ­ t i o n a l h e r b a l cures - one such i s t a n s h i n o n e , an e f f e c t i v e a n t i ­ a n g i n a l compound that has been i s o l a t e d from the r o o t s of S a l v i a m i l t i o r r h i z a Bunge used as Dan Shen i n t r a d i t i o n a l Chinese medicine (69). 2+

2 +

2 +

2 +

1

1

14

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

Conclusion 2+ The currently available slow channel inhibitors (or Ca antagonists) can be conveniently classed as a subgroup of a large group of compounds which block the entry of Ca into cells. Within this subgroup further subclassification is possible, based on tissue specificity. Whilst such a classification does not explain why these substances differ with respect to their tissue specificity, it provides a useful framework for comparing their different modes of action. Literature Cited 1. Fleckenstein, A. Specific inhibitors and promoters of calcium action in the excitation-contraction coupling of heart muscle. In: Calcium and the Heart, eds. Harris, Ρ and Opie, L. Academic Press, London 1970, 135-188. 2. Katz, A.M.; Reuter, H. Am. J. Cardiol. 1979, 44, 188-190. 3. Nayler, W.G. European Heart J. 1980, 1, 225-237. 4. Nayler, W.G.; Grinwald, P. Fed. Proc. 1981, 40, 2855-2861. 5. Christlieb, I.Y.; Clark, R.E.; Nora, J.D.; Henry, P.D.; Fischer, A.E.; Williamson, J.R.; Sobel, B.E. Am. J. Cardiol 1979, 44, 825-831. 6. Reimer, K.A.; Lowe, J.W.; Jennings, R.B. Circ. 1977, 55, 581-587. 7. Nayler, W.G.; Ferrari, R.; Williams, A. Am. J. Cardiol. 1980, 46, 242-248. 8. Krikler, D.M.; Spurrell, R.A.J. Postgrad. Med. J. 1974, 50, 447-453. 9. Zipes, D.P.; Fischer, J.C. Circ. Res. 1974, 34, 184-192. 10. Andreasen, F.; Boye, E.; Christoffersen, D. Europ. J. Cardiol. 1975, 2, 443-452. 11. Gunther, S.; Green, L.; Muller, J.E.; Mudge, G.H.; Grossmann, W. Am. J. Cardiol. 1979, 44, 793-797. 12. Lewis, G.R.J.; Morley, K.D.; Lewis, B.M.; Bones, P.J. New Zealand Med. J. 1978, 612, 351-354. 13. Rosing D.R.; Kent, K.M.; Maron, B.J.; Condit, J.; Epstein, S.E. Chest. 1980, 78, (Suppl.l) 239-247. 14. Fleckenstein, A. Ann. Rev. Pharmacol. Toxicol. 1977, 17, 149-166. 15. Raschack, M. Arzneim. Forsch. 1976, 26, 1330-1333. 16. Kohlhardt, M.; Fleckenstein, A. Naunyn. Schmiedeberg's Arch. Pharmacol. 1977, 298, 267-272. 17. Kohlhardt, M.; Mnich, Z. J. Mol. Cell. Cardiol. 1978, 10, 1037-1054. 18. Rougier, O.; Vassort, G.; Garnier, D.; Gargouil, Y.M.; Coraboeuf, E. Pfluegers Arch. 1969, 308, 91-110. 19. New, W.; Trautwein, W. Pfluegers Arch. 1972, 334, 1-23. 20. Coraboeuf, E. Am. J. Physiol. 1978, 234, 1101-1116. 21. Beeler, G.W. Jr.; Reuter, H. J. Physiol. (Lond) 1970, 207, 191-209.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

2+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch001

1.

NAYLER

Classification

and

Properties

15

22. Reuter, H. Ann. Rev. Physiol. 1979, 41, 413-424. 23. Kass, R.S.; Tsien, R.W. J. Gen. Physiol. 1975, 66, 169-192. 24. Sperelakis, N.; Schneider, J.A. Am. J. Cardiol. 1978, 37, 1079-1085. 25. Langer, G.W. J. Mol. Cell. Cardiol. 1980, 12, 231-240. 26. Solaro, R.J.; Wise, R.M.; Shiner, J.S.; Briggs, F.N. Circ. Res. 1974, 34, 525-530. 27. Nayler, W.G. J. Clin. Orthop. 1966, 46, 157-186. 28. Fabiato, Α.; Fabiato, F. An. New York Acad. Sci. 1978, 307, 491-522. 29. Ringer, S. J. Physiol. 1883, 4, 29-42. 30. Godfraind, T.; Kaba, A. Arch. Int. Pharmacodyn. Ther. 1972, 196, 35-49. 31. Noble, D. Oxford Clarendon Press. 1975. 32. Shine, K.I.; Serena, S.D.; Langer, G.A. Am. J. Physiol. 1971, 221, 1408-1417. 33. Kohlhardt, M.; Haastert, H.P.; Krause, H. Pfluegers Arch. fur die gesamte Physiologic. 1973, 342, 125-136. 34. Kohlhardt, M.; Mnich, Z.; Haap, K. J. Mol. Cell. Cardiol. 1979, 12, 1227-1244. 35. Bassingthwaighte, J.B.; Fry, C.H.; McGuigan, J.A.S. J. Physiol. 1976, 262, 15-37. 36. Beeler, G.W.; Reuter, H. J. Physiol. 1970, 207, 165-190. 37. Shigenobu, K.; Sperelakis, N. J. Mol. Cell. Cardiol. 1971, 3, 271-286. 38. Pappano, A.J. Circ. Res. 1970, 27, 379-390. 39. Schneider, J.Α.; Sperelakis, N. J. Mol. Cell. Cardiol. 1975, 7, 249-273. 40. Reuter, H.; Scholz, H. J. Physiol. 1977, 264, 49-62. 41. Watanabe, A.B.; Besch, H.R. Circ. Res. 1974, 35, 316-324. 42. Ikemoto, Y.; Goto, M. J. Mol. Cell. Cardiol. 1977, 9, 313-326. 43. Schneider, J.Α.; Brooker, G.; Sperelakis, N. J. Mol. Cell. Cardiol. 1975, 7, 867-876. 44. Van Nueten, J.M.; Wellens, D. Arch. Int. Pharm. Ther. 1979, 242, 329-331. 45. Harder, D.R.; Belardinelli, L.; Sperelakis, N.; Rubio, R.; Berne, R.M. Circ. Res. 1979, 44, 177-182. 46. Shen, A.C.; Jennings, R.B. Am. J. Path. 1979, 67, 417-440. 47. Nayler, W.G.; Szeto, J. Cardiovasc. Res. 1972, 6, 120-128. 48. Ebner, F.; Donath, M. Mode of action and efficacy of nifed­ ipine in 4th International Adalat Symposium. ed. P. Peuch, R. Krebs. Publ. Excerpta Medica. Amsterdam, 1980, 25-34. 49. Rowland, E.; Evans, T.; Krikler, D. Brit. Heart. J. 1979, 42, 124-127. 50. Kusukawa, R.; Kinoshita, M.; Shimoto, Y.; Tomonaga, G.; Hoshina, T. Arzneim. Forsch. 1977, 21, 878-883. 51. Henry, P.D.; Shuchleib, R.; Borda, L.R.; Roberts, R.; Williamson, J.R.; Sobel, B.E. Circ. Res. 1978, 43, 372-380. 52. Towart, R.; Kazda, S. Brit. J. Pharmacol. 1979, 67, 409P.

16

53. 54. 55. 56. 57. 58. 59. 60.

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61. 62. 63. 64. 65. 66. 67. 68. 69. 70.

C A L C I U M R E G U L A T I O N BY C A L C I U M

ANTAGONISTS

Towart, R.; Perzborn, E. Europ. J. Pharm. 1981, 69, 213-215. Kohlhardt, M.; Wais, V. J. Mol. Cell. Cardiol. 1979, 11, 917-922. Payet, M.D.; Schanne, O.F.; Ruiz-Ceretti, E.; Demers, J.M. J. Mol. Cell. Cardiol. 1980, 12, 187-200. Payet, M.D.; Schanne, O.F.; Ruiz-Ceretti, E. J. Mol. Cell. Cardiol. 1980, 12, 635-638. Spedding, M. Brit. J. Pharm. 1982, 75, 25P. Bostrom, S.L.; Ljung, B.; Mardh, S.; Forsen, S.; Thulin, E. Nature. 1981, 292, 777-778. Henry, P.D. Amer. J. Cardiol. 1980, 46, 1047-1058. Takenaga, H.; Magaribuchi, T.; Nakajima, H. Japan J. Pharm. 1979, 28, 457-464. Fairhurst, A.S.; Whittaker, M.L.; Ehlert, F.J. Biochem. Pharm. 1980, 29, 155-162. Glossmann, H.; Hornung, R. Mol. Cell. Endrocrinol. 1980, 19, 243-251. Atlas, D.; Adler, M. Proc. Natl. Acad. Sci. 1981, 78, 1237-1241. Nayler, W.G.; Thompson, J.E.; Jarrott, B. J. Mol. Cell. Cardiol. In Press, 1982. Cavey, D.; Vincent, J.P.; Lazdunski, M. FEBS letters. 1977, 84, 110-115. Takenaga, H.; Magaribuchi, T.; Nakajima, H. Japan. J. Pharm. 1979, 28, 457-464. Holk, M.; Thorens, S.; Haeusler, G. European J. Pharm. In press, 1982. Vogel, S.; Crampton, R.; Sperelakis, N. J. Pharm. Exp. Therap. 1979, 210, 378-385. Patmore, L.; Whiting, R.L. Brit. J. Pharm. 1982, 75, 149P. Ikeda, N.; Kodama, I.; Shibata, S.; Kondo, N.; Yamada, K. Cardiovasc. Pharm. 1982, 4, 70-75.

RECEIVED July 14, 1982.

2 Chemical Pharmacology of Calcium Antagonists D. J. TRIGGLE

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch002

State University of New York, School of Pharmacy, Department of Biochemical Pharmacology, Buffalo, NY 14260

The comparative chemical pharmacology of a structurally diverse group of Ca + antagonists in­ cluding verapamil/D600, diltiazem, nifedipine, cinnarizine, flunarizine and l i d o f l a z i n e i s re­ viewed. These agents, the Ca +-channel antagon­ i s t s or Ca + entry blockers, appear to function by blocking Ca + entry through defined Ca2+ channels of the plasma membrane. The existence of structure-activity relationships, particularly for the verapamil/D600 and the nifedipine 1,4-dihydropyridine series, including stereoselectivity of action i n smooth and cardiac muscle preparations i s consistent with specific l o c i of action. Although these l o c i remain to be defined much evidence, in­ cluding the demonstration of specific binding of 3H-nitrendipine (a nifedipine analog) to smooth and cardiac muscle membranes,isindicative of a membrane site of action. Consistent with their chemical heterogeneity, the pharmacologic proper­ ties of these compounds are not uniform which suggests that a m u l t i p l i c i t y of sites and mechan­ isms of action exist to impede Ca2+ entry. Of par­ t i c u l a r importance i n this respect i s the existence of tissue s e l e c t i v i t y . 2

2

2

2

2 +

The g r o u p o f compounds v a r i o u s l y r e f e r r e d t o a s C a a n t a g o n i s t s , Ca + channel , slow channel o r Ca + e n t r y b l o c k e r s has a c h i e v e d c o n s i d e r a b l e p h a r m a c o l o g i c and t h e r a p e u t i c a t t e n ­ t i o n . T h i s g r o u p o f compounds w h i c h i n c l u d e s v e r a p a m i l , d i l t i a ­ zem, and n i f e d i p i n e ( F i g . 1) amongst i t s most p r o m i n e n t members was l i n k e d t o C a + m e t a b o l i s m i n t h e p i o n e e r i n g work o f F l e c k e n ­ s t e i n who showed t h a t t h e s e a g e n t s m i m i c k e d i n many r e s p e c t s t h e e f f e c t s o f C a + w i t h d r a w a l i n smooth a n d c a r d i a c m u s c l e a n d r e p r e s e n t e d a new p h a r m a c o l o g i c a p p r o a c h t o t h e c o n t r o l o f e x ­ c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n t h e s e t i s s u e s 0-J3) · Amongst 2

2

2

2

0097-6156/ 82/0201 -0017$06.50/0 © 1982 American Chemical Society

18

C A L C I U M R E G U L A T I O N B Y C A L C I U M ANTAGONISTS

*\

CH

M e O — f \ - C - (CH ) Ν CH CH — 79» 80). Cardiac c e l l s that develop calcium dependent a c t i o n p o t e n t i a l s may a l s o demonstrate spontaneous o r t r i g g e r e d (dependent on a p r i o r i n i t i a t i n g a c t i o n p o t e n t i a l ) abnormal automaticity, delayed conduction patterns, and prolonged r e f r a c t o r i n e s s . A l l of these f a c t o r s are important i n the development of cardiac arrhythmias (80-83). Independent of disease induced e f f e c t s , pharmacologic i n t e r v e n t i o n s ( d i g i t a l i s , barium, catecholamines) can p o t e n t i a t e the slow inward C a current i n otherwise normal cardiac t i s s u e s and may t r i g g e r membrane o s c i l l a t i o n s which lead to abnormal c e l l u l a r automaticity and c a r d i a c arrhythmias (81). Drugs that demonstrate calcium i n h i b i t o r y a c t i v i t y d i s p l a y d i f f e r e n t c a r d i a c membrane e f f e c t s dependent upon t h e i r s t r u c t u r e , p h y s i c a l p r o p e r t i e s and c o n c e n t r a t i o n . Compounds which i n t e r f e r e with C a i n f l u x are expected to prolong the d u r a t i o n of the a c t i o n p o t e n t i a l . T h i s p r e d i c t i o n i s supported by evidence that decreased i n t r a c e l l u l a r C a a c t i v i t y may decrease the l a t e increase i n K conductance and p o t e n t i a l l y prolong the d u r a t i o n o f r e p o l a r i z a t i o n (phase 3) (69)· The f i r s t substances reported t o i n h i b i t I~± without i n f l u e n c i n g rapid d e p o l a r i z a t i o n (phase 0) were Mn2+, N i and C o (16, 17)» The e f f e c t s of the chemically complex calcium i n h i b i t o r y compounds upon I and a c t i o n p o t e n t i a l duration, however, are d i f f i c u l t to p r e d i c t because they may not act by a s i n g l e e l e c t r o p h y s i o l o g i c mechanism. F i n a l l y , i t has been suggested that the N a - C a exchange mechanism may be important i n p r o v i d i n g calcium f o r cardiac c o n t r a c t i o n (33.). M e t a l l i c cations (Mn , % , C o ) are known t o d i s p l a c e membrane bound which p a r t i c i p a t e s i n N a - C a exchange (34, 3 9 ) · The e f f e c t of the m a j o r i t y of calcium i n h i b i t o r y compounds on the Na -Ca exchange process i s u n c e r t a i n . +

+

+

+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

2 +

2 +

2 +

2 +

+

2 +

+

2+

g

i

2

+

2 +

2 +

+

+

2 +

2 +

2 +

3.

MUIR

Inhibitory

Compounds

and

the

Cardiovascular

System

E l e c t r o p h y s i o l o g i c E f f e c t s of Calcium I n h i b i t o r y Agents i n Normal and Diseased Hearts E f f e c t s Upon A c t i o n P o t e n t i a l C o n f i g u r a t i o n and Automaticity* The one e l e c t r o p h y s i o l o g i c e f f e c t that a l l calcium i n h i b i t o r y compounds apparently share i n common i s i n h i b i t i o n of I j _ . The magnitude of t h i s e f f e c t i s dependent upon the potency and dose of the drug being i n v e s t i g a t e d . Furthermore, a number of compounds with calcium i n h i b i t o r y a c t i v i t y demonstrate m u l t i p l e membrane e f f e c t s dependent upon o p t i c a l isomerism. The (+)-isomers of racemic verapmil, D-600, and prenylamine demonstrate predominantly f a s t - c h a n n e l ( l f l ) i n h i b i t o r y e f f e c t s whereas, the (-)-isomers of these same compounds are predominantly i n h i b i t o r s of I j _ (45, 84, 85.)· I n t e r e s t i n g l y , only those calcium i n h i b i t o r y compounds which demonstrate e f f e c t s upon I * j are u s e f u l c l i n i c a l l y as a n t i a r r h y t h m i c s (45, 86-89;» Because of d i f f e r e n c e s i n drug potency, dose, and f o r m u l a t i o n , d e s c r i p t i v e experiments report that calcium i n h i b i t o r y compounds have v a r i a b l e e f f e c t s upon a c t i o n p o t e n t i a l c h a r a c t e r i s t i c s and c o n f i g u r a t i o n (^1, 60, 62, 90, 91, 92). In g e n e r a l , s e v e r a l statements can be made about compounds e x h i b i t i n g calcium i n h i b i t o r y a c t i v i t y . At drug concentrations, which begin to r e s u l t i n negative i n o t r o p i c e f f e c t s i n i s o l a t e d heart muscle p r e p a r a t i o n s , most calcium i n h i b i t o r y compounds cause l i t t l e or no change i n a c t i o n p o t e n t i a l amplitude, maximum upstroke v e l o c i t y of phase 0 ( V ) , or a c t i o n p o t e n t i a l d u r a t i o n at 100 percent r e p o l a r i z a t i o n . As drug c o n c e n t r a t i o n i s increased, gradual changes i n a t r i a l , v e n t r i c u l a r myocardium and P u r k i n j e f i b e r a c t i o n p o t e n t i a l c o n f i g u r a t i o n are observed. In a t r i a l and v e n t r i c u l a r myocardium, these changes i n c l u d e i n c r e a s e s i n the slope of the p l a t e a u (phase 2), a decrease i n a c t i o n p o t e n t i a l d u r a t i o n at 25 and 50 percent r e p o l a r i z a t i o n (ADP25 and A P D ^ Q ) and decreases, no change, or i n c r e a s e s i n A P D ^ Q Q . At s t i l l higher drug c o n c e n t r a t i o n s , the demarcation between phases 2 and 5 becomes i n d i s t i n g u i s h a b l e and A P D i s decreased at a l l p o i n t s during r e p o l a r i z a t i o n . A s s o c i a t e d with the changes observed i n A P D ] _ Q Q are s i m i l a r , but not p r o p o r t i o n a l , changes i n the e f f e c t i v e r e f r a c t o r y period (ERP); decreases i n A P D ^ Q Q r e s u l t i n i n c r e a s e s i n the E R P / A P D . In a d d i t i o n , s e v e r a l compounds (2-n-propyl and 2-n-butyl MDl, racemic verapamil, D-600, prenylamine) decrease V (45, 84, 91)* S i m i l a r but more pronounced changes are observed i n a c t i o n p o t e n t i a l s recorded from P u r k i n j e f i b e r s . Although s t u d i e s u s i n g voltage clamp techniques suggest that s e v e r a l of the calcium i n h i b i t o r y compounds (verapamil, D-600, and manganese) may a f f e c t background inward c u r r e n t s c a r r i e d by Na or time-dependent outward c u r r e n t s c a r r i e d by K (both of which could a f f e c t a c t i o n p o t e n t i a l d u r a t i o n ) , the changes that occur i n a c t i o n p o t e n t i a l c o n f i g u r a t i o n could be caused by v a r i a t i o n s i n the degree of i n t r a c e l l u l a r C a i n h i b i t i o n (95, 94)· Drugs which have profound depressant e f f e c t s upon i n t r a c e l l u l a r C a a c t i v i t y would be expected to s

+

a

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

s

a

m a x

m a x

+

+

2 +

2 +

CALCIUM

52

REGULATION

BY

CALCIUM

ANTAGONISTS

d r a m a t i c a l l y abbreviate phase 2 l e a d i n g to premature a c t i v a t i o n of the outward K c u r r e n t s and marked reductions i n A P D during a l l phases of r e p o l a r i z a t i o n . Compounds which have poor i n t r a c e l l u l a r Ca^ i n h i b i t o r y a c t i v i t y and which act p r i m a r i l y at the c e l l membrane by modulating the i n f l u x of C a 2 \ i n d i r e c t l y reduce i n t r a c e l l u l a r C a r e l e a s e from sarcoplasmic r e t i c u l u m . T h i s r e s u l t s i n minimal a b b r e v i a t i o n of phase 2, reductions i n A P D 2 5 and A P D ^ Q » d delayed a c t i v a t i o n of outward K c u r r e n t s , thereby prolonging APD^QO* Recently, we have had the opportunity to examine the i n v i t r o e l e c t r o p h y s i o l o g i c e f f e c t s of two calcium antagonists which are b e l i e v e d to act i n t r a c e l l u l a r l y (2-n-propyl and 2-n-butyl MDl) (55.) · A d d i t i o n of e i t h e r of these compounds at concentrations ranging from 10~7 to 10~5 M to Tyrode's s o l u t i o n superfusing canine P u r k i n j e f i b e r s r e s u l t s i n a c t i o n p o t e n t i a l changes c h a r a c t e r i s t i c of calcium i n h i b i t i o n , i n c l u d i n g decreases i n A P D 2 5 , A P D 5 0 and A P D ] _ o o * I* separate experiments, s i m i l a r concentrations of a quaternary d e r i v a t i v e of 2-n-butyl MDl were added to the s u p e r f u s i o n medium r e s u l t i n g i n s i g n i f i c a n t reductions i n A P D 2 5 , v a r i a b l e changes i n A P D ^ Q and increases i n A P D 1 0 0 (Figure 4 ) · These experiments suggest that the e l e c t r o p h y s i o l o g i c changes caused by calcium i n h i b i t o r y compounds are i n p a r t dependent upon i n h i b i t i o n of and the c u r r e n t s r e s p o n s i b l e f o r r e p o l a r i z a t i o n . Because of the n e g l i g i b l e e f f e c t s of 2-n-butyl MDl and i t s quaterinary d e r i v a t i v e upon phase 4 d e p o l a r i z a t i o n (discussed below), i t i s u n l i k e l y that i t i n t e r f e r e s with a c t i o n p o t e n t i a l r e p o l a r i z a t i o n by a l t e r i n g K e f f l u x , although t h i s aspect r e q u i r e s f u r t h e r study. +

+

2 +

a n

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

+

1

+

E f f e c t Upon Normal Automaticity 2 +

The e f f e c t s of many C a i n h i b i t o r y compounds on s i n o a t r i a l , a t r i o v e n t r i c u l a r , and P u r k i n j e f i b e r a u t o m a t i c i t y have been examined (45> 46, 95-100)» In general, i n v i t r o s t u d i e s suggest that the a d d i t i o n of C a i n h i b i t o r y compounds to s o l u t i o n s superfusing i s o l a t e d t i s s u e s r e s u l t s i n no change or a decrease i n spontaneous a c t i v i t y (98, 100). As p r e v i o u s l y s t a t e d , time-dependent outward c u r r e n t s c a r r i e d by K , and a c t i v a t i o n of an inward current c a r r i e d by Na and C a exert a s i g n i f i c a n t i n f l u e n c e on SA and AV node spontaneous a c t i v i t y (74)· Tyrodes superfused i n r a b b i t and guinea p i g and blood perfused canine sinus node preparations d i s p l a y the most pronounced negative chronotropic e f f e c t s to compounds which have the a b i l i t y to i n h i b i t Na and C a transmembrane f l u x . Verapamil, D-600, d i l t i a z e m and n i l u d i p i n e , i n a d d i t i o n to i n h i b i t i n g of I j _ induce changes i n Na and, i n d i r e c t l y i n K transmembrane f l u x r e s u l t i n g i n pronounced decreases i n spontaneous r a t e (46_, 91 > 102). In c o n t r a s t to t h e i r e f f e c t s upon SA and AV a u t o m a t i c i t y , calcium i n h i b i t o r y compounds do 2 +

+

+

+

2 +

+

s

2 +

+

MUIR

Inhibitory

Compounds

and the Cardiovascular

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

3.

53

System

2200V/sec

M200V/sec ^ O m V 50msec Figure 4. Action potentials recorded from Purkinje fibers before (A) and after 1 Χ ΙΟ Μ (Β), 1 χ 10~ M (C), and 1 X 10' M (D) 2-n-butyl MDl (top) and quaternary 2-n-butyl MDl (bottom). Note the obvious prolongation in APD in C(bottom). 6

5

4

100

CALCIUM

54

REGULATION

BY CALCIUM

ANTAGONISTS

not a f f e c t , or only minimally suppress, spontaneous a c t i v i t y i n normal Tyrodes o r blood superfused Purkinje f i b e r s (51, 60, 61, 62, 102). T h i s f i n d i n g i s not s u p r i s i n g i n view of the lack of importance of the inward C a current i n determining spontaneous a c t i v i t y i n normal Purkinje f i b e r s . Those calcium i n h i b i t o r y compounds that do e x h i b i t the a b i l i t y to depress spontaneous normal automaticity i n P u r k i n j e f i b e r s a l s o e x h i b i t e f f e c t s upon membrane N a conductance. N i f e d i p i n e , a compound b e l i e v e d to a c t p r i m a r i l y by i n h i b i t i n g C a i n f l u x , has no e f f e c t upon P u r k i n j e f i b e r automaticity (60). Verapamil, a calcium i n h i b i t o r y compound with both N a and C a inhibitory a c t i v i t y , has been reported to cause no change or decreases i n P u r k i n j e f i b e r a u t o m a t i c i t y (62). Both 2-n-propyl and 2-n-butyl MDl, and AHR-2666 demonstrate i n s i g n i f i c a n t e f f e c t s upon the slope of phase 4 but depress a u t o m a t i c i t y i n Purkinje fibers. Calcium antagonists that depress spontaneous a c t i v i t y without a f f e c t i n g phase 4 d e p o l a r i z a t i o n presumably a c t by s h i f t i n g the threshold f o r a c t i v a t i o n of the inward N a current i n a p o s i t i v e d i r e c t i o n (51)· 2 +

+

2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

+

2 +

+

E f f e c t s Upon Abnormal Automaticity In c o n t r a s t to t h e i r n e g l i g i b l e e f f e c t s upon normal automatic mechanisms i n P u r k i n j e f i b e r s , calcium i n h i b i t o r y compounds depress abnormal automaticity induced by a v a r i e t y of experimental techniques (51, 60, 61, 62, 99-102). Calcium i n h i b i t o r y compounds are p a r t i c u l a r l y u s e f u l i n suppressing impulse formation due to i n t r i n s i c changes i n membrane conductance which r e s u l t i n c y c l i c a l , pacemaker-like o s c i l l a t i o n s of d i a s t o l i c p o t e n t i a l (51, 80, 91)· This type of a c t i v i t y i s most commonly observed i n depressed, p a r t i a l l y d e p o l a r i z e d f i b e r s and has been termed " d e p o l a r i z a t i o n - i n d u c e d a u t o m a t i c i t y " (105)· Depending upon t h e i r temporal r e l a t i o n s h i p to normally produced a c t i o n p o t e n t i a l s , membrane o s c i l l a t i o n s are c a l l e d e a r l y or delayed a f t e r d e p o l a r i z a t i o n s (104)· Delayed a f t e r d e p o l a r i z a t i o n s are a l s o c a l l e d o s c i l l a t o r y a f t e r p o t e n t i a l s , t r a n s i e n t d e p o l a r i z a t i o n s , low amplitude p o t e n t i a l s and enhanced d i a s t o l i c d e p o l a r i z a t i o n s (104)· A f t e r d e p o l a r i z a t i o n s that are dependent on a p r i o r i n i t i a t i n g a c t i o n p o t e n t i a l , reach threshold and i n i t i a t e rhythmic a c t i v i t y are c a l l e d " t r i g g e r e d " (85, 105)« A f t e r d e p o l a r i z a t i o n s and t r i g g e r e d a c t i v i t y have been recorded from SA and AV nodes, s p e c i a l i z e d a t r i a l and v e n t r i c u l a r (Purkinje) f i b e r s , a t r i o v e n t r i c u l a r valve l e a f l e t s , coronary sinus t i s s u e , and v e n t r i c u l a r myocardium (51, 75, 80, 105, 106). In a recent review, i t was pointed out that membrane o s c i l l a t i o n s can be produced by s t r e t c h , ischemia, hypoxia, c o o l i n g , drugs (catecholamines, d i g i t a l i s , C a , B a , a c o n i t i n e , v e r a t r i n e ) , elevated P 02> a l t e r a t i o n s i n the e l e c t r o l y t e content of s u p e r f u s i o n s o l u t i o n s , and spontaneously 2 +

C

2 +

3.

MUIR

Inhibitory

Compounds

and

the

Cardiovascular

System

o c c u r r i n g disease (75)· The calcium i n h i b i t o r y compounds which have demonstrated the a b i l i t y to reduce or e l i m i n a t e membrane o s c i l l a t i o n s i n c l u d e m e t a l l i c ions ( M n , N i , C o ) , verapamil, D-600, n i f e d i p i n e , n i l u d i p i n e , d i l t i a z e m , p e r h e x i l i n e , AHR 2666, 2-n-propyl and 2-n-butyl MDl and many others (51, 60, 61, 62, 99-108). Recent evidence suggests that t r a n s i e n t r e l e a s e of i n t r a c e l l u l a r C a from sarcoplasmic reticulum i s r e s p o n s i b l e f o r the membrane conductance changes which produce membrane o s c i l l a t i o n s (104)· I f t h i s theory i s c o r r e c t , the e f f i c a c y of calcium i n h i b i t o r y compounds i n reducing and a b o l i s h i n g membrane o s c i l l a t i o n s can be l i n k e d to t h e i r a b i l i t y to l i m i t i n t r a c e l l u l a r calcium r e l e a s e , transmembrane C a i n f l u x , or both. Since p a r t i a l a c t i v a t i o n of the background inward Na current i s b e l i e v e d to be somewhat r e s p o n s i b l e f o r membrane o s c i l l a t i o n s , calcium i n h i b i t o r y compounds which i n h i b i t Na transmembrane f l u x (verapamil, D-600, p e r h e x i l i n e , AHR 2666, 2-n-propyl and 2-n-butyl MDI's) may be p a r t i c u l a r l y u s e f u l i n a b o l i s h i n g abnormal rhythm disturbances due to membrane o s c i l l a t i o n s (79)· 2+

2 +

2 +

2 +

2 +

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+

+

E f f e c t s Upon Conduction Diseased Hearts

of the Cardiac Impulse i n Normal and

Cardiac a c t i v a t i o n i s dependent upon the proper timing and sequencing of c a r d i a c e x c i t a t i o n . A l t e r a t i o n s i n the pathways or conduction v e l o c i t y which r e s u l t i n normal c a r d i a c a c t i v a t i o n could lead to abnormal conduction p a t t e r n s and disturbances i n c a r d i a c rhythm (75)· The r e l a t i v e potencies of calcium i n h i b i t o r y compounds i n depressing c a r d i a c conduction v e l o c i t y i n i s o l a t e d t i s s u e s i s dependent upon dose and the type of t i s s u e being i n v e s t i g a t e d . Normal a t r i a l and v e n t r i c u l a r muscle, and P u r k i n j e f i b e r s demonstrate l i t t l e or no response to the negative dromotropic e f f e c t s of calcium i n h i b i t o r y agents unless exposed to extremely l a r g e drug concentrations (62, 100, 102, 109)· Tyrode's or blood superfused SA and AV nodal t i s s u e s demonstrate dose dependent decreases i n conduction v e l o c i t y when exposed to calcium i n h i b i t o r y compounds (96-100, 102). Recordings from the upper and middle p o r t i o n s of the AV node i n d i c a t e that calcium i n h i b i t o r y compounds cause a r e d u c t i o n i n a c t i o n p o t e n t i a l amplitude and upstroke v e l o c i t y and i n c r e a s e the e f f e c t i v e r e f r a c t o r y p e r i o d (102)· The i o n i c mechanisms r e s p o n s i b l e f o r the e f f e c t s of calcium i n h i b i t o r y compounds upon conduction v e l o c i t y i n SA and AV nodal t i s s u e s remain undetermined although i n h i b i t i o n of I i i s conjectured to be r e s p o n s i b l e f o r the m a j o r i t y of changes based upon r e v e r s a l of these e f f e c t s by increasing extracellular C a or catecholamine a d m i n i s t r a t i o n (46, 102, 104)· s

2 +

The e l e c t r o p h y s i o l o g i c c o r r e l a t e s of the e f f e c t s of calcium i n h i b i t o r y agents upon conduction v e l o c i t y i n i s o l a t e d

55

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

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c a r d i a c t i s s u e s are observed i n i s o l a t e d whole heart preparations (electrograms), and s p e c i f i c i n t e r v a l s of the electrocardiogram recorded from i n t a c t animals (4-8, 95, 96, 98, 99, 110-115). Drug e f f e c t s are dependent upon d i v e r s e hemodynamic, metabolic, autonomic and r e f l e x changes. For example, i n v i t r o s t u d i e s i n d i c a t e that n i f e d i p i n e i s twice as potent as verapamil i n slowing AV nodal conduction when both are administered at equal negative chronotropic concentrations (95)* In v i v o s t u d i e s , on the other hand, i n d i c a t e that n i f e d i p i n e i n c r e a s e s heart rate and does not a f f e c t AV nodal conduction, while verapamil prolongs AV nodal conduction and produces second degree AV block (89, 111, 115)* Furthermore, verapamil exerts i t s negative dromotropic e f f e c t s i n both innervated and denervated hearts ( i l l ) . Studies i n p a t i e n t s i n sinus rhythm support these f i n d i n g s by i n d i c a t i n g that while verapamil has no e f f e c t upon the R-R, QRS and Q-T i n t e r v a l s of the electrocardiogram, i t d r a m a t i c a l l y prolongs both the a t r i a l to His (A-H) time and the P-R i n t e r v a l (59, 111)· Similar s t u d i e s using n i f e d i p i n e i n r e s t i n g human p a t i e n t s are unable to demonstrate d i s c e r n a b l e e f f e c t s upon SA and AV nodal or His P u r k i n j e conduction (96). The calcium i n h i b i t o r y compounds, d i l t i a z e m and p e r h e x i l i n e , demonstrate e f f e c t s i n i n t a c t hearts midway between those of verapamil and n i f e d i p i n e (_6, 98). S e v e r a l i n v e s t i g a t o r s a t t r i b u t e the d i f f e r e n c e s caused by calcium i n h i b i t o r y compounds i n i n t a c t animals to the magnitude of t h e i r in v i t r o f a s t (Na ) versus slow channel ( C a ) i n h i b i t o r y a c t i v i t y and t h e i r a b i l i t y to i n i t i a t e r e f l e x autonomic e f f e c t s (114, 115)* For example, verapamil i n h i b i t s both I j j and I j _ i n v i t r o , but demonstrates minimal autonomic e f f e c t s i n v i v o (62, 111?"» N i f e d i p i n e , however, depresses I j _ i n v i t r o and e l i c i t s strong r e f l e x sympathetic a c t i v i t y jln v i v o TÎ14)· Studies of these calcium i n h i b i t o r y compounds which are b e l i e v e d to act i n t r a c e l l u l a r l y (2-n-propyl and 2-n-butyl MDI's) upon a u t o m a t i c i t y and conduction i n SA and AV nodes and ischemic myocardium remain to be done. c

+

a

2 +

s

s

The e f f e c t s of calcium i n h i b i t o r y agents upon conduction v e l o c i t y and delayed a c t i v a t i o n i n normal hearts may be e n t i r e l y d i f f e r e n t from that observed i n ischemic myocardium. In c o n s t r a s t to depressant I i dependent conduction e f f e c t s i n SA and AV nodes of normal hearts, verapamil has demonstrated the a b i l i t y to reduce the degree of ischemia-induced conduction delay i n anesthetized dogs a f t e r l i g a t i o n of t h e i r l e f t a n t e r i o r descending coronary a r t e r y (109, 110). Recent s t u d i e s using n i f e d i p i n e , d i l t i a z e m , n i s o l d i p i n e and n i l u d i p i n e have y i e l d e d r e s u l t s suggesting a s i m i l a r e f f e c t upon ischemiainduced conduction delay (109, 116-124)· These r e s u l t s imply that calcium i n h i b i t o r y compounds f a v o r a b l y a f f e c t myocardial oxygen supply and demand presumably by improving oxygen supply to ischemic areas v i a coronary c o l l a t e r a l blood flow. Furthermore, i t has been argued that the a d m i n i s t r a t i o n of s

3.

MUIR

Inhibitory

Compounds

and

the

Cardiovascular

System

57

calcium i n h i b i t o r y compounds p r i o r to i n f a r c t i o n improves recovery of myocardial f u n c t i o n a f t e r coronary a r t e r y l i g a t i o n by decreasing i n t r a c e l l u l a r C a and energy demand during the ischemic p e r i o d (78, 122, 125). The r e l a t i v e potency of the v a r i o u s calcium i n h i b i t o r y compounds i n producing t h i s myocardial " s p a r i n g " or " s a l v a g i n g " e f f e c t has not been determined. 2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

Antiarrhythmic A c t i v i t y Calcium i n h i b i t o r y compounds are used as antiarrhythmic drugs because of t h e i r d i v e r s e e f f e c t s upon c a r d i a c e l e c t r i c a l a c t i v i t y and experimental evidence which suggests that the slow inward current i s important i n the genesis of c a r d i a c arrhythmias (126). A v a r i e t y of p o t e n t i a l l y t o x i c compounds i n c l u d i n g a c o n i t i n e , barium, d i g i t a l i s and catecholamines have been used to produce abnormal e l e c t r i c a l a c t i v i t y i n i s o l a t e d t i s s u e p r e p a r a t i o n s i n order to demonstrate the e f f e c t i v e n e s s of calcium i n h i b i t o r y compounds i n suppressing abnormal a u t o m a t i c i t y . Experimental s t u d i e s i n i n t a c t animals i n d i c a t e that calcium i n h i b i t o r y compounds possess v a r i a b l e a n t i arrhythmic e f f e c t s against a v a r i e t y of c h e m i c a l l y - ( d i g i t a l i s , calcium, catecholimines) and mechanically- (coronary o c c l u s i o n ) induced v e n t r i c u l a r arrhythmias (79, 81, 109, 110, 127, 128, 129» ) · The importance of I j _ i n the genesis of v e n t r i c u l a r arrhythmias a s s o c i a t e d with these pharmacologic and mechanical manipulations i s assumed but remains s p e c u l a t i v e . The c l i n i c a l value of the v a r i o u s calcium i n h i b i t o r y compounds i n e f f e c t i v e l y c o n t r o l l i n g c a r d i a c arrhythmias caused by t o x i c concentrations of d i g i t a l i s and n a t u r a l l y - o c c u r r i n g disease (ischemia, i n f e c t i o n , hypoxia) i s a l s o disputed (1-26, 150)· I t i s noteworthy that calcium i n h i b i t o r y compounds with the a b i l i t y to i n h i b i t Ifl + possess the most potent antiarrhythmic a c t i v i t y against both experimental and c l i n i c a l arrhythmias (126). For example, n i f e d i p i n e exerts minimal, i f any, antiarrhythmic e f f e c t against ischemic induced arrhythmias i n i n t a c t animals (_6, 87, 1 5 1 ) · D i l t i a z e m and p e r h e x i l i n e , on the other hand, demonstrate v a r i a b l e a n t i a r r h y t h m i c e f f e c t s dependent upon t h e i r dose and the mechanism r e s p o n s i b l e f o r arrhythmia p r o d u c t i o n (ischemia, hypoxia, d i g i t a l i s , a c o n i t i n e ) (102, 126, 151, 152)» Only verapamil, which has been i n v e s t i g a t e d i n the l a r g e s t number of experimental and c l i n i c a l t r i a l s , has demonstrated c o n s i s t e n t a n t i a r r h y t h m i c a c t i v i t y against c a r d i a c arrhythmias r e g a r d l e s s of cause ( 1 5 5 ) · C l i n i c a l e l e c t r o p h y s i o l o g i c s t u d i e s i n d i c a t e that verapamil i s e f f e c t i v e i n combating s u p r a v e n t r i c u l a r arrythmias i n c l u d i n g a t r i a l premature d e p o l a r i z a t i o n s , paroxysmal a t r i a l t a c h y c a r d i a , AV nodal re-entrant paroxysmal s u p r a v e n t r i c u l a r t a c h y c a r d i a , a t r i a l f i b r i l l a t i o n and a t r i a l f l u t t e r ( 1 5 4 ) » Verapamil i s a l s o p o t e n t i a l l y b e n e f i c i a l i n e l i m i n a t i n g s

a

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

58

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ANTAGONISTS

recurrent s u p r a v e n t r i c u l a r t a c h y c a r d i a i n p a t i e n t s with accessory pathways and p r e - e x c i t a t i o n (135)· Verapamil i s most e f f e c t i v e i n c o n t r o l l i n g c i r c u s movement t a c h y c a r d i a i n v o l v i n g accessory pathways when retrograde conduction i s involved r a t h e r than when conduction i s antegrade. Verapamil w i l l not slow v e n t r i c u l a r r a t e during a t r i a l f i b r i l l a t i o n o r antegrade conduction over the accessory pathway. In a d d i t i o n , s i n c e l a r g e doses of verapamil may shorten the c a r d i a c a c t i o n p o t e n t i a l and the e f f e c t i v e r e f r a c t o r y p e r i o d , i n c r e a s e s i n v e n t r i c u l a r r a t e have been reported ( 1 5 4 , 135)· Recent experimental evidence using 2-n-propyl and 2-n-butyl MDl i n d i c a t e s that these compounds possess antiarrhythmic potency equivalent to that of verapamil without producing bradycardia, ECG changes or a t r i o v e n r i c u l a r block (52, 1 5 6 ) . I t w i l l be i n t e r e s t i n g to determine i f the MDI's are e q u a l l y as e f f e c t i v e i n combating arrhythmias i n p a t i e n t s with n a t u r a l l y o c c u r r i n g heart disease as they are i n experimental animals. In summary, compared to d i l t i a z e m , n i f e d i p i n e , and p e r h e x i l i n e , verapamil demonstrates the most c o n s i s t e n t r e s u l t s i n the c o n t r o l of both experimental and c l i n i c a l arrhythmias. In none of the s t u d i e s reported to date, however, has verapamil been uniformly s u c c e s s f u l i n r e - e s t a b l i s h i n g normal sinus rhythm i n a l l animals o r p a t i e n t s although there i s a marked r e d u c t i o n i n the frequency of e c t o p i c v e n t r i c u l a r d e p o l a r i z a t i o n s (126, 1 5 4 ) · The important d i r e c t antiarrhythmic e f f e c t s of the various calcium i n h i b i t o r y agents are the r e s u l t of a combined i n h i b i t i o n of both I f l a ^si (126). A d d i t i o n a l explanations f o r the d i r e c t antiarrhythmic e f f e c t s of calcium i n h i b i t o r y agents i n c l u d e h y p e r p o l a r i z a t i o n of the c a r d i a c c e l l membrane thereby reducing calcium c e l l u l a r i n f l u x and membrane o s c i l l a t i o n s (51, 60, 62, 1 1 0 ) , i n c r e a s e s i n calcium uptake or decreased calcium r e l e a s e from sarcoplasmic r e t i c u l u m or mitochondria (7_, 7 8 , 1 0 0 ) , s t i m u l a t i o n of the sodium-calcium pump promoting calcium e f f l u x ( 5 5 ) , i n t e r f e r e n c e with the i n t r a c e l l u l a r calcium receptor of c o n t r a c t i l e p r o t e i n s , and binding to i n t r a c e l l u l a r r e g u l a t o r y p r o t e i n s (M.T.Piascik et a l , manuscript i n p r e p a r a t i o n ) . E q u a l l y as important, and p o t e n t i a l l y more c l i n i c a l l y r e l e v a n t , are the i n d i r e c t mechanisms r e s p o n s i b l e f o r the antiarrhythmic a c t i v i t y of calcium i n h i b i t o r y agents, i n c l u d i n g coronary v a s o d i l a t i o n and subsequent i n c r e a s e s i n coronary a r t e r y blood flow ( 4 7 , 114, 124, 1 5 7 ) ; decreased c a r d i a c c o n t r a c t i l i t y , metabolism, and a r t e r i a l v a s o d i l a t i o n r e s u l t i n g i n decreases i n myocardial oxygen consumption and w a l l t e n s i o n ; (92) and r e f l e x a l t e r a t i o n s i n autonomic r e g u l a t i o n of heart rate~~(overdrive supression) and AV conduction ( 1 1 4 ) . +

a

n

d

3.

MUiR

Inhibitory

Compounds

and

the

Cardiac and Smooth Muscle Mechanical

Cardiovascular

System

Activity

Cardiac Muscle. Calcium i n h i b i t o r y agents may i n t e r f e r e with e x c i t a t i o n - c o n t r a c t i o n coupling processes i n myocardial or v a s c u l a r smooth muscle c e l l s by a number of mechanisms including: l ) i n h i b i t i o n of the slow inward current through d i r e c t competition f o r slow channels or i n t e r f e r e n c e with the membrane binding of C a ; 2) i n t e r f e r e n c e with the r e l e a s e and uptake of calcium by the various i n t r a c e l l u l a r o r g a n e l l e s ; and 5) a l t e r a t i o n of the a c t i v i t y of s p e c i f i c r e g u l a t o r y p r o t e i n s ( t r o p o n i n , calmodulin). I t i s i n t e r e s t i n g to note that most calcium i n h i b i t o r y agents are from 5 to 10 times more e f f e c t i v e i n reducing smooth muscle c o n t r a c t i o n than i n reducing the f o r c e of myocardial c o n t r a c t i o n . Furthermore, u n l i k e c a r d i a c muscle where tropomyosin and t r o p o n i n are b e l i e v e d to be the important r e g u l a t o r y p r o t e i n s , a substance c a l l e d calmodulin serves a predominant r o l e i n smooth muscle

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

2 +

(158,

139).

Experiments u t i l i z i n g i s o l a t e d superfused and blood perfused c a r d i a c t i s s u e p r e p a r a t i o n s and i s o l a t e d r a t , r a b b i t , and cat whole hearts a l l demonstrate the potent, c o n c e n t r a t i o n dependent, negative i n o t r o p i c a c t i v i t y of the calcium i n h i b i t o r y compounds ( 4 5 , 5 0 , 9 0 - 9 5 , 112, 115, 114, 118, 140, 141)· Studies comparing the r e l a t i v e negative i n o t r o p i c e f f e c t s of s e v e r a l of the calcium i n h i b i t o r y compounds i n d i c a t e that those compounds e x h i b i t i n g the most potent calcium i n h i b i t o r y e f f e c t s i n v i t r o are the most e f f e c t i v e i n reducing c a r d i a c c o n t r a c t i l i t y i n v i v o ( 6 ) . Of those compounds most f r e q u e n t l y compared, n i f e d i p i n e and n i l u d i p i n e e x h i b i t the most profound negative i n o t r o p i c a c t i v i t y followed by verapamil (levo isomer), d i l t i a z e m and p e r h e x i l i n e ( 8 8 , 8 9 - 1 4 2 ) . In i s o l a t e d guinea p i g , r a t , r a b b i t and cat p a p i l l a r y muscle p r e p a r a t i o n s , the a d d i t i o n of a calcium i n h i b i t o r y compound to the superfusing s o l u t i o n decreases i s o m e t r i c or i s o t o n i c t e n s i o n long before measurable changes i n the c a r d i a c a c t i o n p o t e n t i a l occur ( 5 0 , 8 9 , 9 2 , 118, 1 4 2 ) . The subsequent a d m i n i s t r a t i o n of catecholamines or i n c r e a s e s i n the extracellular C a c o n c e n t r a t i o n ( 2 . 5 to 5 · 0 mM) w i l l p a r t i a l l y reverse these e f f e c t s and o r i g i n a l l y served as the b a s i s f o r the hypothesis that calcium i n h i b i t o r y compounds l i m i t sarcolemmal C a f l u x v i a slow channels ( l ) . As p r e v i o u s l y suggested, i n t r a c e l l u l a r mechanisms are important i n d e t e r mining the c o n t r a c t i l e e f f e c t s of calcium i n h i b i t o r y compounds. Experiments conducted i n our l a b o r a t o r y , studying the e f f e c t s of 2-n-propyl or 2-n-butyl MDl i n i s o l a t e d Tryrodes superfused canine p a p i l l a r y muscle p r e p a r a t i o n s , demonstrate t h e i r a b i l i t y to uncouple e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g at drug concentrations which do not reduce a c t i o n p o t e n t i a l c h a r a c t e r i s t i c s i n c l u d i n g a c t i o n p o t e n t i a l amplitude, r e s t i n g p o t e n t i a l , d u r a t i o n at 2 5 , 5 0 , 90 percent r e p o l a r i z a t i o n and the rate of r i s e of phase 0 ( d V / d t ) . I n t e r e s t i n g l y , at drug concentrations g r e a t e r than 5 X 10"-> M, v e n t r i c u l a r muscle 2 +

2 +

m a x

59

CALCIUM

60

REGULATION

BY

ANTAGONISTS

a c t i o n p o t e n t i a l d u r a t i o n s a c t u a l l y increased (Figure 5)· The e x p l a n a t i o n f o r t h i s l a t t e r e f f e c t i s u n c e r t a i n , although s i m i l a r changes appear to be evident upon c l o s e i n s p e c t i o n of f i g u r e s reported by other i n v e s t i g a t o r s using a v a r i e t y of calcium i n h i b i t o r y compounds ( l , 92). I t i s e s t a b l i s h e d that C a * and K are involved i n maintenance and t e r m i n a t i o n of the p l a t e a u phase of the cardiac action potential. Furthermore, i n t r a c e l l u l a r calcium c o n c e n t r a t i o n c o n t r o l s membrane K p e r m e a b i l i t y v i a the v a r i o u s conductance components f o r K (gK^, gK2, g l ) (69)· It i s also e s t a b l i s h e d that a c t i o n p o t e n t i a l d u r a t i o n and myocardial t e n s i o n development are i n t e g r a l l y r e l a t e d (13)· In view of previous observations and explanations f o r the e x c i t a t i o n c o n t r a c t i o n c o u p l i n g process and the e f f e c t s of calcium i n h i b i t o r y compounds upon the cardiac a c t i o n p o t e n t i a l of v e n t r i c u l a r muscle and P u r k i n j e f i b e r s , one p o s s i b l e e x p l a n a t i o n f o r the e f f e c t s observed i n v e n t r i c u l a r muscle preparations i s that low concentrations of calcium i n h i b i t o r y compounds reduce the amount of i n t r a c e l l u l a r f r e e calcium i n the v i c i n i t y of the K channel, thereby changing the channel's c o n f i g u r a t i o n r e s u l t i n g i n a r e d u c t i o n i n gK and delayed r e p o l a r i z a t i o n of the v e n t r i c u l a r muscle a c t i o n p o t e n t i a l . When elevated c o n c e n t r a t i o n s of calcium i n h i b i t o r y compounds are used, the p l a t e a u phase of the a c t i o n p o t e n t i a l i s d r a m a t i c a l l y abbreviated r e s u l t i n g i n reductions i n a c t i o n p o t e n t i a l d u r a t i o n during a l l phases of r e p o l a r i z a t i o n . Depending upon the c a r d i a c f i b e r type (Purkinje f i b e r or v e n t r i c u l a r muscle) and c o n c e n t r a t i o n of the calcium i n h i b i t o r y compound under i n v e s t i g a t i o n , q u a n t i t a t i v e and q u a l i t a t i v e changes i n a c t i o n p o t e n t i a l plateau and r e p o l a r i z a t i o n are to be expected, although c o n t r a c t i l e f o r c e i s i n v a r i a b l y reduced. Smooth Muscle. A l l known calcium i n h i b i t o r y compounds decrease smooth muscle tone i n both the coronary and peripheral c i r c u l a t i o n s (113, 114, 115, 140, 143-147)» Comparative s t u d i e s using dogs suggest that t h i s e f f e c t v a r i e s i n i n t e n s i t y but surpasses negative i n o t r o p i c a c t i v i t y (114, 145)» N i f e d i p i n e , verapamil and p e r h e x i l i n e e x h i b i t t h e i r most pronounced v a s o d i l a t o r e f f e c t s upon femoral a r t e r i a l blood flow followed by coronary, r e n a l , and mesenteric beds (44, 47, 115)· D i l t i a z e m and n i s o l d i p i n e p r e f e r e n t i a l l y d i l a t e the coronary bed (47)· The coronary smooth muscle r e l a x i n g e f f e c t s of the 2 - s u b s t i t u t e d aminoindenes c l o s e l y resemble that produced by verapamil and prenylamine (53, 113)· Two-n-butyl MDl, i n p a r t i c u l a r , demonstrates a 4 - f o l d g r e a t e r coronary a r t e r i a l v a s o d i l a t i n g than negative i n o t r o p i c e f f e c t ( 1 1 3 ) · E q u a l l y as i n t e r e s t i n g and p o t e n t i a l l y of g r e a t e r c l i n i c a l s i g n i f i c a n c e than t h e i r a b i l i t y to d i l a t e coronary a r t e r i e s , i s the a b i l i t y of s e v e r a l calcium i n h i b i t o r y compounds to a f f e c t r e g i o n a l myocardial p e r f u s i o n i n such a way as to i n c r e a s e blood flow to ischemic myocardium (47, 148, 149· 150). 2

+

+

+

x

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CALCIUM

+

+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

3.

MUiR

Inhibitory

Compounds

and

the

Cardiovascular

System

Figure 5. Action potentials and developed tension recorded from canine papillary muscle before (A) and after 1 χ JO Μ (Β) and 1 X 10 M (C) 2-n-butyl-MDl. Developed tension decreased by 25% (B) and 80% (C). 6

s

61

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62

CALCIUM

REGULATION

BY

CALCIUM

ANTAGONISTS

N i s o l d i p i n e , n i f e d i p i n e and verapamil have r e c e n t l y been shown to i n c r e a s e t o t a l coronary c o l l a t e r a l transmural blood flow to ischemic areas of myocardium (47)· Only d i l t i a z e m d i d not produce an i n c r e a s e i n t o t a l transmural blood flow w i t h i n the ischemic zone, although the r a t i o of blood flow i n the subendocardium versus that i n the subepicardium was s i g n i f i c a n t l y i n c r e a s e d . Recent s t u d i e s e v a l u a t i n g the e f f e c t s of n i f e d i p i n e i n dogs with experimentally induced acute myocardial ischemia and i n f a r c t i o n i n d i c a t e that the dosage of calcium i n h i b i t o r y compound u t i l i z e d may be c r i t i c a l i n determining whether or not a b e n e f i c i a l e f f e c t i s obtained (122). Dogs r e c e i v i n g n i f e d i p i n e , 13 yg/kg, demonstrated a 30 percent f a l l i n a o r t i c pressure, a 12 percent r i s e i n heart r a t e , and an extension of t h e i r i n f a r c t zone. Dogs r e c e i v i n g n i f e d i p i n e , 1 ug/kg, demonstrated a 12 percent f a l l i n a r t e r i a l blood pressure, no change i n heart r a t e , and an improvement of r e g i o n a l myocardial p e r f u s i o n suggesting l i m i t a t i o n of i n f a r c t s i z e . Together these s t u d i e s i n d i c a t e that drug, drug dose, dosage rate and method of a d m i n i s t r a t i o n are c r i t i c a l i n determining whether or not a favorable r e d i s t r i b u t i o n of blood flow to ischemic myocardium i s obtained. Calcium I n h i b i t o r y Compounds and Myocardial

Ischemia

Regardless of the e f f e c t s of calcium i n h i b i t o r y compounds upon t o t a l myocardial p e r f u s i o n and the d i s t r i b u t i o n of transmural blood flow, s e v e r a l groups of i n v e s t i g a t o r s have demonstrated that verapamil and d i l t i a z e m can prevent the consequences of myocardial ischemia, p a r t i c u l a r l y m i t o c h o n d r i a l s w e l l i n g and d e s t r u c t i o n (150-153)· Myocardial calcium l e v e l s i n ischemic dog hearts i n c r e a s e to 10 to 20 times normal l e v e l s (78, 154)« Increased i n t r a c e l l u l a r calcium i n c r e a s e s i n t r a c e l l u l a r energy u t i l i z a t i o n , decreases ATP, and impairs m i t o c h o n d r i a l f u n c t i o n (78). Recent evidence suggests that ischemia of myocardial t i s s u e r e s u l t s i n an i n c r e a s e i n i n o r g a n i c phosphate c o n c e n t r a t i o n which induces m i t o c h o n d r i a l s w e l l i n g and uncoupling of o x i d a t i v e phosphorylation mechanisms by s t i m u l a t i n g e n e r g y - d i s s i p a t i n g i n t r a m i t o c h o n d r i a l c y c l i n g of calcium (148). Furthermore, a d i r e c t r e l a t i o n s h i p e x i s t s between the accumulation of m i t o c h o n d r i a l C a and the development of c o n t r a c t u r e i n myocardial muscle s t r i p s and the degree of v e n t r i c u l a r muscle s t i f f n e s s i n i n t a c t hearts (155)· Pretreatment of t i s s u e p r e p a r a t i o n s or i s o l a t e d h e a r t s with a v a r i e t y of calcium i n h i b i t o r y compounds i n c l u d i n g verapamil, d i l t i a z e m , n i f e d i p i n e , prenylamine, f e n d i l i n e and bencyclane prevents m i t o c h o n d r i a l s w e l l i n g and uncoupling of o x i d a t i v e phosphorylation and reduces i n t r a c e l l u l a r calcium (155, 156, 157)· The net r e s u l t s of these e f f e c t s are an i n c r e a s e i n adenine n u c l e o t i d e s , t i s s u e ATP, c r e a t i n e phosphate and improvement i n c a r d i a c f u n c t i o n (78). 2 +

3.

MUIR

Inhibitory

Calcium I n h i b i t o r y

Compounds

and

Compounds and

the

Cardiovascular

System

Hemodynamics

A p p l i c a t i o n of the knowledge of the e l e c t r o p h y s i o l o g i c , negative i n o t r o p i c , and v a s c u l a r smooth muscle e f f e c t s of calcium i n h i b i t o r y compounds i n i s o l a t e d t i s s u e preparations to normal and diseased animals or human p a t i e n t s i s d i f f i c u l t . The net hemodynamic e f f e c t s of calcium i n h i b i t o r y compounds vary considerably depending upon t h e i r d i v e r s e pharmacologic a c t i v i t i e s independent of C a i n h i b i t i o n , t h e i r dose response c h a r a c t e r i s t i c s , and t h e i r a b i l i t y to i n i t i a t e or i n h i b i t c e n t r a l nervous system r e f l e x mechanisms. The a d m i n i s t r a t i o n of calcium i n h i b i t o r y compounds to anesthetized or conscious animals and humans with a v a r i e t y of c a r d i a c diseases i s associated with a dose dependent negative i n o t r o p i c e f f e c t and decrease i n myocardial oxygen consumption (88, 89, 95, 112, 114, 158, 159, 160). In general and at appropriate dosages, indexes of myocardial f u n c t i o n i n c l u d i n g l e f t v e n t r i c u l a r pressure, l e f t v e n t r i c u l a r end d i a s t o l i c pressure, l e f t v e n t r i c u l a r stroke work, the f i r s t d e r i v a t i v e of l e f t v e n t r i c u l a r pressure ( d P / d t ) , maximum v e l o c i t y of c o n t r a c t i o n ( V ) , and a d i a s t o l i c r e l a x a t i o n constant are decreased, whereas coronary sinus oxygen s a t u r a t i o n , l e f t v e n t r i c u l a r end s y s t o l i c diameter, and cardiac output are increased (95_, 114, 159, 161, 162, 163) · Heart rate changes are unpredictable (161, 162). Various review a r t i c l e s have reported that calcium i n h i b i t o r y compounds cause an increase, decrease or no change i n heart rate (4, 6_ 95^, 151» 160). As the dose of calcium i n h i b i t o r y compound increases, however, tachycardia g e n e r a l l y develops (161)· The hemodynamic changes described can be r e l a t e d to the a b i l i t y of calcium i n h i b i t o r y compounds to produce p e r i p h e r a l v a s o d i l a t i o n ( a f t e r l o a d reduction) and the i n t e r p l a y between d i r e c t drug mediated and r e f l e x events. Calcium i n h i b i t o r y compounds demonstrate l i t t l e or no e f f e c t on venous capacitance. The a d m i n i s t r a t i o n of n i f e d i p i n e i n t o the l e f t coronary a r t e r y of normal human volunteers, f o r example, produces decreases i n d P / d t , maximum v e l o c i t y of c o n t r a c t i o n , and l e f t v e n t r i c u l a r stroke work (161). In c o n t r a s t , the intravenous a d m i n i s t r a t i o n of n i f e d i p i n e i n t o the i n t a c t c i r c u l a t i o n of conscious dogs or normal human v o l u n t e e r s f a i l s to produce major changes i n l e f t v e n t r i c u l a r stroke work, d P / d t or mean a o r t i c blood pressure since v a s o d i l a t i o n i s r e f l e x l y counteracted by i n c r e a s e s i n cardiac output and heart rate (159, 162). Studies conducted i n human p a t i e n t s with congestive heart f a i l u r e i n d i c a t e that the d i r e c t negative i n o t r o p i c and chronotropic e f f e c t s of n i f e d i p i n e are masked by r e f l e x sympathetic a c t i v a t i o n (165)· The hemodynamic e f f e c t s of verapamil are reportedly more v a r i a b l e than those e l i c i t e d by n i f e d i p i n e . Studies conducted i n dogs and humans r e v e a l i n o t r o p i c and v a s o d i l a t o r e f f e c t s s i m i l a r to those of n i f e d i p i n e except that r e f l e x t a c h y c a r d i a

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2 +

m a x

m a x

9

m a x

m a x

63

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64

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REGULATION

BY

CALCIUM

ANTAGONISTS

i s l e s s l i k e l y to occur (95, 156, 159, 164). The reduced l i k e l i h o o d of verapamil induced t a c h y c a r d i a i s a t t r i b u t e d to a more prominent d i r e c t negative chronotropic e f f e c t (164)» Heart r a t e e f f e c t s s i m i l a r to those reported f o r verapamil have been reported to occur a f t e r the intravenous a d m i n i s t r a t i o n of d i l t i a z e m to human p a t i e n t s and i n dogs administered verapamil, d i l t i a z e m , and 2 - s u b s t i t u t e d aminoindenes (5£, 95_, 140, 165) » The s i d e e f f e c t s reported i n p a t i e n t s r e c e i v i n g calcium i n h i b i t o r y compounds are an extension of the hemodynamic a c t i v i t y of t h i s group of compounds and g e n e r a l l y r e l a t e to drug potency and d i l a t i o n of r e g i o n a l v a s c u l a r beds (56, 151, 160). Depending upon dose, p o s t u r a l hypotension, d i z z i n e s s , headache, AV conduction disturbances, pulmonary edema, heart f a i l u r e and c o n s t i p a t i o n can occur (_6, 151, 166). Cardiac arrhythmias are a p o t e n t i a l problem and i f already present, could become more severe (161). The m a j o r i t y of these s i d e e f f e c t s are most f r e q u e n t l y reported f o l l o w i n g n i f e d i n e a d m i n i s t r a t i o n although a g r e a t e r long-term experience with a l l the calcium i n h i b i t o r y compounds i s required before a d e f i n i t e c o n c l u s i o n can be made regarding t h e i r s a f e t y . Despite the l i m i t e d knowledge concerning the usage of calcium i n h i b i t o r y compounds i n p a t i e n t s , s e v e r a l authors have adopted g e n e r a l p o l i c i e s regarding t h e i r a d m i n i s t r a t i o n . I t i s suggested that calcium i n h i b i t o r y compounds not be used or administered with c a u t i o n to p a t i e n t s with s i g n i f i c a n t SA or AV node disease, l e f t v e n t r i c u l a r outflow o b s t r u c t i o n , low s y s t o l i c blood pressure, paroxysmal n o c t u r n a l dyspnea or orthopnea (166). T h i s l i s t of c o n t r a i n d i c a t i o n s i s c e r t a i n to i n c r e a s e as the calcium i n h i b i t o r y compounds become more popular. In c o n c l u s i o n , calcium membrane f l u x e s and v a r i a t i o n s i n i n t r a c e l l u l a r calcium a c t i v i t y play a p i v o t a l r o l e i n maintaining normal smooth muscle and myocardial c e l l f u n c t i o n . The slow inward current ( l i ) c a r r i e d p r i n c i p a l l y by C a serves as the mechanism whereby e x t r a c e l l u l a r Ca^ can enter the myocardial c e l l to i n i t i a t e or t r i g g e r e x c i t a t i o n c o n t r a c t i o n c o u p l i n g . Although the mechanism(s) of calcium i n h i b i t o r y compounds are disputed, the net e f f e c t of t h e i r a d m i n i s t r a t i o n i s a r e d u c t i o n i n i n t r a c e l l u l a r calcium a c t i v i t y l e a d i n g to a v a r i e t y of b e n e f i c i a l and p o t e n t i a l l y d e l e t e r i o u s e f f e c t s . Because of the a b i l i t y of calcium i n h i b i t o r y compounds to regulate calcium a c t i v i t y and, t h e r e f o r e , a v a r i e t y of c e l l u l a r f u n c t i o n s , they are p o t e n t i a l l y b e n e f i c i a l as therapy f o r a n g i o s p a s t i c angina (167, 168), angina due to mechanical coronary a r t e r y o c c l u s i o n ( p l a t e l e t aggregation) (169), c a r d i a c arrhythmias (126), a r t e r i a l hypertension (170), acute or chronic l e f t v e n t r i c u l a r f a i l u r e (166), myocardial ischemia and i n f a r c t i o n (158, 165), myocardial s a l v a g i n g ( c a r d i o p l e g i a ) (155), cardiomyopathy (171) and v a r i o u s v a s o s p a s t i c syndromes (172). Compounds demonstrating calcium i n h i b i t o r y a c t i v i t y r e q u i r e f u r t h e r development and refinement 2 +

S

+

3.

MUIR

Inhibitory

Compounds

and

the Cardiovascular

System

before current understanding of the e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g process and the t o t a l usefulness of t h i s group of compounds as t h e r a p e u t i c agents can be f u l l y r e a l i z e d .

Literature Cited 1. 2. 3.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Fleckenstein, A. "Calcium and the Heart", Academic Press, New York, 1971, pp 135-188. Fleckenstein, A. Annu. Rev. Pharmacol. Toxicol. 1977, 17, 149-166. Church, J.; Zsoter, T. T. Can. J. Physiol. Pharmacol. 1980, 58, 254-264. Henry, P.D. "Trace Metals in Health and Disease", Raven Press, New York, 1979, pp 227-233. Zsoter, T. T. Am. Heart J. 1980, 99, 805-810. Henry, P. D. Am. J. Cardiol. 1980, 46, 1047-1058. Nayler, W. G.; Poole-Wilson, P. Basic Res. Cardiol. 1981, 76, 1-15. Fabiato, Α.; Fabiato, F. Annu Rev. Physiol. 1974, 41, 473-484. Langer, G. A. J. Mol. Cell. Cardiol. 1980, 12, 231-239. Ringer, S. J. Physiol. (Lond) 1882, 4, 29-42. McDonald, T. F.; Dieter, P.; Wolfgang, T. Circ. Res. 1981, 49, 576-583. New, W.; Trautwein, W. Pfluegers Arch. 1972, 334, 24-38. Trautwein, W.; McDonald, T. F.; Tripath, O. Pfluegers Arch. 1975, 354, 55-74. Fabiato, Α.; Fabiato, F. Circ. Res. 1977, 40, 119-129. Fabiato, Α.; Fabiato, F. Ann. Ν. Y. Acad. Sci. 1978, 307, 491-522. Langer, G. Α.; Frank, J. S. J. Cell. Biol. 1972, 54, 441-455. Langer, G. Α.; Frank, J. S. Am. J. Physiol. 1979, 237, H-239-H246. Fabiato, Α., Fabiato, F. Nature 1979, 281, 146-148. Thorens, S.; Endo, M. Proc. Japan Acad. 1975, 51, 473-478. Frank, J. S.; Langer, G. Α.; Nudd, L. M.; Seraydarian, Κ. Circ. Res. 1977, 41, 702-714. Langer, G. Α.; Frank, J. S.; Philipson, Κ. D. Circ. Res. 1981, 49, 1289-1299. Beeler, G. W. Jr.; Reuter, H. J. Physiol. 1970, 207, 211-229. Gibbons, W. R.; Fozzard, H. A. J. Gen. Physiol. 1975, 65, 367-384. Reuter, H. J. Physiol. 1967, 192, 479-192. Wollenberger, Α.; Will, H. Life Sci. 1978, 22, 1159-1178. Wray, H. L.; Gray, R. R. Biochim. Biophys. Acta 1977, 461, 441-459.

65

66

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

27.

Cheung, W. Y. Science 1980, 207, 19-27.

28.

Katz, S.; Remtulla, M. A. Biochem. Biophys. Res. Commun.

29.

B i l e z i k j i a n , L. M.; Kranias, E. G.; P o t t e r , J . D.; Schwartz, A. Fed. Proc. 1980, 1663, A b s t r a c t . B i l e z i k j i a n , L. M.; Kranias, E. G.; P o t t e r , J . D.; Schwartz, A. C i r c . Res. 1981, 49, 1356-1362. C o l l i n s , J . H.; Kranias, E. G.; Reeves, A. S.; Bilezikjian, L.M.; Schwartz, A. Biochem. Biophys. Res. Commun. 1981, 99, 796-803.

1978,

30. 31.

32.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

33. 34. 35. 36. 37. 38. 39.

8 3 , 1373-1379.

LePeuch, C. J . ; Haiech, J . ; Demaille, J . G. Biochemistry 1979, 18, 5150-5157. G l i t s c h , H. G.; Reuter, H.; Scholz, H. J. P h y s i o l . (Lond) 1970, 209, 25-43. Reuter, H. C i r c . Res. 1974, 34, 599-605. Coraboeuf, E.; Deroubaix, E.; Hoerter, J. Supp.1, C i r c . Res. 1976, 38, I92-I98. Diamond, J . M.; Wright, Ε . M. Annu. Rev. P h y s i o l . 1969, 1, 581-646. Kohlhardt, M.; Bauer, B.; Krause, H.; F l e c k e n s t e i n , A. P f l u e g e r s Arch. 1972, 335, 309-322. Payet, M. D.; Schanne, O. F.; R u i z - C e r e t t i , E.J.Mol. Cell. C a r d i o l . 1980, 12, 635-638. Rosenberger, L.; T r i g g l e , D. J. "Calcium in Drug A c t i o n " ; Weiss, G.B., Ed.; Plenum Press: New York, 1978; 3-31.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

S a n g u i n e t t i , M. C.; West, T. C. J . Pharmacol. Exp. Ther. 1981, 219, 715-722. Bristow, M. R.; Green R. D. Eur. J. Pharmacol. 1981, 45, 267-279. O'Hara Naoki; Ono H.; Oguro K,; Hashimoto, K. J. Cardiovasc. Pharmacol. 1981, 3, 251-268. Kaufmann, R. Munch. Med. Wochenschr. 199 Suppl 1977, 1, 6-11. Nagao, T, Sato, M, Nakajima, H, Kiyomoto, A. Chem. Pharm. Bull. 1973, 21, 92-97. F l e c k e n s t e i n , Α.; Tritthart, H. S.; Doring, H. J . ; Byon, Y. K. Arzneim. Forsch. 1972, 22, 22-33. Kodama, I . ; H i r a t a , Y.; Toyama, J . ; Yamada, K. J. Cardiovas. Pharmacol. 1980, 2, 145-153. W a r l t i e r , D. C.; M e i l s , C. M.; Gross, G. J.; Brooks, H. L. J . Pharmacola. Exp. Ther. 1981, 218, 296-302. Ledda, F.; M a n t e l l i , L.; Manzini, S.; Amerini, S.; M u g e l l i , A. J. Cardiovasc. Pharmacol. 1981, 3, 1162-1173. Gmeiner, R.; Ng, C. K. J . Cardiovasc. Pharmacol. 1981, 3, 237-250. Sutko, J . L.; W i l l e r s o n , J . T. C i r c . Res. 1980, 46, 332-343. S i e g a l , M S.; Gliklich, J . I.; Mary-Robine, L.; Hoffmann, B. F. J. Pharmacol. Exp. Ther. 1979, 211, 606-614.

3.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

52.

MUIR

Inhibitory

Compounds

and

the Cardiovascular

System

Piascik, M. F.; Piascik, M. T.; Witiak, D. T.; Rahwan, R. G. Can. J. Physiol. Pharmacol. 1979, 57, 1350-1358. 53. Rahwan, R. G.; Piascik, M. F.; Witiak, D. T. Can. J. Physiol. Pharmacol. 1979, 57, 443-460. 54. Zsoter, T. T. Am. Heart J. 1980, 99, 805-810. 55. Rahwan, R. G.; Faust, M. M.; Witiak, D. T. J. Pharmacol. Exp. Ther. 1977, 201, 126-137. 56. Sugiyama, S.; Kitazawa, M.; Kotaka, K.; Miyazaki, Y.; Ozawa, T. J. Cardiovasc. Pharmacol. 1981, 3, 801-806. 57. van Breemen, C.; Hwang, O.; Meisheri, K. D. J. Pharm. Exp. Ther. 1981, 218, 459-463. 58. Walus, K. M.; Fondacaro, J. D.; Jacobson, E. D. Circ. Res. 1981, 48, 692-700. 59. Wiggins, J. R. Circ. Res. 1981, 49, 718-725. 60. Dangman, Κ. H.; Hoffman, B. F. Am. J. Cardiol. 1980, 46, 1059-1067. 61. Danilo Jr, P.; Hordof, A. J.; Reder, R. F.; Rosen, M. R. J. Pharm. Exp. Ther. 1980, 213, 222-227. 62. Rosen, M. R.; Ilvento, J. P.; Gelband, H.; Merker, C. J. Pharm. Exp. Ther. 1974, 189, 414-422. 63. Ehara, T.; Kaufman, R. J. Pharmacol. Exp. Ther. 1978, 207, 49-55. 64. Reuter, H. Annu. Rev. Physiol. 1979, 41, 413-424. 65. Eckert R.; Tillotson D. L.; Brehm P. Fed. Proc. 1981, 40, 2226-2232. 66. Ito, S.; Surawicz B. Am. Physiol. Soc. 1981, H139-H144. 67. Lee, C. O.; Fozzard, H. A. Am. Physiol. Soc. 1979, C156-C165. 68. Colatsky, Τ J.; Hogan, P. M. Circ. Res. 1980, 46, 543-552. 69. Isenberg, G. Pfluegers Arch. 1977, 374, 77-85. 70. Siegal, M. S.; Hoffman, B. F. Circ. Res. 1980, 46, 227-236. 71. Noble, D.; Tsien, R. W. J. Physiol. 1968, 195; 185-214. 72. DiFrancesco, D. J. Physiol. 1981, 314, 377-393. 73. DiFrancesco, D.; Ohba, M.; Ojeda, C. J. Physiol. 1979, 297, 135-162. 74. Brown, H.; DiFrancesco, D. J. Physiol. 1980, 308, 331-351. 75. Singer, D. H.; Baumgarten, C. M.; Teneick, R. E. Prog. in Cardiovascl. Dis. 1981, 24, 97-156. 76. Bredikis, J.; Bukauskas, F.; Veteikis, R. Circ. Res. 1981, 49, 815-820. 77. DeMello, W. C. J. Physiol. 1976, 250, 171-197. 78. Watts, J. Α.; Koch, C. D.; LaNoue, K. F. Am. Physiol. Soc. 1980, H909-H916. 79. Rosen, M R.; Danilo Jr, P. Circ. Res. 1980, 46, 117-124. 80. Vassalle, M.; Mugelli, A. Circ. Res. 1981, 48, 618-631. 81. Cranefield, P. F.; Aronson, R. S. Circ. Res. 1974, 34, 477-481.

67

68

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82.

CALCIUM

REGULATION

BY

CALCIUM

ANTAGONISTS

Ten Eick, R. E.; Baumgarten, C. M.; Singer, D. H. Prog. in Cardiovascl. Dis. 1981, 24, 157-188. 83. Wit, A. L.; Cranefield, P. F.; Gadsby, D. C. Circ. Res. 1981, 49, 1029-1042. 84. Bayer, R.; Henekes, R.; Kaufman, R.; Mannhold, R. Naunyn-Schmiedeberg's Arch. Pharmacol. 1975, 290, 49-68. 85. Satoh, K.; Yanagisawa, T.; Taira, Ν. J. Cardiovasc. Pharmacol. 1980, 2, 309-318. 86. Endoh, M.; Yanagisawa, T.; Taira, Ν. NaunynSchmiedeberg's Arch. Pharmacol. 1978, 302, 235-238. 87. Gutovitz, A. L.; Cole, B.; Henry, P. D.; Sobel, Β. E.; Roberts R. Circulation 1977, 56, Suppl III, 111-179. 88. Raschack, M. Naunyn-Schmiedeberg's Arch. Pharmacol. 1976, 294, 285-291. 89. Raschak, M. Arzneim. Forsch (Drug Res.), 1976, 26, 1330-1333. 90. Bayer, R.; Rodenkirchen, R.; Kaufman, R.; Lee, J. H.; Hennekes R. Naunyn-Schmiedeberg's Arch Pharmacol, 1977, 301, 29-37. 91. Cranefield, P. F.; Aronson, R. S.; Wit, A. L. Circ. Res. 1974, 34, 204-213. 92. Nabata, H. Japan J. Pharmacol. 1977, 27, 239-249. 93. Kass, R. S.; Tsien, R. W. J. Gen. Physiol. 1975, 66, 169-192. 94. Nawrath, H.; Ten Eick, R. E.; McDonald, T. F.; Trantwein, W. Circ. Res. 1977, 40, 408-414. 95. Gibson, R.; Driscoll, D.; Gillette, P.; Hartley, C. Dev. Pharmacol. Ther. 1981, 2, 104-116. 96. Kawai, C.; Konishi, T.; Matsuyama, E.; Entman, M. L. "Adalat. New Experimental and Clinical Results"; Excerpta Medica: Amsterdam, 1979, 5-6. 97. Narimatsu, Α.; Taira, Ν. Naunyn-Schmiedeberg's Arch. Pharmacol. 1976, 294, 169-177. 98. Ono, H.; O'Hara, N. J. Cardiovas. Pharmacol. 1981, 3, 446-454. 99. Taira, Ν.; Narimatsu, A. Naunyn-Schmiedeberg's Arch. Pharmacol. 1975, 290, 107-112. 100. Zipes, D. P.; Fischer, J. C. Circ. Res. 1974, 34, 184-92. 101. Ono, H.; Himori, N.; Taira, Ν. Tohoku. J. Exp. Med. 1977, 121, 383-390. 102. Wit, A. L.; Cranfield, P. F. Circ. Res. 1977, 35, 413-425. 103. Katzung, B. G.; Morgenstern, J. A. Circ. Res. 1977, 40, 105-111. 104. Cranefield, P. F. Futura, 1975. 105. Wit, A. L.; Cranfield, P. F. Circ. Res. 1976, 38, 85-98. 106. Cranefield, P. F. Circ. Res. 1977, 41, 415-423. 107. Hordof, A. J.; Edie, R.; Malm, J. R. Hoffman, B. F.; Rosen, M. R. Circulation 1976, 54, 774-779. 108. Mary-Rabine, L.; Hardof, A. J.; Danilo, P. Jr; Malm, J.R.; Rosen, M.R. Circ. Res. 1980, 47, 267-277.

3. MUiR

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

109.

Inhibitory Compounds and the CardiovascularSystem69

Elharrar, V.; Gaum, W. E.; Zipes, D. P. Am. J. Cardiol. 1977, 39, 544-549. 110. Dersham, G. H.; Han, J. J. Pharmacol. Exp. Ther. 1981, 216, 261-264. 111. Mangiard, L. M.; Hariman, R. J.; McAllister Jr, R. G.; Bhargava, V.; Surawicz, B.; Shabetas, R. Circulation 1978, 7, 366-372. 112. Newman, R. K.; Bishop, V. S.; Peterson, D. F.; Leroux, Ε. J.; Horwitz, L. D. J. Pharmacol. Exp. Ther. 1977, 201, 723-730. 113. Piascik, M. F.; Rahwan, R. G.; Witiak, D. T. J. Pharmacol. Exp. Ther. 1979, 210, 141-146. 114. Gross, R.; Kirchheim, H.; von Olshausen, K. Drug Res. 1979, 29, 1361-1368. 115. Ogawa, K.; Wakamasu, Y.; Ito T.; Suzuki, T.; Yamazaki, N. Drug Res. 1981, 31, 770-773. 116. Bush, L. R.; Li, Y. P.; Shlafer, M.; Jolly, S. R.; Lucchesi, B. R. J. Pharm. Exp. Ther. 1981, 218, 653-661. 117. Fujimoto, T.; Peter, T.; Hamamoto, H.; Mandel, W. J. Am. J. Card. 1981, 48, 851-857. 118. Hattori, S.; Weintraub, W. W.; Agarwal, J. B.; Bodenheimer, M. M.; Banka, V. S.; Helfant, R. H. Am. J. Cardiol. 1980, 45, 485, Abstract. 119. Henry, P. D. "Ischemic Myocardium and Antianginal Drugs"; Winbury M. M., Abiko Y., Eds.; Raven Press: New York, 1979; 143-153. 120. Millard, R W. Chest 1980, 78, 193-199. 121. Peng, C.; Kane, J. J.; Straub, K. D.; Murphy, M. L. J. Cardiovas. Pharmacol. 1980, 2, 45-54. 122. Selwyn, A. P.; Welman, E.; Fox, K.; Horlock, P.; Pratt, T.; Klein, M. Circ. Res. 1979, 44, 16-23. 123. Sherman, L. G.; Liang, C.; Boden, W. E; Hood, W. B. Circulation 1979, 29, 60, Suppl II, Abstract. 124. Weishaar, R.; Ashikawa, K.; Bing, R. J. Am. J. Cardiol. 1979, 43, 1137-1143. 125. Sperelakis, N.; Schneider, J. A. Am. J. Cardiol. 1976, 37, 1079-1085. 126. Kirkler, D. M.; Rowland, E., "Calcium Antagonismus"; Fleckenstein A., Roskamm H, Eds.; Springer-Verlag: Berlin, 1980; 55-61. 127. El-Sherif, N.; Lazzarra, R. Circulation 1979, 60, 605-615. 128. Fandacaro, J. D.; Han, J.; Yoon, M. S. Am. Heart J. 1978, 98, 81-86. 129. King, R. M.; Zipes, D. P.; DeBnicoll, A. Circulation 1974, 50, Suppl III, 111-183, Abstract. 130. Schamroth, L. Cardiovasc. Res. 1971, 5, 419-424. 131. Ellrodt, G.; Chew, C. Y. C.; Singh, Β. N. Circulation 1980, 62, 669-679. 132. Pickering, T. G.; Goulding, L. Br. Heart Jr. 1978, 40, 851-855.

70

133. 134. 135. 136. 137.

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138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154.

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

Singh, Β. N.; Elldrodt, G.; Peter, C. Drugs 1978, 15, 169-197. Waxman, H. L.; Myerberg, R. J.; Appel, R, Sung, R. J. Am. J. Cardiol. 1980, 45, 482, Abstract. Spurrell, R. A. J.; Krikler, D. M.; Santon, Ε. Br. Heart J. 1974, 36, 256-264. Lynch, J. J.; Rahwan, R. G.; Witiak, D. T. J. Cardiovasc. Pharmacol. 1981, 3, 49-60. Gotsman, M. S.; Lewis, B. S.; Bakst, Α.; Mitha, A. S. Afr. Med. J. 1972, 46, 2017-2019. Hashimoto, K.; Taira, Ν.; Chiba, S.; Hashomoto Jr., K.; Endoh, M.; Kokobun, M.; Kokobun, H.; Iijima, T.; Kimura, T.; Kubota, K.; Oguro, K. Arzneim. Forsch. 1972, 22, 15-21. Adelstein, R. S.; Hathaway, D. R. Am. J. Cardiol. 1979, 44, 783-787. Franklin D,; Millard, R. W; Nagao, T. Chest 1980, 78, 200-204. Mannhold, R.; Zierden, P.; Bayer, R.; Rodenkirchen, R.; Steiner, R. Drug Res. 1981, 31, 773-780. Himori, N.; Ono, H.; Taira, Ν. Jpn. J. Pharmacol. 1976, 26, 427-435. Guazzi, M.; Olivari, M. T.; Polese, A.;Fiorentini, C.; Magrini, F.; Moruzzi, P. Clin. Pharmacol. Ther. 1977, 22, 528-532. Ito, Y.; Kuriyama, H.; Suzuki, H. Br. J. Pharmacol. 1978, 64, 503-510. Schmier, J.; Bruckner, V. B.; Mittman, V.; Wirth, R. K. The Third International Adalat Symposium. Amsterdam: Excerpta Medica, 1976, 42-9. Schmier, J.; VanAckern, K.; Bruckner, U. "The First Nifedipine Symposium"; Hashimoto, K.; Kimura, E.; Kobayashi, T., Eds.; Tokyo Press: Tokyo, 1975, 45-52. Zsoter, T. T.; Wolchinsky, C.; Henein, N. F.; Hol, C. Cardiovas. Res. 1977, 11, 353-357. Nagao, T.; Matlib, Μ. Α.; Franklin, D.; Millard, R. W.; Schwartz, A. J. Mol. Cell. Cardiol. 1980, 12, 29-43. Nayler, W. G.; Grau, Α.; Slade, A. Cardiovasc. Res. 1976, 10, 650-662. Smith, H. J.; Goldstein, R. Α.; Griffith, J. M.; Kent, Κ. M.; Epstein, S. E. Circulation, 1976, 54, 629-635. Cohen, L.; Gilula, Z.; Meier, P.; Lazaron, B.; Herbstman, D. J. Cardiovasc. Pharmacol. 1981, 3, 581-597. Magee, P. G.; Flaherty, J. T.; Bixler, T. J.; Glower, D.; Gardner, T. J.; Burley, B. H.; Goh, V. L. Circulation. 1979, 60, Suppl I, 151-157. Nayler, W. G.; Ferrari, R.; Williams, A. Am. J. Cardiol. 1980, 46, 242-248. Shen, A. C.; Jennings, R. B. Am. J. Pathol. 1972, 67, 417-440.

3. MUIR

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch003

155.

71 System Inhibitory Compounds and the Cardiovascular

Henry, P. D. "Ischemic Myocardium and Antianginal Drugs"; Winbury M. M.; Abiko Y., Eds.; Raven Press: New York, 1979, 143-153. 156. Vaghy, P. L.; Bor, P.; Szekeres, L. Biochem. Pharacol. 1980, 29, 1385-1389. 157. Bor, P.; Pataricza, J.; Vaghy, P. L.; Szekeres, L. Proc. Int. Union Physiol. Sci. 1980, 14, 333. 158. Atterhog, J. E.; Ekelung, L. G. Eur. J. Clin. Pharmacol. 1975, 8, 317-322. 159. Carlens, P. J. Cardiovasc. Pharmacol. 1981, 3, 1-10. 160. Stone, P. H.; Antman, E. M.; Muller, J. E.; Braunwald, E. Ann. Intern. Med. 1980, 93, 886-904. 161. Hollman, W.; Rost, R.; Liesen, H.; Emirkanian, O. "The Second Int. Adalat Symposium"; Lochner W.; Braasch W.; Kroneberg G., Eds.; Springer-Verlag: New York, 1976, 243-247. 162. Lichtlen, P. "The First Nifedipine Symposium"; Hashimoto, K; Kimura, E; Kobayashi, T, Eds.; Tokyo Press: Tokyo, 1975, 114-119. 163. Matsumoto, S.; Ito, T.; Sada, T.; Takanashi, M.; Su, Κ. M.; Ueda, Α.; Okabe, F.; Sato, M.; Sekine, I.; Ito, Y. Am. J. Cardiol. 1980, 46, 476-480. 164. Lewis, B. S.; Mitha, A. S.; Gofsman, M. S. Cardiology 1975, 60, 366-376. 165. Kusukawa, R.; Kinoshita, M.; Shimono, Y.; Tomonaga, G.; Hosino, T. Arzneim. Forsch. 1977, 27, 878-887. 166. Epstein, S. E.; Rosing, D. R. Circulation 1981, 64, 437-441. 167. Freedman, B.; Dunn, R. F.; Richmond, D. R.; Kelly, D. T. Circulation 1979, 60, Suppl II, 249, Abstract. 168. Hosada, S.; Kimura, E. "The Third International Adalat Symposium"; Jatene A. D.; Lichtlen P. R., Eds.; Excerpta Medica: Amsterdam, 1976, 174, 195-199. 169. Kaltenbach, M. "The First Nifedipine Symposium"; Hashimoto K.; Kimura E.; Kobayashi T., Eds.; Tokyo Press: Tokyo, 1975, 189, 126-135. 170. Blaustein M. P. Am. J. Physiol. 1977, 232, C165-C173. 171. Kaltenbach, M.; Hopf, R.; Kober, G.; Bussmann, W. D.; Keiler, M.; Petersen, Y. Br. Heart J. 1979, 42, 35-42. 172. Webb, R. C.; Bohr, D. V. Prog. Cardiovasc. Dis. 1981, 24, 213-242. RECEIVED June 16, 1982.

4 C o n t r o l of Intracellular C a l c i u m i n Smooth Muscle

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch004

Ε. E. DANIEL, A. K. GROVER, and C. Y. KWAN McMaster University Health Science Centre, Department of Neurosciences, Hamilton, Ontario, Canada L8N 3Z5 Control of intracellular calcium in smooth muscle cells is essential for control of the contractile state of the cells. Elevation of intra-cellular Ca is achieved by opening membrane channels for transport of Ca down its electrochemical gradient. The nature of these channels and their interaction with drugs including Ca antagonists are briefly considered. In some smooth muscles, elevation of intra-cellular Ca can occur by release of Ca sequestered in the cell; the role of this process in initiation of contraction is unclear. Lowering of intracellular Ca to maintain or allow relax­ ation may utilize a variety of mechanisms. The major focus of this article is to summarize and eval­ uate the data showing that the plasma membrane plays a major role in lowering intracellular Ca . This evidence has been obtained by isolating and purify­ ing plasma membrane vesicles and studying their trans­ port properties. They possess a vectorial ATP-depen­ dent Ca transport system capable of accumulating 1000-fold or higher Ca gradients. The properties of this system are described. Plasma membrane ve­ sicles also possess a Na - Ca exchange system and ATP-independent binding mechanisms. The direction of future research to evaluate the contributions of these systems to control of intra-cellular Ca is discussed. 2+ 2+ Control of intracellular Ca , [Ca ^], in uterine and other smooth muscles is essential for control of tension production. Studies of smooth muscles with plasma membranes damaged by glyce­ rol (1) or non-ionic detergents (2,3_,4) or of isolated contractile protein_^rom smo_oth muscle (5^,_6, 7^) all suggest that with lejss thgiji 10 M Ca . no active tension is produced while at 10 M Ca . or perhaps less, maximum active tension is produced. 0097-6156/82/0201-0073$06.00/0 © 1982 American Chemical Society 2+

2+

2+

2+

2+

2+

2+

2+

2+

+

2+

2+

74

CALCIUM

REGULATION

BY CALCIUM

ANTAGONISTS

E l e v a t i o n of f r e e Mg i n c r e a s e s the Ca . c o n c e n t r a t i o n r e q u i r e ment s l i g h t l y (4). Intermediate c o n c e n t r a t i o n s lead to graded changes i n t e n s i o n . £ljie obvious problem i s how the smooth muscle c e l l s can r e g u l a t e Ca ^ over t h i s range. 2+ E l e v a t i o n of [Ca .] Increases i n [Ca ±] a r e , at f i r s t g l a n c e , easy to achieve s i n c e the e x t e r n a l C a * c o n c e n t r a t i o n [Ca i l i s about 10 M, 100 to 10,000-fold higher than i n t e r n a l C a (< 10 M i n relaxed muscle) and the transmembrane e l e c t r i c a l gradient i s -40 to -60mv, i n s i d e negative. Opening of an inward Ca leak down tjj 150 mM and [ K ] i s about 5 mM, tljie e l e c t r o c h e m i c a l e q u i l i b r i u m f o r a membrane passing mostly Κ c u r r e n t s occurs at about -SjO mv. H y p e r p o l a r i ­ z a t i o n a c t i v a t e d by e l e v a t i o n of i n t e r n a l Ca i s a widespread phenomenon i n natur, 1,000-fold gradients of Ca across the v e s i c l e s membrane have been estimated. Since a trgijisvesicle membrane p o t e n t i a l was not knowingly produced, the Ca accumulations may represent the gradients p o s s i b l e with v o l t a g e operated channels open. On the other hand, these channels may ha/^e been damaged o r i n a c t i v a t e d during membrane i s o l a t i o n . Ca accumulated i n s i d e p r o p e r l y o r i e n t e d ves i c l e s i s slowly r e l e a s e d when they are d i l u t e d i n t o Ca-free or Ca-free m ^ i a ; i t i s r a p i d l y , almost instantaneously r e l e a s e d when the Ca - s e l e c t i v e ionophores (A23187, ionomycin, o r X537A) are added (31,35,36,39). In unpublished s t u d i e s we have found the Ca ATP-dependent transport by plasma membrane v e s i c l e s to be enhanced by calmodulin and i n h i b i t e d by phenothiazines and to be stimulated by c-AMP dependent p r o t e i n k i n a s e s . The mechanisms o f a c t i o n of these modulating systems and the p h y s i o l o g i c a l f u n c t i o n s remain to be determined.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch004

2

Na

+

- Ca^*" Exchange

These plasma membrane v e s i c l e s , a t Iç^st i n tljie two cases examined t o 95%

l

Vein:

Canine G a s t r i c Corpus: ( c i r c u l a r muscle)

^ 60%

31, 35 , 46, H-

H

70 - 80%

36

70 - 80%

33,

70 - 80%

38

70 - 80%

33,

70 - 80%

37

- 80%

39

^70

40

40

No f r a c t i o n s of endoplasmic r e t i c u l u m were so f a r obtained. > 50% p u r i t y . I ι Most of the impurity

i n t h i s f r a c t i o n appears to be a t t a c h ­

ed c o n t r a c t i l e p r o t e i n (31, 46). Table I I I P h y s i c a l P r o p e r t i e s of Rat Myometrium Plasma Membrane References 1.08 - 1.03 g/ml

Density: Size:

Smooth surface v e s i c l e s with c r o s s - s e c t i o n a l d i a ­ meters of 0.05 - 0.5 ym

Trapping Volume:

1 - 3 yl/mg p r o t e i n using sucrose and 4 - 5 yl/mg using i n u l i n (unpublished)

31

47^

Protein: Phospholipid

^ _1 (w/w) Ratio: 20% broken

Orientation: 40%

sealed

rightside-out

40%

sealed

inside-out

unpublished 47

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch004

84

CALCIUM

REGULATION

BY

CALCIUM

ANTAGONISTS

provide a complete c o n t r o l (49,5()) but may p a r t i c i p a t e i n regu­ l a t i n g i t by a f f e c t i n g Ca f l u x e s (30,51). Iiji the i s o l a t e d plasma membrane v e s i c l e s which we s t u d i e d , the Na -pump was not a c t i v a t e d (no ATP present) and the Na gradients were s h o r t l i v e d (1 to 3 minutes). A l s o , these i s o l a t e d v e s i c l e s may have no transmembrane p o t e n t i a l d i f f e r e n c e u n l i k e the i n t a c t c e l l ; so the t o t a l e l e c t r o c h e m i c a l gradient was le_ss. Furthermore, they probably have an increased leak of both Na and Ca through t h e i r membrane. The absence of ATP may have reduced the a f f i n i t y of exchange s i t e s f o r Ca . A l l these u n c e r t a i n t i e s l i m i t the conclusions about the p o t e n t i a l r o l e of Na - Ca ex­ change that should be drawn^at t h i s stage. Nevertheless, the maximum i n i t i a ^ r a t e of Ca transport (from a medium c o n t a i n i n g 40 μΜ f r e e Ca ) d r i v e n by the Na gradient was comparable to that e f f e c t e d by the^^TP-dependent Ca pump (from a medium con­ t a i n i n g 1 μΜ f r e e Ca ) (34). T h i s r a t e was obtained with a Na gradient of about 2 0 - f o l d . In the i n t a c t c e l ^ the Na -gradient i s probably about 10-fold and the i n t e r n a l C a concentration during r e l a x a t i o n i s 0.1 μΜ; so t h i s Na ~2§. exchange system may operate r a p i d l y only where i n t e r n a l Ca concentrations are elevated. 2+ 2 +

a

+

A f i n a l judgment cannot be made u n t i l Na - Ca exchange has been studied under optimal c o n d i t i o n s i n the presence of a maintained Na gradient i n i s o l a t e d plasma mem^r_anes and i n t a c t cells. Tljie stoichiometry, e l e c t r o g e n i c i t y , Ca -gradient depen­ dence, Na -gradient dependence, as w e l l as i n f l u e n c e of ATP and v a r i o u s r_egulat^.ng f a c t o r s need to be determined. V a r i a t i o n be­ tween Na - Ca exchange i n plasma membranes of d i f f e r e n t smooth muscles i s to be expected, adding to present u n c e r t a i n t y . A s i m ^ a r suggestion has been made that i n t e r n a l s e q u e s t r a ­ t i o n of Ca by mitochondria occurs onj-y at high i n t e r n a l Ca concentrations, based on t h e i r low Ca content i n s t u d i e s using x-ray d i f f r a c t i o n a n a l y s i s of s e c t i o n s r a p i d l y £r_ozen to minimize t r a n s l o c a t i o n s (52,53) and on s t u d i e s of the Ca dependence f o r Ca transport (54,55). In humgiji myometrium, however, i s o l a t e d mitochondria could transport Ca from concentrations l e s s than 1 μΜ (56). Endoplasmic Reticulum

2+ and Regulation of Ca .

Based l a r g e l y on analogy to seriated muscle, i t has become a dogma that the major s i t e of Ca s e q u e s t r a t i o n and transport i n smooth muscle i s i n the endoplasmic r e t i c u l u m (ER). The best p o s i t i v e evidence f o r t h i s c o n s i s t s of s t u d i e s by e l e c t r o n probe of r a p i d l y frozen muscles (52,53,57) ; so f a r only large_ blood v e s s e l s of guinea p i g have been studied and i n them Ca was r e ­ ported to be accumulate^ i n ER under c o n d i t i o n s when i n t r a c e l l u l a r Ca was increased by Κ - d e p o l a r i z a t i o n and even i n relaxed muscle. This method has important l i m i t a t i o n s ; i t would not

4.

DANIEL E T A L .

Intracellular

Calcium

in Smooth

Muscle

85

2+ de£e_ct a Ca transport f u n c t i o n by plasma membrane s i n c e the Ca i s t r a n s p o r t e ^ i n t o the e x t r a c e l l u l a r space. Also i t s a b i l i t y to r e s o l v e Ca bound to one surface of a b i o l o g i c a l mem-^^ brane i s not e s t a b l i s h e d . F i n a l l y no q u a n t i t a t i v e study of Ca t r a n s l o c a t i o n s during preparations o f specimens i s a v a i l a b l e . Other problems e x i s t f o r a p p l i c a t i o n of t h i s evidence to provide general support f o r the dogma. F i r s t , many smooth muscle c e l l s , e s p e c i a l l y small a r t e r i e s , v e i n s , and some gut muscles c o n t a i n a small volume o£ ER, i n s u f f i c i e n t to provide f o r accumulation of the necessary Ca . Second, t o f u n c t i o n as a major f u n c t i o n a l s i t e f o r Ca accumulâtio^ during r e p e t i t i v e c o n t r a c t i o n c y c l e s , ER must e i t h e r r e l e a s e Ca again a f t e r accumulation or i n some way t r a n s l o c a t e i t to the e x t e r i o r . No d i r e c t evidence f o r e i t h e r of these processes e x i s t s i n f u l l y studied smooth muscles (see above). T h i r d , no one has been able t o ^ ^ s o l a t e a h i g h l y p u r i f i e d ER f r a c t i o n with the appropriate Ca transport properties. Instead, crude microsomal f r a c t i o n s c o n t a i n i n g a l a r g e amount (probably i t s major £), a s i m i l a r mechanism of a c t i o n by p h y s i o l o ­ gical platelet agonists, e.g. ADP, epinephrine, thrombin, thromboxane A2, e t c . had not been demonstrated. 4 5

2 +

2

2 +

2 +

2 +

2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

2 +

2

4

ADP-stimulated Ca " " m o b i l i z a t i o n . On t h i ^ b a s i s , we i n v e s ­ tigated! ân increase Tn i n t r a c e l l u i a r C a as a p o s s i b l e mechanism of p l a t e l e t a c t i v a t i o n induced by ADP. In these studies i t was f i r s t demonstrated that ADP-stimulated shape change and aggregation i s not associated with a transmembrane Ca influx (20). This observation was subsequently confirmed by Massini and Luscher {21). Based on these f i n d i n g s i t was concluded that an e l e v a t i o n i n c y t o s o l i c C a in response to ADP must be derived from i n t r a - p l a t e l e t storage s i t e s . In order to pursue t h i s question we developed a technique which permitted the measurement of intracellular Ca movements i n i n t a c t p l a t e l e t s (22). This was accomplished through the use of a C a ^ - s e n s i t i v e fluorescent probe, c h l o r t e t r a c y c l i n e (CTC), and a photon counting m i c r o s p e c t r o fluorometer. CTC had been p r e v i o u s l y used as a means of monitoring changes in i n t r a c e l l u l a r C a b i n d i n g i n many c e l l systems i n c l u d i n g sarcoplasmic r e t i c u l u m (23), red blood c e l l s (24) and mitochon­ d r i a ( 2 5 ) . CTC i s known to form a h i g h l y f l u o r e s c e n t pH i n s e n sitive~~[pH 6 . 0 - 8 . 5 ) adduct when chelated with d i v a l e n t c a t i o n s bound to b i o l o g i c a l membranes (26). Even though CTC c h e l a t e s both M g and Ca , i t was p r e v i o u s l y reported using red blood c e l l s (24) and confirmed by our measurements i n p l a t e l e t s (22) that the observed fluorescence stems almost e n t i r e l y from the Ca-CTC adduct. The c h a r a c t e r i s t i c which makes CTC a v a l u a b l e i n t r a c e l l u l a r C a ? probe i s that the fluorescence i n t e n s i t y of the Ca-CTC complex markedly decreases when the i s released from the membrane to a more p o l a r environment, e . g . the c y t o s o l (27_). Consequently, r e l a t i v e changes i n c e l l u l a r fluorescence 2 +

2 +

2 +

2 +

+

2 +

2 +

+

156

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

can be used as an index of t r a n s i e n t a l t e r a t i o n s i n the a v a i l ­ a b i l i t y of i n t r a p l a t e l e t i o n i c c a l c i u m . Using t h i s procedure we demonstrated that the a d d i t i o n of ADP (1 μΜ) to p l a t e l e t r i c h plasma r e s u l t e d i n a m o b i l i z a t i o n of platelet C a (as evidenced by a decrease i n fluorescence) which occurred c o i n c i d e n t with a change in platelet shape (Figure 1). In order to e l i m i n a t e the p o s s i b i l i t y that the observed change in fluorescence represented alterations in C a * binding to the p l a t e l e t external membrane, the external Ca pool was e l i m i n a t e d by p r i o r a d d i t i o n of EGTA (3 mM). Furthermore, when the p l a t e l e t s were p r e t r e a t e d with ATP, which acts as a s p e c i f i c ADP antagonist (28), p l a t e l e t shape change and the change i n Ca-CTC fluorescence were both i n h i ­ bited. It can also be seen that s t i m u l a t i o n of p l a t e l e t shape change by the d i v a l e n t c a t i o n ionophore, A23187, was a s s o c i a t e d with a s i m i l a r r e d i s t r i b u t i o n of i n t r a p l a t e l e t C a . These f i n d i n g s provided evidence that p l a t e l e t a c t i v a t i o n induced by ADP i s mediated through an i n t e r n a l r e l e a s e of membrane bound Ca . T h i s notion was supported by a d d i t i o n a l studies i n which platelet water was totally exchanged with deuterium oxide (D2O) (29). D2O had been p r e v i o u s l y shown to block the r e l e a s e of C a from the sarcoplasmic r e t i c u l u m of barnacle muscle and inhibit muscular contraction (30). Since the p l a t e l e t DTS i s thought to f u n c t i o n i n a manner analogous to the sarcoplasmic reticulum i n muscle, we examined the p o s s i b i l i t y that D2O would have a s i m i l a r i n h i b i t o r y e f f e c t i n p l a t e l e t s . T h i s was indeed found to be the case, i . e . , D2O i n h i b i t e d both shape change and aggregation stimulated by ADP (29). Other studies have suggested that D?0 treatment (2^60%) can r e s u l t i n enhanced aggregation (31). In these experiments, however, aggregation was induced with the d i v a l e n t cation ionophore A23187, r a t h e r than ADP. Since i t i s known that D2O will also facilitate Ca-stimulated muscle contraction (32,33) the effects of D2O on ionophore-induced platelet a c t i v a t i o n would be c o n s i d e r a b l y d i f f e r e n t from i t s e f f e c t s on ADP-induced p l a t e l e t a c t i v a t i o n . Thus^ with A23187, D 0 would augment the e f f e c t s of a v a i l a b l e C a whereas with ADP, D2O would i n h i b i t C a release. 2 +

2

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

2

2 +

2 +

2

2

2

2

Epinephrine-stimulated C a * i n f l u x . Our r e s u l t s with D2O a l s o provided evidence that a d i f f e r e n t p l a t e l e t a g o n i s t ^ i . e . epinephrine, does not act through the same mechanism of C a m o b i l i z a t i o n as does ADP. In t h i s regard, D2O was not found to be effective in blocking epinephrine-induced platelet a c t i v a t i o n (29). T h e r e f o r e , we i n v e s t i g a t e d the possibility that epinephrine, in contrast to ADP, increases platelet membrane p e r m e a b i l i t y to C a . Using t r a c e amounts of Ca i t was demonstrated that epinephrine does i n f a c t induce a transmembrane C a i n f l u x which i s concomitant with the 2

2

2 +

2 +

4 5

9.

L E BRETON ET AL.

Cyclic

Nucleotides,

Τ 1

Ca, and Platelets

157

' % c h o n g e m light scatter (90°)

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

Δ

Δ23Ι87 I ' M F (sec) Figure 1. Relationship between light scattering ( ) and fluorescence ( ) of platelets containing chlortetracycline. (Reproduced, with permission, from Ref. 22. Copyright 1976, Biochemical and Biophysical Research Communications.) Human platelets were incubated at 25°C with 50 μΜ CTC for 40 min. The plasma was supplemented with 3 mM EGTA to prevent aggregation. Platelets from a portion of the plasma were then pelleted through silicone oil, andfluorescencewas determined in a microspectrofluorometer (400 nm excitation: 530 nm emission). A: ADP (1 μΜ) was added to the plasma, and samples were pelleted and analyzed forfluorescenceat 60, 130, and 220 s; each time point represents the mean ± SEM of 25 samples from three different blood donors. Platelet shape change in response to ADP (1 μΜ) was separately determined by measuring the intensity of scattered light at 90°C. The change in platelet shape represents a typical platelet response in the presence of 50 μΜ CTC. B: Inhibition of ADP induced shape change and plateletfluorescencechange by treatment with ATP (10 Μ). ADP (1 Μ) was added 60 s after ATP. Thefluorescencevalues at each time point represent the mean ± SEM of 16 samples from two different blood donors. C: Relationship between platelet shape change induced by A23187 (1 μΜ) and fluorescence change. The fluorescence values at each point represent the mean ± SEM of eight samples from two blood donors. μ

μ

CALCIUM

158

R E G U L A T I O N BY C A L C I U M

ANTAGONISTS

4 b

aggregation response (34_) (Figure 2 ) . This C a uptake was dependent upon the dose of epinephrine employed (10-0.1 μΜ) and was not stimulated by d-epinephrine which is biologically i n a c t i v e ( 3 A ) . Evidence that a change in p l a t e l e t membrane permeability to Ca was specifically mediated through a receptor i n t e r a c t i o n was provided by the f i n d i n g that the α - r e c e p t o r a n t a g o n i s t , phentolami ne, blocked C a uptake as well as p l a t e l e t aggregation (34). F i n a l l y , pretreatment of the platelets with v e r a p a m i l , "which selectively blocks Ca channels in c a r d i a c c e l l s , i n h i b i t e d both e p i n e p h r i n e - s t i m u l a t e d ^ C a uptake ( F i g u r e 3) and p l a t e l e t aggregation but d i d not i n h i b i t aggregation s t i m u l a t e d by ADP (34). The i n h i b i t i o n b^ verapamil could in turn be reduced by external Ca supplementation. These f i n d i n g s provided evidence that the mechanism by which epinephrine stimulates p l a t e l e t f u n c t i o n a l change i s through enhanced membrane p e r m e a b i l i t y to c a l c i u m . This e f f e c t of epinephrine appears to be in marked c o n t r a s t to the mechanism of A D P - s t i m u l a t i o n which i s presumably mediated through i n t e r n a l Ca release. 2 +

4 5

2 +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

2

2 +

ADP-stimulated Na* i n f l u x . Although the nature of the ADP i n t e r a c t i o n with p l a t e l e t receptors to induce calcium r e d i s t r i ­ bution i s unknown, i t i s c l e a r t h a t ADP does not penetrate the membrane (35). I t i s p o s s i b l e that the A D P - p l a t e l e t i n t e r a c t i o n involves the f l u x of other ions as part of the s t i m u l u s - t r a n s f e r pathway. In t h i s r e s p e c t , p l a t e l e t s , l i k e other c e l l s , maintain low i n t r a c e l l u l a r Na l e v e l s and high K l e v e l s (36J; presum­ a b l y , as a consequence of an Na e f f l u x pump. We therefore investigated the possibility that ADP-induced platelet a c t i v a t i o n i s associated with an a l t e r e d f l u x of N a . ADP added to r e s t i n g p l a t e l e t s induced a sudden r i s e i n N a uptake, an increase i n p l a t e l e t N a , and a decrease i n K (Figure 4) (37). E a r l i e r s t u d i e s had shown that p l a t e l e t water volume does not increase a f t e r ADP-induced aggregation (38), and using C 1 , we found no increase i n C 1 uptake (37). An uptake of Na was not seen after GDP a d d i t i o n . Furthermore, the uptake of N a occurred s i m u l t a n e o u s l y w i t h the onset of shape change and aggregation (37^,39^,40). On the other hand, epinephrine-induced aggregation, shown e a r l i e r to induce Ca uptake, had no e f f e c t to induce Na uptake (Figure 5) (39^,40). Consequently i t appears that c a t i o n i n f l u x i s agonist s p e c i f i c , at l e a s t with respect to a c t i v a t i o n by ADP or by epinephrine. Whether the uptake of Na associated with ADP-induced shape change and aggregation i s the primary s t i m u l u s - t r a n s f e r pathway of a c t i v a t i o n has not been e s t a b l i s h e d . Platelets maintain a membrane p o t e n t i a l (negative inside) (41), and a c t i v a t i o n may ensue i n a s s o c i a t i o n with a chancje i n membrane p o t e n t i a l and an increase i n p e r m e a b i l i t y to Na . A sudden +

+

+

+

2 2

+

+

3 6

3 6

+

2 2

4 5

2 +

2 2

+

9.

L E BRETON E T AL.

Cyclic

Nucleotides,

Ca, and Platelets

42

159

Epi ( I 0 M ) 6

a

Q-

JADP(IO" M) 6

o

Saline

σ O

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

60

150

240

Time (sec) Figure 2. or ADP.

Ca uptake associated with primary aggregation induced by epinephrine (Reproduced, with permission, from Ref. 34. Copyright 1980, The American Physiological Society.) 2+

Aspirin (1 mg/mL) or indomethacin (20 μΜ) treated human blood platelets were isolated by albumin density gradient centrifugation and resuspended in Tyrode buffer (pH 7.4) in which the ionic calcium concentration had been adjusted to 100 μΜ with CaCl . Thirty min after CaCl addition, the platelet suspension was supplemented with fibrino­ gen (0.1 mg/mL), I-human serum albumin, and Ca. Three min later, aggregation was induced by epinephrine (1 μΜ), or ADP (1 μΜ), and 1 mL aliquots of the platelet suspension" were withdrawn at 60, 150, and 240 s following the addition of aggregating agent. The platelets were then analyzed for Ca * uptake. Each point represents the mean ± SEM of 16 samples from four blood donors. 2

2

125

45

2

Figure 3. Inhibition of epinephrine-induced Ca uptake by verapamil in resus­ pended platelets. (Reproduced, with permission, from Ref. 34. Copyright 1980, The American Physiological Society.) 2+

Platelets were isolated and Ca uptake was determined (conditions were as stated in Figure 2). Resuspended platelets were incubated with verapamil (50 μΜ) for 1 min before addition of epinephrine (1 μΜ). Each point represents mean ± SEM of 28 samples from seven separate blood donors. 2+

CALCIUM REGULATION BY CALCIUM

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

160

ANTAGONISTS

Figure 4. Effect of ADP on platelet Na and K and Na 'space.' Platelet rich plasma was incubated at 37°C in the absence (panel A) or presence (panel B) of ouabain (1 μΜ). The arrow indicates the time of addition of ADP to an aliquot of the sample. Key: Φ, Na+ 'space'; Δ , Na content; and O , K content of the unstimulated platelets. The separate symbols aligned with the arrow depict the same measurements obtained 60 s after the addition of ADP. Samples were taken at slightly different times in four experiments; the time ranges are shown by the horizontal bars. Each point represents the mean ± SEM. (Reproduced, with permission, from Ref. 37. Copyright 1977, Elsevier Biomedical Press B.V.) +

22

+

+

22

+

+

9.

L E BRETON ET AL.

Cyclic

Nucleotides,

Ca, and Platelets

161

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

AN OVA TABLE SS DF MS F ADP 250 3 0833 BetweenEPI , OU 3 0038 ADP-14 124 t. ADP

Within EPI

620 105

005E

.567 124

004*

Epi -0828

ω Q) Q.

σ Φ c

Q.

Control t 60" Treatment

120"

180

Time after Agonist Figure 5. Effect of epinephrine on Ν a* uptake by platelets. Uptake of Na* (neq/ JO platelets) is based on an average value of 165 meq/1 of Ν a in cit rated plasma. Aggregation initiated by ADP 10 μΜ) or by epinephrine (fâ, 10 μΜ); values represent mean ± SEM of 10 experiments. (Reproduced, with permission, from Ref. 40. Copyright 1981, Elsevier Biomedical Press B.V.) 8

162

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

+

r i s e i n p l a t e l e t N a l e v e l s could f u n c t i o n to a c t i v a t e p l a t e ­ l e t s by d i s p l a c i n g C a ^ from bound s i t e s . This p o s s i b i l i t y i s c o n s i s t e n t with the f i n d i n g s of S t e i n e r and T a t e i s h i (42J that Ca^ s e q u e s t r a t i o n by p l a t e l e t membranes i s i n h i b i t e d by N a and enhanced by K . In t h i s connection we found that ouabain, which increases the Na level in p l a t e l e t s , induces an increased s e n s i t i v i t y to both ADP and to epinephrine a c t i v a t i o n (39). +

+

+

+

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

+

Involvement of Ca^* i n p o t e n t i a t e d aggregation. I t has been known f o r some time that Tôw doses of epinephrine wi 11 p o t e n t i a t e p l a t e l e t aggregation induced by ADP ( 4 2 , 4 4 ) . We next i n v e s t i g a t e d whether these same d i s t i n c t mechanisms of ADP and epinephrine are o p e r a t i v e during p o t e n t i a t i o n of aggregation by each agonist ( 4 5 ) . On t h i s b a s i s we examined whether the mechanism of epinephrine p o t e n t i a t i o n i s due to a l t e r a t i o n s i n membrane p e r m e a b i l i t y to C a ^ . I t was found that a requirement for epinephrine p o t e n t i a t i o n i s indeed a transmembrane Ca f l u x , since the p o t e n t i a t e d response was completely a b o l i s h e d by verapamil. This i n h i b i t i o n was i n turn p a r t i a l l y reversed by Ca supplementation. Based on these r e s u l t s , i t was oroposed that low doses of epinephrine induce an uptake of C a ^ which promotes the a b i l i t y of ADP to r e d i s t r i b u t e C a from i n t e r n a l Ca stores. This p o t e n t i a t i o n could proceed through a process of c a l c i u m - i n d u c e d - c a l c i u m r e l e a s e as has been suggested to occur i n the sarcoplasmic r e t i c u l u m of muscle c e l l s ( 4 6 ) . Support f o r t h i s notion was provided in a study which i n v e s ­ t i g a t e d the e f f e c t s of low dose epinephrine on i n t r a - p l a t e l e t C a ^ b i n d i n g . Using i n t a c t p l a t e l e t s and the C a - s e n s i t i v e probe CTC, we found that sub-aggregatory doses of epinephrine can indeed lead to a m o b i l i z a t i o n of i n t r a p l a t e l e t C a ^ , s t o r e s (47) (Figure 6 ) . An e s s e n t i a l feature of t h i s " e p i n e p h r i n e stimulated C a ^ , r e d i s t r i b u t i o n " was a transmembrane Ca^ f l u x , since the m o b i l i z a t i o n process was blocked by v e r a p a m i l . Thus, i t appears that an i n i t i a l increase i n i n t r a p l a t e l e t C a , as a consequence of C a ^ uptake, can i n turn promote the release of C a from i n t e r n a l b i n d i n g s i t e s . The s i g n i ­ f i c a n c e of the i n t e r n a l C a ^ r e l e a s e observed with epinephrine i s u n c l e a r . Presumably i t does not c o n s t i t u t e a major source of the increase i n c y t o s o l i c C a ^ , s i n c e D^O which would be expected to s t a b i l i z e the DTS does not block epinephrine-induced aggregation. Rather, this ability of C a to stimulate additional Ca^ release may serve as an amplification mechanism by which the e f f e c t of C a ^ i n f l u x i s enhanced. +

2 +

2 +

+

2 +

2 +

+

2 +

+

+

+

2 +

+

2 +

+

+

2 +

+

+

d

Ca m o b i l i z a t i o n by metabolites of arachadonic a c i d . One p o s s i b l e mechanism by which t h i s a m p l i f i c a t i o n process could proceed i s through C a ^ a c t i v a t i o n of enzymes involved i n arachadonic a c i d (AA) metabolism. In t h i s regard, i t i s known that blood p l a t e l e t s metabolize endogenous AA to the potent +

9.

LE BRETON ET AL.

Cyclic

Nucleotides,

Ca, and

163

Platelets

platelet stimulant thromboxane A2 (TXA?) (48). Thus, AA which i s e s t e r f i e d i n the phospholipid pool of the p l a t e l e t i s l i b e r a t e d by the a c t i o n of phospholipase C (or A?) (49,50). The f r e e AA i s then acted upon by a e y e l o - o x y g é n a s e r e s u l t i n g i n the production of the p r o s t a g l a n d i n endoperoxide, p r o s t a g l a n d i n G2 ( P G G 2 ) . PGG2 i s i n t u r n r a p i d l y converted to p r o s t a g l a n d i n H2 ( P G H 2 ) which serves as substrate f o r the enzyme thromboxane synthetase. The rate l i m i t i n g step i n T X A 2 prod u c t i o n appears to be at the l e v e l of the phospholipase. Since phospholipase C or A 2 are C a - a c t i v a t e d enzymes (49,50J, it i s p o s s i b l e that the a b i l i t y of C a to cause a d d i t i o n a l " ( H r r e l e a s e may, at l e a s t i n p a r t , be mediated through C a - s t i m u lated TXA2 production. The T X A 2 so produced may then act to d i r e c t l y release C a from the p l a t e l e t DTS (51). 2 +

2 +

2

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

2 +

T h i s notion was examined by measuring"the e f f e c t s of the cyclo-oxygenase i n h i b i t o r , i ndomethacin, on e p i n e p h r i n e stimulated Ca redistribution. It was found (47), that i n h i b i t i o n of endogenous AA metabolism did i n f a c t s u b s t a n t i a l l y reduce the C a m o b i l i z a t i o n process (Figure 7). Consequently, it appears that at least a portion of the observed Ca release stimulated by epinephrine i s mediated through TXA2 production. Furthermore, d i r e c t evidence to support the notion that TXA2 is capable of causing Ca release within the p l a t e l e t was provided by the f i n d i n g that a d d i t i o n of the s t a b l e TXAg "mimetic", U46619, to intact platelets causes a m o b i l i z a t i o n of i n t e r n a l C a stores (Figure 8). In summary, i t appears that p h y s i o l o g i c a l stimulants of platelet function, e.g. ADP, epinephrine and T X A 2 all act through a Ca mediated process. Recently, Feinstein (52) demonstrated that the a b i l i t y of thrombin to cause plateTêt s e c r e t i o n i s a l s o r e l a t e d to the r e l e a s e of i n t r a p l a t e l e t membrane bound C a . On t h i s b a s i s , i t would seem that i n t r a platelet cytosolic Ca as a second messenger is directly l i n k e d to the state of p l a t e l e t a c t i v a t i o n . 2 +

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Inhibition of platelet monophosphate (cAffFT

function

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One of the f i r s t i n d i c a t i o n s that cAMP might be i n v o l v e d in the regulation of platelet function was provided by experiments of Marcus and Zucker (53). In these studies i t was shown that the addition of cAMF" to platelets resulted in i n h i b i t i o n of p l a t e l e t aggregation. T h i s notion was supported by subsequent studies of A r d l i e et a l . (54) who demonstrated that phosphodiesterase inhibitors a l s o "THocked ADP-induced p l a t e l e t aggregation. It now appears that many of the agents, in particular the prostaglandins, which inhibit platelet a c t i v a t i o n , produce t h e i r e f f e c t through i n c r e a s e s i n p l a t e l e t cAMP l e v e l s . Thus, numerous i n v e s t i g a t o r s have demonstrated that p r o s t a g l a n d i n (PGE^) i s a potent stimulant of the

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

164

Figure 6. Effect of verapamil on Ca * mobilization in response to epinephrine. 2

Human platelets were incubated at 25°C with 50 Μ CTC for 40 min. CTC-treated platelets were incubated for 180 s with saline or verapamil (25 μ Μ ) alone, saline plus epinephrine (0.1 μΜ), or verapamil (25 μΜ) plus epinephrine (0.1 Μ). Platelets were then pelleted through silicone oil, and pellet fluorescence was determined as in Figure 1. A decrease influorescencecounts relative to control indicates mobilization of platelet Ca *. The fluorescence values are represented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

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Figure 7. Effect of indomethacin on Ca * mobilization in response to epine­ phrine. 2

Platelets were incubated with CTC and fluorescence determined as described in Figure 6. CTC-treated platelets were incu­ bated with saline or indomethacin (20 Μ) alone, saline plus epinephrine (0.1 μΜ), or indomethacin (20 Μ) plus epinephrine (0.1 μΜ). A decrease influorescencecounts relative to control indicates mobilization of platelet Ca *. Fluorescence values are repre­ sented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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Platelets were incubated with CTC and fluorescence determined as described in Figure 6. Platelets were then treated with saline (control), ADP (0.5 Μ), or U46619 (0.5 μΜ). A decrease influorescencecounts relative to control indicates mobilization of platelet Ca \ The fluorescence change in response to the ADP is indicated for com­ parative purposes. Fluorescence values are represented as counts/s and are the mean ± SEM of 16 samples from four separate blood donors.

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

9.

LE BRETON ET AL.

Cyclic

Nucleotides,

Ca, and

165

Platelets

p l a t e l e t adenylate cyclase (55-59). Furthermore, more recent studies have i n d i c a t e d that tïïe i n h i b i t o r y properties of prostaglandin D2 (PGD2) and p r o s t a c y c l i n ( P G I 2 ) are also p r i m a r i l y due to s t i m u l a t i o n of adenylate c y c l a s e a c t i v i t y (60,6^,62^. A d d i t i o n a l evidence to support t h i s concept i s provided by the f i n d i n g , that the e f f e c t s of the i n h i b i t o r y prostaglandins are markedly p o t e n t i a t e d by phosphodiesterase i n h i b i t o r s (63). F i n a l l y , the i n f l u e n c e of cAMP on p l a t e l e t r e a c t i v i t y i s c l e a r l y demonstrated by the f i n d i n g , that an increase of only 25% i n p l a t e l e t cAMP l e v e l s i s associated with a complete i n h i b i t i o n of maximal aggregation induced by e i t h e r AA or U46619 ( 6 4 ) . At present then, there i s a s u b s t a n t i a l body of evidence which suggests that increases i n p l a t e l e t cAMP l e v e l s are d i r e c t l y l i n k e d to i n h i b i t i o n of p l a t e l e t f u n c t i o n . In s p i t e of t h i s , however, the mechanism by which these i n h i b i t o r y e f f e c t s are produced remains u n c l e a r . 2

R e l a t i o n s h i p between C a * and cAMP There have been two t h e o r i e s proposed which attempt to e x p l a i n the u n d e r l y i n g mechanism by which cAMP i n h i b i t s blood platelet function. The f i r s t model suggests that cAMP i s d i r e c t l y involved i n the r e g u l a t i o n of c y t o s o l i c C a levels. Using an i s o l a t e d p l a t e l e t membrane f r a c t i o n r i c h i n the DTS, Kaser-Glanzmann et a l . (65) demonstrated that the C a accumu­ l a t i n g a c t i v i t y of the i s o l a t e d v e s i c l e s was markedly s t i m u l a t e d by cAMP. These r e s u l t s provided evidence that cAMP may act to promote the movement of C a from the p l a t e l e t c y t o s o l i n t o the platelet DTS, and were c o n s i s t e n t with the previous suggestion of White et à]_. ( 1 3 ) . The net e f f e c t of t h i s uptake mechanism would be a decrease i n the a v a i l a b i l i t y of free C a f o r the s t i m u l a t i o n of shape change, aggregation and s e c r e t i o n . D i r e c t support f o r t h i s concept was provided by experiments measuring C a binding in intact p l a t e l e t s . I t was found that a l t e r a t i o n s i n p l a t e l e t cAMP l e v e l s , induced by e i t h e r PGE^ or P G I 2 » were d i r e c t l y r e l a t e d to i n t r a p l a t e l e t C a seques­ t r a t i o n (47) (Figure 9) as i n d i c a t e d by CTC f l u o r e s c e n c e . Furthermore, the e f f e c t s of PGE^ or P G I on cAMP l e v e l s and Ca b i n d i n g were augmented by pretreatment of the p l a t e l e t s with the phosphodiesterase i n h i b i t o r RO 1724. In a d d i t i o n , i t was found that the increases i n cAMP, stimulated by e i t h e r PGEi or P G I 2 , were associated with an i n h i b i t i o n of C a m o b i l i z a t i o n induced by e p i n e p h r i n e , A23187 or U46619 ( F i g u r e 10). Thus, i t appears that one consequence of increased cAMP production i s a " s t a b i l i z a t i o n " of i n t r a p l a t e l e t C a pools. In t h i s c o n n e c t i o n , however, these experiments d i d not d i f f e r e n ­ t i a t e between i n h i b i t e d C a r e l e a s e or enhanced Ca? r e s e questration. Consequently, i t i s p o s s i b l e that cAMP prevents the discharge of C a from the DTS i n a manner analogous to the proposed mechanism of i n h i b i t i o n by l o c a l a n e s t h e t i c s ( 6 6 ) , 2 +

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

166

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

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centrations of A23187 (68-72). Consequently^ i f the rate of C a release J S greater than the rate of Ca^ resequestration, cytosolic C a l e v e l s w i l l r i s e . 3) In F i g u r e 10 i t can be seen that PGE^ or PGI treatment actually increased C a binding above c o n t r o l levels. If cAMP were only a c t i n g to block the release of C a i n the stimulated p l a t e l e t , such i n c r e a s e s i n C a binding would not be expected. Furthermore, in the presence of PGE^ or PGI plus a phosphodiesterase inhibitor, epinephrine (which increases platelet membrane permeability to C a ) induced a further increase in Ca binding (Figure 10). Thus, Ca influx induced by epinephrine would provide a d d i t i o n a l C a f o r cAMP promoted C a sequestion w i t h i n the p l a t e l e t . 4) Low doses of p l a t e l e t a g o n i s t s , e . g . ADP, AA, U46619, e t c . induce r e v e r s i b l e aggregation. Consequently, a mechanism f o r the r e s e q u e s t r a t i o n of C a (or C a e f f l u x ) must e x i s t i n the p l a t e l e t (13,73). z +

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

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In t h i s c o n n e c t i o n , i t has been shown that PGT^ not only i n h i b i t s p l a t e l e t aggregation but a l s o reverses the aggregation process (74). T h i s e f f e c t can be r e a d i l y explained on the b a s i s t h a £ cAMP-stimulated C a r e s e q u e s t r a t i o n exceeds the rate of Ca^ release. Furthermore, i t has a l s o been demonstrated that the specific TXA /PGH receptor a n t a g o n i s t , 13-azaprostanoic a c i d (13-APA) (75), causes r e v e r s a l of both p l a t e l e t shape change and aggregation i n response to AA or U46619 (76,77). Since 13-APA i s capable of b l o c k i n g the r e l e a s e of Ca^ " i n response to TXA i n isolated platelet vesicles (78), the a b i l i t y of 13-APA to reverse p l a t e l e t a c t i v a t i o n i s presumably due to a sudden inhibition of TXA -stimulated Ca release. This inhibition, i n the face of continued C a resequestration, ultimately leads to a lowering of i n t r a p l a t e l e t C a levels (Figure 11). That the mechanisms of p l a t e l e t d e a c t i v a t i o n by 13-APA and PGI are indeed separate, i s demonstrated by the f i n d i n g that the a b i l i t y of 13-APA to cause deaggregation is p o t e n t i a t e d by PGI (79). Consequently, the combined e f f e c t of each i n d i v i d u a l mechanism of i n a c t i v a t i o n , i . e . i n h i b i t e d C a * rel ease with 13-APA, and cAMP promoted Ca^ r e s e q u e s t r a t i o n with P G I , i s more e f f e c t i v e i n reducing c y t o s o l i c Ca l e v e l s than e i t h e r mechanism alone. z +

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These observations strongly support the concept that cAMP i n h i b i t i o n of p l a t e l e t f u n c t i o n i s mediated through enhanced

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Figure 10. Effect of PGE or PGI on Ca * mobilization in response to epinephrine, A23187 or Ό46619. {Reproduced, with permission, from Ref. 47. Copyright 1981, The American Physio­ logical Society.)

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LE BRETON ET AL.

Cyclic

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Platelets

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Ca sequestration. Althouah the mechanism by which cAMP may promote the b i n d i n g of C a ^ w i t h i n the p l a t e l e t i s unknown, t h i s uptake mechanism has been a s s o c i a t e d with the phosphoryla­ t i o n of a s p e c i f i c 22,000 dalton p r o t e i n ( 7 2 , 8 0 ) . In t h i s regard, i t has been p o s t u l a t e d (80) that p l a t e l e t s c o n t a i n an ATP-dependent C a pump, and furthermore, t h a t the e f f i c i e n c y of t h i s pump i s enhanced through phosphorylation of a 22,000 molecular weight p r o t e i n by a cAMP dependent p r o t e i n k i n a s e . An a l t e r n a t i v e mechanism by which cAMP may act to i n h i b i t p l a t e l e t f u n c t i o n was proposed by Hathaway et a l . (81). In t h i s model (Figure 12), i t i s suggested that an increase i n cAMP r e s u l t s i n i n h i b i t i o n of myosin phosphorylation and consequent i n h i b i t i o n of p l a t e l e t c o n t r a c t i l e a c t i v i t y ( s i n c e unphosphorylated myosin cannot i n t e r a c t with a c t i n ) . Thus, i t was proposed that cAMP causes the a c t i v a t i o n of a p r o t e i n kinase which i n turn phosphorylates myosin k i n a s e . In the phosphoryl a t e d form, myosin kinase i s less capable of binding calmodulin and therefore i s not as e f f e c t i v e i n phosphorylating myosin. Although t h i s model would e x p l a i n the a b i l i t y of cAMP to i n t e r f e r e with p l a t e l e t a c t i v a t i o n , the r e s u l t s to date were obtained using p u r i f i e d p l a t e l e t myosin k i n a s e . Consequently, more c o n c l u s i v e proof to support t h i s mechanism of i n h i b i t i o n would r e q u i r e the demonstration that cAMP dependent phosphoryla­ t i o n of myosin kinase occurs under more p h y s i o l o g i c a l c o n d i t i o n s , e . g . in i n t a c t p l a t e l e t s or p l a t e l e t membrane fragments. In a d d i t i o n to the p o s s i b l e i n t e r a c t i o n s of C a and cAMP o u t l i n e d above, c e r t a i n experimental r e s u l t s have also suggested that C a may a c t u a l l y r e g u l a t e cAMP p r o d u c t i o n . In t h i s connection, i t was demonstrated by Rodan and F e i n s t e i n (82) that Ca i s a potent i n h i b i t o r of adenylate c y c l a s e ( K i = 16 μΜ) in i s o l a t e d p l a t e l e t membranes. Support f o r t h i s concept comes from two l i n e s of evidence: 1) agents which increase c y t o s o l i c Ca l e v e l s e . g . ADP, e p i n e p h r i n e , TXA?, e t c . , reduce the e l e v a t i o n i n cAMP s t i m u l a t e d by e i t h e r PGEi or P G I (59,83); and 2) the C a antagonist TMB-8 i n h i b i t s the a b i l i t y of T X A 2 to lower cAMP ( 8 3 ) . The p h y s i o l o g i c a l s i g n i f i c a n c e of these f i n d i n g s i s u n c l e a r . However, previous r e s u l t s have suggested that C a may be involved i n other p o s i t i v e feedback systems enhancing p l a t e l e t r e a c t i v i t y , e . g . C a - i n d u c e d Ca release or C a a c t i v a t i o n of phospholipase C or A ^ . The reported a b i l i t y of C a to i n h i b i t adenylate c y c l a s e a c t i v i t y may therefore represent an a d d i t i o n a l amplification mechanism, whereby s l i g h t increases i n c y t o s o l i c C a levels lead to a d d i t i o n a l net C a mobilization. In t h i s case, Ca inhibition of cAMP production would promote platelet a c t i v a t i o n by e i t h e r decreasing the r a t e of C a resequest r a t i o n or decreasing phosphorylation of myosin k i n a s e . +

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

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CALCIUM REGULATION BY CALCIUM

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ANTAGONISTS

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

shape change, aggregation

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Possible mechanism of calcium release and sequestration.

PROTEIN KINASE (inactive)
P 0 - MYOSIN KINASE (inactive) 4

ADP > P O 4 - MYOSIN Scheme according to Hathaway et al. (8 1 ).

171 Cyclic Nucleotides, Ca, and Platelets

9. LE BRETON ET AL.

Conclusions The above findings thereforç suggest that there is an intimate relationship between Ca and cAMP in the human blood platelet. In this respect, evidence has been presented which indicates that many, if not all, physiological platelet agonists mediate their effects through a common intracellular messenger, i.e. ionic Ca . Furthermore, it is clear that the mechanisms for this intraplatelet Ca mobilization are indeed complex. Thus, an initial increase in intraplatelet Ca , whether as a consequence of enhanced Ca membrane permeability induced by epinephrine or direct Ca release by U46619, results in additioiial Ca mobilization by activation of AA metabolism or by Ca -induced-Ca release. The ability of cAMP to modulate platelet function also appears, at least in part, to be due to regulation of cytosolic Ca levels, possibly through direct stimulation of Ca^ sequestration. 2

2+

2+

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Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch009

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+

Acknowledgements This work was supported in part by a Grant-in-Aid from the American Heart Association. The expert typing services of the Word Processing Center are greatly appreciated. Literature Cited 1. Holmsen, H.; Day, H.J.; Storm, E. Biochim. Biophys. Acta 1969, 186, 254-266. 2. Moore, S.; Pepper, D.S.; Cash, J.D. Biochim. Biophys. Acta 1975, 379, 370-384. 3. White, J.G. "Platelet Aggregation"; Caen, J., Ed.; Masson et Cie: Paris, 1971; 15-52. 4. Behnke, O. J. Ultrastruct. Res. 1965, 13, 469-477. 5. White, J.G. "The Circulating Platelet"; Johnson, S.A., Ed.; Academic Press: New York, 1971; 45-121. 6. Behnke, O. Anat. Rec. 1967, 158, 121-8. 7. White, J.G.: Am. J. Pathol. 1972, 66, 295-312. 8. Grette, K. Acta Physiol. Scand. 1962, 56 Supplement 195, 1-93. 9. Gordon, J.L.; Milner, A.J. "Platelets in Biology and Pathology"; Dingle, J.T., Ed.; North-Holland: New York, 1976; 3-22. 10. Holmsen, H. "Platelets, Production, Function, Transfusion and Storage"; Baldini, M. and Ebbe, S., Eds.; Grune and Stratton: New York, 1974; 207-220. 11. Massini, P; Luscher, E.F. Biochim. Biophys. Acta 1974, 372, 109-121. 12. Feinman, R.D.; Detwiler, T.C. Nature London 1974, 249, 172-173.

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172

CALCIUM REGULATION BY CALCIUM ANTAGONISTS 13. White, J.G.; Rao, G.H.R.; Gerrard, J.M. Am. J. Path. 1974, 77, 135-149. 14. Worner, P.; Brossmer, R. Thrombosis Res. 1975, 6, 295-305. 15. Feinstein, M.B.; Fraser, C. J. Gen. Physiol. 1975, 66, 561-581. 16. Murer, Ε.H.; Stewart, G.J.; Rausch, Μ.Α.; Day, H.J. Thrombos. Diathes. Haemorrh. 1975, 34, 72-82. 17. Kinlough-Rathbone, R.L.; Cahil, Α.; Packham, M.A.; Reimers, H.J.; Mustard, J.F. Thrombosis Res. 1975, 7, 435-449. 18. Pressman, B.C. Fed. Proc. 1973, 32, 1698-1703. 19. Reed, P.W.; Lardy, H.A. J. Biol. Chem. 1972, 247, 6970-7. 20. Le Breton, G.C.; Feinberg, H. Pharmacologist l974, 16, 699. 21. Massini, P.; Luscher, E.F. Biochim. Biophys. Acta 1976, 436, 652-663. 22. Le Breton, G.C.; Dinerstein, R.J.; Roth, L.J.; Feinberg, H. Biochem. Biophys. Res. Comm. 1976, 71, 362-370. 23. Caswell, A.H.; Warren, S. Biochem. Biophys. Res. Comm. 1972, 46, 1757-1763. 24. Hallet, M.; Schneider, A.S.; Carbone, Ε. J. Memb. Biol. 1972, 10, 31-44. 25. Luchra, M.; Olson, M.S. Biochim. Biophys. Acta 1976, 440, 744-758. 26. Caswell, A.H. J. Memb. Biol. 1972, 7, 345-364. 27. Caswell, A.H.; Hutchinson, J.D. Biochem. Biophys. Res. Comm. 1971, 42, 43-9. 28. Macfarlane, D.E.; Mills, D.C.B. Blood 1975, 46, 309-320. 29. Le Breton, G.C.; Sandler, W.C.; Feinberg, H. Thrombosis Res. 1976, 8, 477-485. 30. Kaminer, B.; Kimura, J. Science 1972, 176, 406-7. 31. Menche, D.; Israel, Α.; Karpatkin, S. J. Clin. Invest. 1980, 66, 284-291. 32. Eastwood, Α.Β.; Grundfest, H.; Brandt, P.W.; Reuben, J.P. J. Membrane Biol. 1975, 24, 249-263. 33. Sandow, Α.; Pagala, M.K.D.; Sphicas, E.C. Biochim. Biophys. Acta 1976, 440, 733-743. 34. Owen, N.E.; Feinberg, H.; Le Breton, G.C. Am. J. Physiol. 1980, 239, H483-8. 35. Born, G.V.R.; Feinberg, H. J. Physiol. 1975, 251, 803-816. 36. Moake, J.L.; Ahmed, K.; Sachur, N.R.; Gutfreund, D.E. Biochim. Biophys. Acta 1970, 211, 337-344. 37. Feinberg, H.; Sandler, W.C.; "Scorer, M.; Le Breton, G.C.; Grossman, B.; Born, G.V.R. Biochim. Biophys. Acta 1977, 470, 317-324. 38. Feinberg, H.; Michael, H.; Born, G.V.R. J. Lab. Clin. Med. 1974, 84, 926-934.

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9. LE BRETON ET AL.

Cyclic Nucleotides, Ca, and Platelets 173

39. Sandler, W.; Le Breton, G.C.; Feinberg, H. Biochim. Biophys. Acta 1980, 600, 448-455. 40. Feinberg, H.; LeBreton, G.C. "Cell Membrane in Function and Dysfunction of Vascular Tissue"; Godfraind, T. and Meyer, P., Eds.; Elsevier/North Holland: New York, 1981; 129. 41. Wencel, J.; Feinberg, H. Circ. 1979, 58, supplement 2, 124. 42. Steiner, M.; Tateishi, T. Biochim. Biophys. Acta 1974, 367, 232-246. 43. Ardlie, N.G.; Glew, G.; Schwartz, C.J. Nature 1966, 212, 415-7. 44. Born, G.V.R.; Mills, D.C.B.; Roberts, G.C.K. Proc. Physiol. Soc. 1967, 17, 43-4. 45. Owen, N.E.; Le Breton, G.C. Thrombosis Res. 1980, 17, 855-863. 46. Fabiato, Α.; Fabiato, F. Nature 1979, 281, 146-8. 47. Owen, N.E.; Le Breton, G.C. Am. J. Physiol. 1981, 241, H613-9. 48. Hamberg, M.; Svensson, J.; Samuelsson, B. Proc. Natl. Acad. Sci. 1975, 72, 2994-8. 49. Rittenhouse-Simmons, S. J. Clin. Invest. 1979, 63, 580-7. 50. Lapetina, E.G.; Cuatrecasas, P. Biochim. Biophys. Acta 1979, 573, 394-402. 51. Gerrard, J.M.; Butler, A.M.; Graff, G.; Stoddard, S.F.; White, J.G. Prostaglandins and Med. 1978, 1, 373-385. 52. Feinstein, M.D. Biochem. Biophys. Res. Comm. 1980, 93, 593-600. 53. Marcus, A.J.; Zucker, M.B. "The Physiology of Blood Platelets"; Grune and Stratton, Inc.: New York, 1965. 54. Ardlie, N.G.; Glew, G.; Schultz, B.G.; Schwartz, C.J. Thrombos. Diathes. Haemorrh. 1967, 18, 670-3. 55. Robison, G.A.; Arnold, Α.; Hartmann, R.C. Pharmacol. Res. Comm. 1969, 1, 325-332. 56. Wolfe, S.M.; Shulman, N.R. Biochem. Biophys. Res. Comm. 1969, 35, 265-272. 57. Zieve, P.D.; Greenough, W.B. Biochem. Biophys. Res. Comm. 1969, 35, 462-473. 58. Marquis, N.R.; Vigdahl, R.L.; Tavormina, P.A. Biochem. Biophys. Res. Comm. 1969, 36, 965-972. 59. Salzman, E.W.; Levine, L. J. Clin. Invest. 1971, 50, 131-141. 60. Mills, D.C.B.; Macfarlane, D.E. Thrombosis Res. 1974, 5, 401-412. 61. Gorman, R.R.; Bunting, S.; Miller, O.V. Prostaglandins 1977, 13, 377-388. 62. Best, L.C.; Martin, T.J.; Russel, R.G.G.; Preston, F.E. Nature 1977, 267, 850-2. 63. Mills, D.C.B.; Smith, J.B. Biochem. J. 1971, 121, 185-196.

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64. Hung, S.C.; Robin, B.M.; Venton, D.L.; Le Breton, G.C. Circulation 1980, 62, 405. 65. Kaser-Glanzmann, R.: Jakabova, M.; George, J.N.; Luscher, E.F. Biochim. Biophys. Acta 1977, 466, 429-440. 66. Feinstein, M.B.; Fiekers, J.; Fraser, C. J. Pharm. Exp. Ther. 1976, 197, 215-228. 67. Charo, I.F.; Feinman, R.D.; Detwiler, T.C. Biochem. Biophys. Res. Comm. 1976, 72, 1462-7. 68. Fox, J.E.B.; Say, A.K.; Haslam, R.J. Biochem. J. 1979, 184, 651-661. 69. Chaiken, R.; Pagano, D.; Detwiler, T.C. Biochim. Biophys. Acta 1975, 403, 315-325. 70. Feinstein, M.B.; Becker, E.L.; Fraser, C. Prostaglandins 1977, 14, 1075-1093. 71. Rittenhouse-Simmons, S.; Deykin, D. Biochim. Biophys. Acta 1978, 543, 409-422. 72. Haslam, R.J.; Lynham, J.A.; Fox, J.E.B. Biochem. J. 1979, 178, 397-406. 73. Statland, B.E.; Heagan, B.M.; White, J.G. Nature 1969, 223, 521-2. 74. Moncada, S.; Gryglewski, R.J.; Bunting, S.; Vane, J.R. Prostaglandins 1976, 12, 715-737. 75. Le Breton, G.C.; Venton, D.L.; Enke, S.E.; Halushka, P.V. Proc. Natl. Acad. Sci. 1979, 76, 4097-4101. 76. Le Breton, G.C.; Venton, D.L. "Advances in Prostaglandin and Thromboxane Research"; Samuelson, B., Ramwell, P.W. and Paoletti, R., Eds.; Raven Press: New York, 1980, 497-503. 77. Parise, L.V.; Venton, D.L.; Le Breton, G.C. Fed. Proc. 1981, 40, 834. 78. Rybicki, J.P.; Venton, D.L.; Le Breton, G.C. Thrombosis and Hemostasis. 1981, 46, 651. 79. Parise, L.V.; Venton, D.L.; Le Breton, G.C. Thrombosis and Hemostasis 1981, 46. 650. 80. Kaser-Glanzmann, R.; Gerber, E.; Luscher, E.F. Biochim. Biophys. Acta 1979, 558, 344-7. 81. Hathaway, D.R.; Eaton, C.R. Adelstein, R.S. Nature 1981, 291, 252-4. 82. Rodan, G.A.; Feinstein, M.B. Proc. Natl. Acad. Sci. 1976, 72, 1829-1833. 83. Gorman, R.R.; Wierenga, W.; Miller, O.V. Biochim. Biophys. Acta 1979, 572, 95-104. RECEIVED June 28, 1982.

10 Suppression of Atherogenesis with a MembraneActive Agent—Nifedipine PHILIP D. HENRY and KAREN BENTLEY

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

Washington University School of Medicine, Cardiovascular Division, Department of Medicine, St. Louis, MO 63110 We tested the effects of the dihydropyridine nifedipine on atherogenesis in rabbits fed a 2% cholesterol diet. The drug was given o r a l l y , 40 mg/day, and control rabbits received placebo. Nifedipine was well tolerated, and evoked peak reduction i n mean a r t e r i a l pressure of less than 12 mm Hg. Plasma t o t a l cholesterol after eight weeks before killing the rabbits was similar i n the placebo and nifedipine-treated groups, averaging 1,903 ± 138 (n = 13) and 1,848 ± 121 mg/dl (n = 13; mean ± SE; Ρ > 0.8). In placebo­ -treated rabbits, aortic lesions stainable with Sudan IV covered 40 ± 5% of the intimal surface. The cholesterol and calcium concentrations i n aortic tissue were 47 ± 5 mg/g protein and 297 ± 18μg/gprotein. In nifedipine-treated rabbits, values for stainable lesions, aortic cholesterol, and aortic calcium were signifi­ cantly depressed (P < 0.005), and averaged 17 ± 3%, 29 ± 2 mg/g protein, and 202 ± 14 μg/g protein. Thus, the dihydropyridine suppressed structural and biochemical changes of athero­ sclerosis without reducing the hypercholesterolemic response to the diet. Current evidence suggests that calcium plays an important pathogenic r o l e i n a t h e r o s c l e r o s i s (1-7.)· In cholesterol-fed r a b b i t s , a n t i c a l c i f y i n g and hypocalcémie a g e n t s s u c h a s d i p h o s p h o n a t e s ( 2 - 5 ) , t h i o p h e n e compounds ( 6 ) , a n d EDTA Ç 7 ) h a v e b e e n demonstrated t o exert a n t i a t h e r o g e n i c e f f e c t s without a p p r e c i a b l y altering circulating lipids. T h i s s t u d y was p e r f o r m e d t o d e t e r ­ mine w h e t h e r n i f e d i p i n e , a c a l c i u m a n t a g o n i s t , s u p p r e s s e s a t h e r o ­ g e n e s i s i n c h o l e s t e r o l - f e d r a b b i t s . N i f e d i p i n e and o t h e r c a l c i u m a n t a g o n i s t s i n h i b i t c a l c i u m u p t a k e b y smooth m u s c l e c e l l s , b u t u n l i k e p r e v i o u s l y u s e d a g e n t s , t h e y h a v e no c a l c i u m c h e l a t i n g 0097-6156/82/0201-0175$06.00/0 © 1982 American Chemical Society

176

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ANTAGONISTS

p r o p e r t i e s (8). R e s u l t s i n d i c a t e t h a t i n t r a c e l l u l a r a c c u m u l a t i o n o f c a l c i u m , a p o s t u l a t e d n o n - s p e c i f i c mechanism o f c e l l d e a t h 09, 1 0 ) , may be i m p o r t a n t i n m e d i a t i n g h y p e r c h o i e s t e r o l e m i c vascular injury.

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

Methods F i f t y - s i x m a l e w h i t e New Z e a l a n d r a b b i t s p u r c h a s e d f r o m t h e same v e n d o r ( B o s w e l l ' s Bunny Farm, P a c i f i c , Mo.) w e i g h i n g 2.3 t o 2.6 k g w e r e h o u s e d i n d i v i d u a l l y u n d e r c o n t r o l l e d c o n d i ­ t i o n s and r a n d o m l y a s s i g n e d t o f o u r d i e t and t r e a t m e n t g r o u p s : a) s t a n d a r d p e l l e t s and p l a c e b o , b ) s t a n d a r d p e l l e t s and n i f e d i ­ p i n e , c ) 2% c h o l e s t e r o l p e l l e t s ( N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , O h i o ) and p l a c e b o , and d) 2% c h o l e s t e r o l p e l l e t s and nifedipine. The d a i l y r a t i o n o f p e l l e t s was 100 g f o r a l l r a b b i t s , and w a t e r was g i v e n ad l i b i t u m . N i f e d i p i n e was f o r c e f e d , two 10 mg c a p s u l e s a t 7 a.m. and 7 p.m. (40 mg/day), and untreated rabbits received i d e n t i c a l capsules without n i f e d i p i n e (placebo). I n each group, f i v e r a b b i t s were s e l e c t e d randomly f o r t h e s t u d y o f a r t e r i a l p r e s s u r e d u r i n g t h e f i r s t and l a s t week o f t h e d i e t p e r i o d . A No. 21 p e d i a t r i c n e e d l e was i n s e r t e d i n t o t h e c e n t r a l e a r a r t e r y and t a p e d t o t h e e a r ( 1 1 ) . W i t h o u t r e ­ s t r a i n i n g t h e r a b b i t , t h e n e e d l e was i n t e r m i t t e n t l y c o n n e c t e d t o a G o u l d P23 Db p r e s s u r e t r a n s d u c e r ( G o u l d , I n c . , I n s t r u m e n t s D i v i s i o n , C l e v e l a n d , O h i o ) , w h i c h was p l a c e d a t m i d - c h e s t l e v e l and a t t a c h e d t o a G o u l d a m p l i f i e r / r e c o r d e r s y s t e m . At the be­ g i n n i n g and a t t h e end o f t h e d i e t p e r i o d , b l o o d s a m p l e s w e r e c o l l e c t e d from t h e c e n t r a l e a r a r t e r y o f a l l r a b b i t s i n t o tubes c o n t a i n i n g Na2EDTA (1 y g / 1 0 0 μ ΐ ) . The s a m p l e s w e r e u s e d f o r t h e d e t e r m i n a t i o n o f t h e m i c r o h e m a t o c r i t , and t h e s e p a r a t e d p l a s m a was a n a l y z e d f o r t o t a l c h o l e s t e r o l , t r i g l y c e r i d e s , p h o s p h a t e , t o t a l p r o t e i n , and a l b u m i n w i t h a Gemini a u t o m a t i c a n a l y z e r ( E l e c t r o - N u c l e o n i c s , I n c . , F a i r f i e l d , N . J . ) . P l a s m a c a l c i u m was measured by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( s e e b e l o w ) . The r a b b i t s w e r e k i l l e d a f t e r e i g h t w e e k s . The t h o r a c i c a o r t a was removed, c l e a n e d o f s u r r o u n d i n g t i s s u e , and h a l v e d w i t h l o n g i t u d i n a l c u t s t h r o u g h t h e a n t e r i o r and p o s t e r i o r w a l l s . E a c h l o n g i t u d i n a l h a l f - a o r t a was b l o t t e d and w e i g h e d . One-half was q u i c k l y f r o z e n and s t o r e d a t -70°C, t h e o t h e r l a i d f l a t , i n t i m a i s i d e u p , on c l e a r p l e x i g l a s . The p r e p a r a t i o n was g l u e d a r o u n d i t s edges t o t h e p l a s t i c w i t h Permabond 910 (Permabond I n t e r n a t i o n a l Corp., E n g l e w o o d , N . J . ) , and s t a i n e d w i t h Sudan IV (Sigma C h e m i c a l Co., S t . L o u i s , Mo.) a s d e s c r i b e d b y Kramsch and Chan, 1978; Chan e t a l . , 1978; and Holman e t a l . , 1958 ( 4 , 6, 1 2 ) . A p l e x i g l a s p l a t e c o v e r e d w i t h t r a n s l u c e n t p a p e r was p l a c e d o n t h e s u r f a c e o f t h e p r e p a r a t i o n . The t r a n s p a r e n t o u t ­ l i n e s and s t a i n e d a r e a s o f t h e v e s s e l w e r e d e l i n e a t e d w i t h b l a c k i n k and a r e a s p l a n i m e t e r e d w i t h a c o m p u t e r i z e d p l a n i m e t e r (Model 9874A, H e w l e t t P a c k a r d Co., P a l o A l t o , C a l i f o r n i a ) . The f r o z e n h a l f - a o r t a was p u l v e r i z e d a t l i q u i d n i t r o g e n t e m p e r a t u r e , and

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

10.

HENRY AND

BENTLEY

Atherogenesis

and

Nifedipine

111

h o m o g e n i z e d i n 10 m l o f c h l o r o f o r m - m e t h a n o l ( 2 : 1 , v o l / v o l ) i n a D u a l l h o m o g e n i z e r ( K o n t e s Co., V i n e l a n d , N . J . ) . Fractions of homogenate w e r e d r i e d a n d u s e d f o r measurements o f c a l c i u m b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y o n a P e r k i n - E l m e r M o d e l 303 a p p a r a t u s ( P e r k i n - E l m e r C o r p . , P h y s i c a l E l e c t r o n i c s D i v . , Eden P r a i r i e , Minn.) as p r e v i o u s l y d e s c r i b e d (13), and f o r e s t i m a t i o n o f p r o t e i n by t h e method o f Lowry ( 1 4 ) . Small weighed t i s s u e s a m p l e s f r o m s e l e c t e d a r t e r i e s w e r e f i x e d i n 10% b u f f e r e d f o r m a ­ l i n , embedded i n p a r a f f i n , a n d s t a i n e d w i t h h e m a t o x y l i n - e o s i n o r Verhoeff-van Gieson s t a i n . The d i f f e r e n c e b e t w e e n s e q u e n t i a l mean v a l u e s i n t h e same g r o u p was e v a l u a t e d b y t h e t - t e s t f o r p a i r e d c o m p a r i s o n s . The d i f f e r e n c e b e t w e e n mean v a l u e s f o r p l a n i m e t e r e d l e s i o n s was a n a l y z e d by W i l c o x o n ' s s i g n e d rank t e s t . V a l u e s r e l a t i n g l e s i o n s t o t i s s u e c h o l e s t e r o l w e r e a s s e s s e d b y t h e same t e s t . D i f f e r ­ e n c e s b e t w e e n g r o u p means f o r v a r i a b l e s o c c u r r i n g i n a l l g r o u p s were e v a l u a t e d by c o m p u t e r i z e d a n a l y s i s o f v a r i a n c e u s i n g t h e General L i n e a r Models procedure (15). Results 1) R e s p o n s e o f t h e R a b b i t s t o t h e D i e t a n d D r u g R e g i m e n s . Two c h o l e s t e r o l - f e d r a b b i t s , one r e c e i v i n g p l a c e b o , t h e o t h e r n i f e d i p i n e , d i e d o f unknown c a u s e . The d i e t a n d d r u g r e g i m e n s w e r e w e l l t o l e r a t e d , a n d w e i g h t g a i n d u r i n g t h e e i g h t week p e r i o d was s i m i l a r i n t h e d i f f e r e n t g r o u p s ( T a b l e I ) . H i g h d o s e d n i f e d i p i n e (30 mg/kg day) h a s b e e n p r e v i o u s l y shown t o be w e l l t o l e r a t e d i n r a t s (16). P e a k e f f e c t s o n mean a r t e r i a l p r e s s u r e a n d h e a r t r a t e a f t e r e a c h d o s e o f n i f e d i p i n e w e r e -11 ± 3 mm Hg a n d 44 ± 18 b e a t s / m i n d u r i n g t h e f i r s t week o f t r e a t m e n t . These e f f e c t s were t r a n s i e n t , v a l u e s r e t u r n i n g t o b a s e l i n e w i t h i n two h o u r s o r l e s s . Hemody­ namic e f f e c t s o f n i f e d i p i n e d i d n o t d i f f e r s i g n i f i c a n t l y between t h e d i e t a r y g r o u p s , n o r was t h e r e a d i f f e r e n c e i n e a c h g r o u p b e t w e e n v a l u e s d u r i n g t h e f i r s t a n d l a s t week o f t r e a t m e n t . 2) B l o o d C h e m i s t r y . R e s u l t s o f b i o c h e m i c a l measurement i n b l o o d a r e shown i n T a b l e I . N i f e d i p i n e t r e a t m e n t h a d no e f f e c t on p l a s m a c h o l e s t e r o l l e v e l s o f r a b b i t s m a i n t a i n e d o n s t a n d a r d d i e t o r 2% c h o l e s t e r o l d i e t . 3) S t r u c t u r a l Changes i n A o r t a . In cholesterol-fed rabbits, the p e r c e n t a g e o f t h e i n t i m a i s u r f a c e covered by S u d a n - p o s i t i v e l e s i o n s a v e r a g e d 40 ± 5% (SE) i n p l a c e b o - t r e a t e d r a b b i t s ( n = 13), and 17 ± 3% i n n i f e d i p i n e - t r e a t e d r a b b i t s ( n = 1 3 ; Ρ < 0.001) (Figure 1). Microscopic evaluation of a o r t i c tissue revealed q u a l i t a ­ t i v e l y s i m i l a r l e s i o n s i n t h e two c h o l e s t e r o l - f e d g r o u p s , b u t a q u a n t i t a t i v e s t r u c t u r a l a n a l y s i s was n o t p e r f o r m e d i n t h i s s t u d y .

25

+

0.2

3.9 +

4 0.4

+

+ 0.2

6

+

5.7 +

6.1

13.5

93

0.5

+

+ 121 + 52)

1.1* 0.5)

+

0.3 0.3

4.1 +

4 6.0 +

5.7 +

0.2

30

+ 138* + 7)

+ +

+ 166 ± 63)

13.6 +

129

1903 (48

35 (41

3058 (2551

PLACEBO ( n = 13)

0.9* 0.6)

± 25

± 121* ± 6)

± ±

± 158 ± 54)

3

3.8 ± 0.2

5.4 ± 0.4

6.0 ±

13.7 ± 0.2

136

1848 (46

34 (42

3150 (2590

NIFEDIPINE ( n = 13)

2% CHOLESTEROL DIET

V a l u e s (mean ± SE) r e f e r t o measurements o b t a i n e d a t t h e end o f t h e d i e t p e r i o d . Values a t the onset of t h e p e r i o d , i n d i c a t e d i n p a r e n t h e s e s , a r e g i v e n o n l y i f they d i f f e r e d from t h e subsequent v a l u e s (p< .05, p a i r e d t - t e s t ) . Simultaneous comparisons between t h e f o u r groups ( a n a l y s i s o f v a r i a n c e , s e e M e t h o d s ) r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s o n l y b e t w e e n c h o l e s t e r o l g r o u p s and s t a n d a r d d i e t g r o u p s ( v a l u e s w i t h *; ρ < . 0 0 1 ) .

4.2 ± 0.3

Albumin g / d l

5.9 ± 0.3 5.9 ± 0.4

mg/dl

Total Protein g/dl

Phosphorus

13.5 ± 0.1

23

41

8

T o t a l C a l c i u m mg/dl

±

41

3268 (2524

NIFEDIPINE (n = 14)

0.6

90

±

±

± 113 ± 68)

T r i g l y c e r i d e s , mg/dl

45

mg/dl

Total Cholesterol

3201 (2591 40

grams

Hematocrit, V o l %

Body w e i g h t ,

PLACEBO (n = 14)

STANDARD DIET

BODY WEIGHT, HEMATOCRIT, AND PLASMA CONSTITUENTS Table I

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

10.

HENRY AND

BENTLEY

Atherogenesis

and

Nifedipine

179

4) B i o c h e m i c a l Changes i n A o r t a . The c h o l e s t e r o l c o n c e n ­ t r a t i o n s i n a o r t a s f r o m r a b b i t s g i v e n s t a n d a r d p e l l e t s w e r e 6.1 ±0.3 mg/g p r o t e i n f o r t h e p l a c e b o g r o u p , a n d 6.3 ± 0.4 mg/g p r o t e i n f o r the n i f e d i p i n e group ( F i g u r e 2 ) . Incholesterol-fed r a b b i t s , v a l u e s f o r t h e p l a c e b o and n i f e d i p i n e groups d i f f e r e d s i g n i f i c a n t l y (P < 0 . 0 0 1 ) , a v e r a g i n g 47 ± 5 a n d 29 ± 2 mg/g protein (Figure 2). Treatment w i t h n i f e d i p i n e a l t e r e d t h e r e l a ­ t i o n s h i p between a o r t i c c h o l e s t e r o l a c c u m u l a t i o n and f o r m a t i o n o f S u d a n - p o s i t i v e l e s i o n s . The r a t i o r e l a t i n g p l a n i m e t e r e d l e s i o n s ( p e r c e n t ) t o t i s s u e c h o l e s t e r o l ( m i l l i g r a m s p e r gram p r o t e i n ) a v e r a g e d 0.85 ± 0.05 and 0.58 ± 0.04% (% · g p r o t / m g ) i n u n t r e a t e d and t r e a t e d r a b b i t s (P < 0.001) ( F i g u r e 3 ) . In r a b b i t s given standard d i e t , calcium concentrations i n a o r t i c t i s s u e f o r t h e p l a c e b o and n i f e d i p i n e g r o u p s w e r e 190 ± 1 1 and 194 ± 14 Ug/g p r o t e i n . In cholesterol-fed rabbits, aortic c a l c i u m f o r t h e n i f e d i p i n e g r o u p was s i g n i f i c a n t l y l o w e r t h a n t h a t f o r t h e p l a c e b o g r o u p , v a l u e s a v e r a g i n g 202 ± 14 a n d 297 ± 18 ug/g p r o t e i n (P < 0.005) ( F i g u r e 4 ) . Discussion C a l c i u m o v e r l o a d a s a p a t h o g e n i c mechanism o f c e l l i n j u r y has b e e n i n c r i m i n a t e d i n v a r i o u s d i s o r d e r s o f c a r d i a c a n d s k e l e t a l muscle i n c l u d i n g catecholamine-induced c a r d i a c n e c r o s i s (8, 1 3 ) , m y o c a r d i a l i s c h e m i a (8, 13), m y o p a t h i e s (9, 17), and m a l i g n a n t hyperthermia (18). Of i n t e r e s t i s t h a t i n t h e m a j o r i t y o f t h e s e pathophysiological e n t i t i e s calcium antagonists e f f e c t i v e l y s u p p r e s s c a l c i u m a c c u m u l a t i o n a n d c e l l n e c r o s i s ( 8 , j^3, _17, 18) . Moreover, myopathic S y r i a n hamsters given a c a l c i u m - d e f i c i e n t d i e t e x h i b i t fewer l e s i o n s i n s k e l e t a l and c a r d i a c muscle ( 1 7 ) . Conversely, f a c i l i t a t i o n o f c a l c i u m uptake w i t h ionophores o r membrane-active t o x i n s such as l y s o p h o s p h o l i p i d s o r m a c r o l i d e a n t i b i o t i c s a c c e l e r a t e n e c r o s i s o f i s o l a t e d s k e l e t a l muscle (19) a n d r a t h e p a t o c y t e s i n c u l t u r e ( 1 0 ) . S t u d i e s showing p r o t e c t i v e e f f e c t s o f c a l c i u m a n t a g o n i s t s i n syndromes a s s o c i a t e d w i t h membrane i n j u r y , c a l c i u m o v e r l o a d , and c e l l n e c r o s i s may p a r t l y e x p l a i n t h e a n t i a t h e r o g e n i c a c t i v i t y o f n i f e d i p i n e . A t h e r o g e n e s i s i s a c c o m p a n i e d by a n a c c u m u l a t i o n o f c a l c i u m i n a r t e r i a l w a l l s ( 1 ~ 7 ) , a n d p r o l i f e r a t i o n o f smooth m u s c l e c e l l s , a k e y p r o c e s s i n l e s i o n f o r m a t i o n , goes hand i n hand w i t h c e l l n e c r o s i s (20, 2 1 ) . N e c r o s i s o f foam c e l l s r e ­ l e a s i n g m e m b r a n e - a c t i v e l i p i d , s u c h a s c h o l e s t e r o l , may a f f e c t t h e membranes o f n e i g h b o r i n g c e l l s a n d a c c e l e r a t e c e l l u l a r d e ­ t e r i o r a t i o n and t u r n o v e r ( F i g u r e 5 ) . Decreased n e c r o s i s a s s o c i ­ a t e d w i t h i n t r a c e l l u l a r r e t e n t i o n o f l i p i d may e x p l a i n why l e s i o n f o r m a t i o n f o r a g i v e n l i p i d a c c u m u l a t i o n was s i g n i f i c a n t l y d e c r e a s e d i n n i f e d i p i n e - t r e a t e d r a b b i t s . We h a v e p r e v i o u s l y demonstrated t h a t a h i g h c h o l e s t e r o l environment s e n s i t i z e s i s o ­ lated a r t e r i e s to the c o n s t r i c t o r effects of calcium, consistent w i t h i n c r e a s e d c a l c i u m u p t a k e a f t e r a c q u i s i t i o n o f membrane

180

CALCIUM REGULATION BY CALCIUM ANTAGONISTS

Publication Date: October 14, 1982 | doi: 10.1021/bk-1982-0201.ch010

LESIONS (Percent of Surface Stained)

Ρ < .001 Figure 1. Effect of nifedipine on the extent of aortic lesions (planimetry of sudanophilic lesions) induced by cholesterol feeding. Key (n — 13): •, placebo; and nifedipine.

50

r-

40

h

30\TISSUE CHOLESTEROL ( mg/g protein )

20h

P