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Membrane actions of antiarrhythmic drugs Au, Tony Long Sang 1978

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MEMBRANE ACTIONS OF ANTIARRHYTHMIC DRUGS by TONY LONG SANG AU B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Pharmacology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1978 © Tony Long Sang Au, 1978 In present ing th i s thes is in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of Pharmacology The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 27th April, 1978 i i ABSTRACT The structural and functional consequences of the interaction of various antiarrhythmics with human erythrocyte membranes, guinea pig brain synap-tosomes and myocardial sarcolemmal membranes were studied at drug concen-trations affecting the stability of intact erythrocytes to hypotonic l y s i s . It was assumed that such stabilization might bear some molecular resemblance to the electrical stabilizing properties of these drugs in excitable tissues. Membrane perturbational actions of these drugs were measured in terms of the specific incorporation of the chromophoric probes, 5,5 1-dithio-bis-(2-nitrobenzoic acid) (DTNB) and trinitrobenzenesulfonic acid (TNBS) into membrane sulfhydryl and amino groups respectively. Most drugs tested, including lidocaine, quinidine, the verapamil analogue D-600 and the quater-nary analogues QX 572 and pranolium, exhibited a concentration-dependent stimulation of DTNB and TNBS incorporation. At drug concentrations producing erythrocyte stabilization, the protein perturbational properties of quinidine, lidocaine, D-600 and QX 572 as viewed in terms of DTNB labelling were equiv-alent while differences were apparent with quinidine, D-600 and lidocaine at high concentrations in the destabilizing range. Most agents, with the excep-tion of pranolium, showed a similar pattern of DTNB incorporation in brain synaptic membranes as in erythrocytes. Studies of the incorporation of TNBS i i i i n t o e r y t h rocyte membranes i n d i c a t e d that antiarrhythmics induce greater s t r u c t u r a l a l t e r a t i o n s i n membrane phospholipids as compared w i t h membrane p r o t e i n s . B r e t y l i u m and p r a c t o l o l , two substances w i t h minimal d i r e c t cardiodepressant p r o p e r t i e s , d i d not enhance DTNB or TNBS in c o r p o r a t i o n s i n t o erythrocyte membranes, although both agents, e s p e c i a l l y p r a c t o l o l , possessed marked a n t i h e m o l y t i c p r o p e r t i e s . I t appeared, t h e r e f o r e , that the membrane p e r t u r b a t i o n a l a c t i o n s of antiarrhythmics as analyzed here by means of g r o u p - s p e c i f i c chemical probes are a b e t t e r index of t h e i r d i r e c t myocardial membrane acti o n s than erythrocyte s t a b i l i z a t i o n . The f u n c t i o n a l consequences of drug-membrane i n t e r a c t i o n as r e f l e c t e d i n the i n h i b i t i o n of membrane-associated enzymes by antiarrhythmics were shown to be c r i t i c a l l y dependent on the drug and membrane i n question. The a c t i v i t y of erythrocyte membrane ouabain-sensitive K + - s t i m u l a t e d p - n i t r o p h e n y l -phosphatase (K +-NPPase) was more r e a d i l y i n h i b i t e d than that of Mg + +-independent and M g + + - s t i m u l a t e d NPPase by most drugs examined. In myocardial sarcolemmal membranes, l i d o c a i n e was s t i m u l a t o r y to the K +-NPPase whereas a l l other agents e x h i b i t e d s t i m u l a t o r y actions only at the lowest drug concentrations. The Ca + +-ATPase system i n the erythrocyte membrane was a l s o i n h i b i t e d by antiarrhythmics with p r o p r a n o l o l , pranolium and l i d o c a i n e showing a r e l a t i v e l y higher degree of i n h i b i t i o n of the high C a + + a f f i n i t y component while q u i n i d i n e and D-600 exerted equal i n h i b i t o r y actions on both high and iv low Ca + + a f f i n i t y components of the enzyme. A comparison of the perturbational actions of antiarrhythmics in isolated erythrocyte membranes, in the membranes of the intact erythrocyte and in brain synaptic membranes was made by analyzing the effects of drugs on the activity of the membrane acetylcholinesterase present in these preparations. Inhibitory actions of a l l drugs tested were comparable in both intact and isolated erythrocyte membranes but differed in the excitable tissue membrane. The nature of the inhibition exerted by the antiarrhythmics on acetylcholinesterase of intact erythrocytes was of a mixed type for most drugs except practolol which inhibited non-competitively. The transmembrane chloride gradient had no influence on the inhibition by bretylium, lidocaine and D-600 of the acetylcholinesterase activity of the intact cells but the inhibition produced by quinidine and propranolol was enhanced when erythrocytes were suspended in a low chloride medium. The foregoing results, therefore, indicate that the membrane perturb-ational actions of antiarrhythmics vary with the agent in question and with the particular membrane system. It is suggested that the molecular mechanisms by which these drugs alter cardiac automaticity may not be identical and may differ in various regions of the myocardium. This in turn may underlie the differing spectra of c l i n i c a l effectiveness exhibited by these pharmacological agents. V TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS 20 METHODS 21 Membrane Preparations 21 Compositional Assays of Membranes 24 Thin Layer Chromatographic Analysis of 24 Membrane Phospholipids Enzyme Assays 25 Chemical Probe Studies 31 Hemolysis Studies 33 RESULTS 34 Antiarrhythmic Agents Investigated 34 Antihemolytic Studies 34 Analysis of Mitochondrial Contamination and 39 Compositional Characteristics Membrane Perturbational Properties of the 46 Antiarrhythmic Agents Effects of Antiarrhythmics on Activity of 66 Membrane-Associated Enzymes Effects of Antiarrhythmics on Activity of 76 Acetylcholinesterase DISCUSSION 94 BIBLIOGRAPHY 111 v i LIST OF TABLES No. T i t l e Page I. Comparison of the a c t i v i t y of suc c i n i c dehydrogenase i n 41 guinea pig myocardial membrane fractions prepared by various procedures. I I . Ouabain-sensitive Mg + +-dependent Na +, K +-stimulated 42 ATPase a c t i v i t y of guinea pig myocardial membrane fr a c t i o n s prepared by the combined procedures of Hui et a l and Sulakhe et a l . I I I . Compositional data of human erythrocyte and guinea p i g br a i n 44 synaptosomal membranes. IV. Rf values and r e l a t i v e proportion of various phospholipids 45 from membranes of human erythrocyte and guinea pig brain synaptosome. V. E f f e c t s of antiarrhythmics on the slope of the l i n e a r 62 re l a t i o n s h i p c haracterizing membrane phospholipid l a b e l l i n g by TNBS as a function of membrane protein l a b e l l i n g . VI. E f f e c t s of antiarrhythmics on a c t i v i t y of basal 68 (Mg + +-independent) and Mg + +-stimulated erythrocyte membrane p-nitrophenylphosphatase. VII. Inhibitory e f f e c t s of antiarrhythmics on the a c e t y l - 85 cholinesterase a c t i v i t y of in t a c t erythrocytes, erythrocyte membranes and br a i n synaptic membranes. VIII. Influence of antiarrhythmics on the enzyme-substrate k i n e t i c s 89 of intact erythrocytes. V l l LIST OF FIGURES No. T i t l e 1. A n t i h e m o l y t i c e f f e c t s of q u i n i d i n e , D-600, l i d o c a i n e and p r a c t o l o l . 2. Antihemolytic e f f e c t s of p r o p r a n o l o l , pranolium, QX 572, and br e t y l i u m . 3. M o d i f i c a t i o n of erythrocyte membrane s u l f h y d r y l groups by 5,5' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB) i n the presence of an t i a r r h y t h m i c s . 4. M o d i f i c a t i o n of erythrocyte membrane s u l f h y d r y l groups by 5,5' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB) i n the presence of ant i a r r h y thmi c s. 5. R e l a t i o n s h i p between the concentration-dependence of a n t i -hemolysis and of the incremental increases i n DTNB m o d i f i -c a t i o n of erythrocyte membranes produced by a n t i a r r h y t h m i c s . 6. R e l a t i o n s h i p between the concentration-dependence of a n t i -hemolysis and of the incremental increases i n DTNB m o d i f i -c a t i o n of erythrocyte membranes produced by pro p r a n o l o l and pranolium. 7. M o d i f i c a t i o n of guinea p i g b r a i n synaptosomal membrane s u l f -h y d r y l groups by 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB) i n the presence of an t i a r r h y t h m i c s . 8. R e l a t i v e increases i n the i n c o r p o r a t i o n of t r i n i t r o b e n z e n e -s u l f o n i c a c i d (TNBS) i n t o p h o s p h o l i p i d and p r o t e i n components of erythrocyte membranes i n the presence of i n c r e a s i n g concen-t r a t i o n s of an t i a r r h y t h m i c s . 9. E f f e c t s of antiarrhythmics on the l a b e l l i n g of p r o t e i n and phospholipid components of erythrocyte membranes. 10. I n h i b i t i o n of bas a l (Mg + +-independent) and Mg + +-dependent p-nitrophenylphosphatase (NPPase) by p r a c t o l o l and pr o p r a n o l o l i n r e l a t i o n to the ant i h e m o l y t i c e f f e c t s of these agents. 11. I n h i b i t i o n of the K + - s t i m u l a t e d component of Mg + +-dependent p-nitrophenylphosphatase (K+-NPPase) by antiarrhythmics i n r e l a t i o n to the anti h e m o l y t i c e f f e c t s of these agents. v i i i No. T i t l e Page 12. Relationship between erythrocyte and myocardial membrane K +- 74 NPPase i n h i b i t i o n by various antiarrhythmics. 13. Relative i n h i b i t i o n of high and low a f f i n i t y C a + +-stimulated 77 Mg + +-dependent ATPase (Ca + +-ATPase) from erythrocyte membranes by various antiarrhythmics. 14. I n h i b i t i o n of intact erythrocyte acetylcholinesterase a c t i v i t y 81 by various antiarrhythmics r e l a t i v e to t h e i r antihemolytic properties. 15. H i l l plot analysis of the i n h i b i t i o n of erythrocyte membrane 83 and synaptic membrane acetylcholinesterase by antiarrhythmics. 16. Eadie plot analysis of the nature of the i n h i b i t i o n of in t a c t 87 erythrocyte acetylcholinesterase a c t i v i t y by antiarrhythmics. 17. E f f e c t of transmembrane chloride gradient on the i n h i b i t i o n of 91 inta c t erythrocyte acetylcholinesterase a c t i v i t y by antiarrhythmics. ix ACKNOWLEDGEMENT I would like to express my gratitude to Dr. David V. Godin for his resourceful and valuable supervision in the preparation of this thesis and to Dr. Michael J.A. Walker for introducing me to antiarrhythmic research. I wish also to thank Mrs. T.W. Ng and Miss M.E. Garnett for their technical assistance and Mr. Glenn Collins for photographic work. Page 1 INTRODUCTION Although a considerable amount of information i s a v a i l a b l e regarding the e l e c t r o p h y s i o l o g i c a l e f f e c t s of antiarrhythmics on i s o l a t e d t i s s u e as w e l l as on the i n t a c t myocardium i n v i t r o or i n v i v o , the molecular mechanisms governing antiarrhythmic drug a c t i o n have yet to be f u l l y e l u c i d a t e d . Thus, the use of a n t i a r r h y t h m i c drugs c l i n i c a l l y i n the treatment of cardiac rhythm di s o r d e r s i s almost e n t i r e l y based on e m p i r i c a l knowledge, e.g. q u i n i d i n e i s p a r t i c u l a r l y u s e f u l i n the treatment of a t r i a l arrhythmias (1) w h i l e l i d o c a i n e i s u s u a l l y employed to t r e a t v e n t r i c u l a r rhythm d i s o r d e r s (1). The e f f e c t i v e -ness of q u i n i d i n e and l i d o c a i n e i n these two d i f f e r e n t c o n d i t i o n s may i n part be r e l a t e d to d i f f e r e n c e s i n the mechanism underlying the rhythm abnormality i n these two regions of the heart (2). However, i t may a l s o be that pharmaco-k i n e t i c p r o p e r t i e s r e l a t i n g to d i s t r i b u t i o n and p r e f e r e n t i a l accumulation may play a c r u c i a l r o l e i n determining t h i s apparent ' s e l e c t i v i t y 1 . The o v e r a l l c l i n i c a l e f f e c t i v e n e s s of these agents may w e l l i n v o l v e some combination of these two p r o p e r t i e s but at present, no s a t i s f a c t o r y e x p l a n a t i o n for t h i s r e l a t i v e s e l e c t i v i t y of antiarrhythmic drug a c t i o n i s a v a i l a b l e . Studies of the mechanistic aspects of a n t i a r r h y t h m i c drug a c t i o n have been hindered g r e a t l y by a number of experimental d i f f i c u l t i e s . Some of the d i f f i c u l t i e s a r i s e from the complex time and voltage-dependent behavior of e x c i t a b l e t i s s u e s i n general, while others are p e c u l i a r to the unique s t r u c t u r a l and f u c t i o n a l p r o p e r t i e s of the heart. A normal c o n t r a c t i o n of the Page 2 heart i n v o l v e s the propagation of an impulse from the sinus node through the s p e c i a l i z e d conduction system of the a t r i a l and v e n t r i c u l a r myocardium i n a s e q u e n t i a l and synchronous p a t t e r n . I t i s important, t h e r e f o r e , to study the actions of antiarrhythmics i n the f u n c t i o n i n g i n t a c t heart. However, the s i t u a t i o n here i s complicated by the f a c t that antiarrhythmics f r e q u e n t l y produce qu i t e d i f f e r e n t e l e c t r o p h y s i o l o g i c a l actions i n one r e g i o n of the heart as compared with another. For example, both q u i n i d i n e and procainamide w i l l decrease the e f f e c t i v e r e f r a c t o r y period of the a t r i o v e n t r i c u l a r node but increase that of the H i s - P u r k i n j e system (3-4), while p r o p r a n o l o l has l i t t l e e f f e c t on the H i s - P u r k i n j e system of normal or diseased subjects (5-7). In other i n s t a n c e s , the e f f e c t s of a p a r t i c u l a r antiarrhythmic such as q u i n i d i n e may i n v o l v e a complex i n t e r p l a y between d i r e c t membrane actions and a l t e r -a t i o n s i n autonomic tone as the r e s u l t of a n t i c h o l i n e r g i c p r o p e r t i e s - both of which may d i f f e r c onsiderably i n various regions of the heart (8-10). Another co m p l i c a t i o n i n the study of antiarrhythmic drug a c t i o n r e l a t e s to the f a c t that ischemic or hypoxic myocardial t i s s u e s i n s i t u o f t e n e x h i b i t q u i t e d i f f e r e n t e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s from normal i s o l a t e d cardiac t i s s u e s (11). I t has been shown, f o r example, that ischemic myocardial t i s s u e s are considerably more s u s c e p t i b l e to the modifying e f f e c t s of c e r t a i n antiarrhythmic drugs than normal t i s s u e s . Thus, the e l e c t r o p h y s i o l o g i c a l e f f e c t s of p r o p r a n o l o l were found to be more pronounced i n the ischemic canine myocardium than i n adequately oxygenated myocardial t i s s u e s (12). The e f f e c t s of p r o p r a n o l o l i n slowing conduction, i n prolonging r e f r a c t o r i n e s s , i n Page 3 reducing a c t i o n p o t e n t i a l d u r a t i o n / e f f e c t i v e r e f r a c t o r y period of the ischemic zones of the heart probably e x p l a i n part of i t s antiarrhythmic a c t i o n s i n acute myocardial ischemia. However, i t i s not c e r t a i n whether these e f f e c t s are the r e s u l t of beta-adrenergic blockade or l o c a l anaesthetic a c t i o n or some combination of both. Hondeghem et a l (13) found that e x c i t a b i l i t y , a c t i o n p o t e n t i a l amplitude and maximum rate of r i s e were a l l decreased to a greater extent by q u i n i d i n e , l i d o c a i n e , procainamide and diphenylhydantoin i n hypoxic guinea p i g p a p i l l a r y f i b e r s as compared w i t h normally oxygenated c e l l s . Such p r e f e r e n t i a l e f f e c t s i n ischemic t i s s u e s may w e l l be important i n determining a n t i a r r h y t h m i c drug a c t i o n i n man. Therefore, e x t r a p o l a t i o n of the r e s u l t s of studies using normal i s o l a t e d myocardial t i s s u e to the c l i n i c a l s i t u a t i o n must be undertaken w i t h considerable c a u t i o n . A f i n a l c o m plication which should be mentioned i s the extreme s e n s i t i v i t y of c e r t a i n a n t i a r r h y t h m i c drug e f f e c t s to the e l e c t r o l y t e composition of the e x t e r n a l medium i n i n v i t r o s t u d i e s . The e l e c t r o p h y s i o l o g i c a l actions of diphenylhydantoin are g r e a t l y i n f l u e n c e d by 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 potassium. When potassium concentration i s low (< 3 mM), diphenylhydantoin may increase the r e s t i n g membrane p o t e n t i a l and a c t i o n p o t e n t i a l amplitude of both a t r i a l and P u r k i n j e f i b e r s , but when the potassium concentration of the medium i s at normal plasma values (4-5 mM) or s l i g h t l y h i g h e r , the drug has a pre-dominantly depressant e f f e c t on the a c t i o n p o t e n t i a l of both a t r i a l and P u r k i n j e f i b e r s (14). S i m i l a r l y , Singh and Vaughan Williams (15) showed that a r e d u c t i o n i n e x t e r n a l potassium concentration from 5.6 mM to 3 mM caused a Page 4 decrease in the ab i l i t y of lidocaine to depress the maximum rate of depolar-ization and responsiveness in rabbit a t r i a l and ventricular tissue, such that a ten-fold greater concentration of lidocaine was required to produce a sub-stantial reduction of maximum rate of depolarization of these tissues. Furthermore, the toxicity of quinidine is also affected by the serum level of potassium. In a study by Brandfonbrener et al (16), hyperkalemic dogs exhibited an increased susceptibility to quinidine toxicity, as reflected in a significantly shorter survival time (132 min) as compared with a group of hypokalemic animals (207 min) following administration of a fatal dose of quinidine. It is proposed that the increased toxicity of quinidine may reflect some synergism between potassium and drug. A primary site of action for local anaesthetic type antiarrhythmic drugs is at the level of excitable membranes and their mode of action as local anaesthetics is relevant to their effects on cardiac muscle (17-19). In nerve, these compounds act to cause a rise in the electrical threshold of excitability, retardation of impulse propagation and reduction of the rate of rise of action potential and at sufficiently high concentrations may abolish impulse conduction (20). A l l these consequences may be explained by the inhibition of the fast depolarizing sodium current across the nerve membrane (21). Local anaesthetics with antiarrhythmic properties have been shown to exhibit similar electrophysiological actions on the cardiac membrane (15,22-24). However, the relative sensitivity of heart muscle and nerve to such membrane depressant effects of a given compound is different, e.g. tetro-Page 5 dotoxin blocks nerve conduction at a concentration of 0.01 mg/1 but concen-t r a t i o n s up to a hundred times greater have no e f f e c t s on the r a t e of r i s e or du r a t i o n of the cardiac a c t i o n p o t e n t i a l (25). In c o n t r a s t , most l o c a l a n aesthetic type antiarrhythmics depress the r a t e of r i s e of the cardiac a c t i o n p o t e n t i a l at concentrations 10-300 times l e s s than those needed to cause a comparable r e d u c t i o n i n the amplitude of the monophasic a c t i o n p o t e n t i a l i n f r o g nerve (19,23-24,26). The e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of l o c a l anaesthetic a n t i a r r h y t h m i c drugs have been l a r g e l y a t t r i b u t e d to t h e i r a b i l i t y to reduce the maximal r a t e of d e p o l a r i z a t i o n i n ca r d i a c muscle (26-27) because such a r e d u c t i o n by ther a p e u t i c concentrations of these drugs has been found to be ass o c i a t e d with (a) an increase i n the th r e s h o l d of e x c i t a b i l i t y ; (b) a depression i n conduction v e l o c i t y ; and (c) a pr o l o n g a t i o n of the e f f e c t i v e r e f r a c t o r y period ( 1 ) . These a l t e r a t i o n s occur without any change i n e i t h e r r e s t i n g membrane p o t e n t i a l or the a c t i o n p o t e n t i a l d u r a t i o n . The f i n d i n g s i n nerve and cardiac muscle may suggest that the mechanism of a c t i o n of l o c a l anaesthetics i s s i m i l a r i n both t i s s u e s . In the case of myocardial membranes, drug-induced a l t e r a t i o n s i n e l e c -t r i c a l c h a r a c t e r i s t i c s of myocardial t i s s u e may occur e i t h e r by v i r t u e of the a b i l i t y of these agents to modify autonomic tone or as the r e s u l t of drug-induced membrane p e r t u r b a t i o n a l e f f e c t s which i n turn a l t e r i o n f l u x e s across the membrane. While these two mechanistic p o s s i b i l i t i e s may be separate and d i s t i n c t , as i n the case of beta-adrenergic antagonists devoid of membrane-s t a b i l i z i n g p r o p e r t i e s or l i d o c a i n e , which does not appr e c i a b l y change Page 6 autonomic tone, a number of antiarrhythmic substances may well act by some combination of these two distinct actions. The antiarrhythmic properties of beta-adrenergic antagonists possessing membrane-stabilizing actions are an example of this latter possibility. In a study of the experimental antiarrhythmic properties of acebutolol, propranolol and practolol, Basil et al (28) suggested that membrane-stabilizing actions of propranolol and acebutolol, rather than the beta-adrenoceptor blockade, were the primary determinant of antiarrhythmic properties in the reversion of ouabain-induced ventricular arrhythmias in anaesthetized dog by infusion of the drug. The findings that the infusion of the d-isomer of propranolol, which is virtually devoid of beta-adrenergic blocking properties, was effective in abolishing ouabain-induced ventricular tachycardia in dog or cat (29), while practolol, a beta-adrenergic antagonist said to be devoid of membrane-stabilizing actions, was ineffective (30) support the above proposal. Several other workers (31-32) also concluded from their experimental observations that the antiarrhythmic efficacy of beta-receptor blocking drugs against arrhythmias induced by di g i t a l i s and myocardial infarction was due to the direct membrane effects of these agents. Although the membrane-stabilizing effects of some beta-receptor blockers seem to adequately explain their action against some experimental arrhythmias, beta-receptor blockade is like l y to be the prime mechanism of antiarrhythmic action in arrhythmias induced by catecholamines or arrhythmias following myocardial infarction (33). There is controversy about the importance of the Page 7 membrane-stabilizing actions of beta-blockers c l i n i c a l l y because the plasma l e v e l s of pr o p r a n o l o l (40-150 ng/ml) during a n t i a r r h y t h m i c therapy i n man are much lower than the drug concentration needed i n v i t r o (>3 ug/ml) to produce d i r e c t e f f e c t s on the transmembrane p o t e n t i a l of normal cardiac c e l l s (33-34). C o l t a r t et a l (35) using racemic p r o p r a n o l o l have found that w h i l e plasma l e v e l s of 40-85 ng/ml may be antia r r h y t h m i c i n some p a t i e n t s , l e v e l s up to 200 ng/ml produced no suppression of arrhythmias i n others. More s i g n i f -i c a n t l y , d-propranolol (which i s devoid of appreciable beta-receptor b l o c k i n g p r o p e r t i e s ) at l e v e l s of 180-310 ng/ml was i n e f f e c t i v e i n the treatment of chronic s t a b l e v e n t r i c u l a r e c t o p i c beats. Therefore the question of the r o l e of membrane actions of beta-receptor blockers i n determining c l i n i c a l a n t i a r r h y t h m i c a c t i o n i s s t i l l unresolved but the foregoing evidence would tend to suggest that t h i s property of c e r t a i n beta-adrenergic antagonists i s not a major determinant of arrhythmic a t i o n i n most c l i n i c a l s i t u a t i o n s . In a d d i t i o n to the p r o p e r t i e s mentioned above, p r o p r a n o l o l a l s o possesses the a b i l i t y to depress cardiac adrenergic nerves (36-37) and to exert c e n t r a l nervous system e f f e c t s (38). I t i s now recognized that the c e n t r a l nervous system may play a c r u c i a l r o l e i n the arrhythmogenic e f f e c t of cardiac g l y c o s i d e s (39), and i t may also be important i n the actions of c e r t a i n a n t i a r r h y t h m i c drugs (39-40). Consequently, the r e c o g n i t i o n of the c e n t r a l nervous system as a p o t e n t i a l s i t e of antiarrhythmic drug a c t i o n may place l i m i t a t i o n s on the importance of information derived from i n v i t r o s t u d i e s . I t has been suggested that the discrepancy between animal studies and c l i n i c a l Page 8 usage as regards the relative importance of beta-adrenergic blockade and membrane stabilization in the antagonism of digitalis-induced arrhythmias by drugs may arise because the level of sympathetic tone in anaesthetized animals is l i k e l y to be considerably less than would be obtained in a c l i n i c a l setting (32). Some have challenged the claim that the central nervous system plays a major role in di g i t a l i s toxicity and have instead implicated the peripheral nervous system (ganglia and the peripheral afferent components of the baro-receptor reflex) (41). As regards antiarrhythmic action, peripheral nerves certainly represent a likely site of action for bretylium. It is accumulated by adrenergic nerve terminals and produces transmission blockade which may be preceded by an i n i t i a l release of transmitter. This observed neural depres-sant property of bretylium was taken as the antiarrhythmic action and might be of c l i n i c a l significance (42). Besides the action of bretylium on the peripheral nervous system, some other drugs (such as chlordiazepoxide, diphenylhydantoin) have antiarrhythmic actions probably related to their effects on the central nervous system. Chlordiazepoxide is able to abolish ventricular arrhythmias caused by digi t a l i s or coronary ligation by acting at the level of the hypothalamus to decrease sympathetic and possibly parasympathetic outflow (40). In a study by Evans and G i l l i s (43), i t was found that diphenylhydantoin was able to prevent both the hyperactivity of sympathetic nerves produced by posterior hypo-thalamic stimulation and the resulting arrhythmias. Such neurodepressant effects of diphenylhydantoin may underlie the beneficial antiarrhythmic Page 9 e f f e c t s of the drug. The neural depressant properties of antiarrhythmic drugs may well involve q u i n i d i n e - l i k e action on neuronal membranes of central or peripheral f i b e r s which i s somewhat analogous to the proposed e l e c t r i c a l s t a b i l i z a t i o n of the myocardial plasma membrane porduced by these agents. The above considerations have introduced yet another complication in analyzing the properties of antiarrhythmic drugs because the exact mechanism or mechanisms by which a p a r t i c u l a r antiarrhythmic exerts i t s e f f e c t s are dependent upon the nature of the arrhythmogenic stimulus. Arrhythmias a r i s i n g under conditions of hypoxia which may be associated with a decreased membrane poten t i a l i n a t r i a l and Purkinje fibers respond p a r t i c u l a r l y well to verapamil ( 4 4 ) . This ' s e l e c t i v i t y ' is presumed to arise from the fact that verapamil i s able to block the slow inward calcium current which i s responsible for the plateau phase of cardiac action p o t e n t i a l ( 4 5 ) . Indeed, various arrhythmias such as a t r i a l and a t r i o v e n t r i c u l a r j unctional arrhythmias are believed to ar i s e from abnormal impulses mediated by such calcium currents which remain functional despite the reduced membrane p o t e n t i a l i n diseased or hypoxic areas of the heart ( 4 6 - 4 7 ) . Such e l u c i d a t i o n of molecular mechanisms underlying arrhythmogenesis should ultimately provide a more r a t i o n a l basis for choosing the most appropriate antiarrhythmic agent i n a p a r t i c u l a r c l i n i c a l s i t u a t i o n . Due to the successful c l i n i c a l use of various antiarrhythmics i n the management of d i f f e r e n t types of rhythm disorders, attempts have been made to c l a s s i f y these drugs into groups based on studies of the e f f e c t s of these agents on the e l e c t r o p h y s i o l o g i c a l properties of the myocardium studied both Page 10 i n v i t r o and i n v i v o . Hoffman and Bigger (48) have c l a s s i f i e d various antiarrhythmics i n t o two major groups based p r i m a r i l y on t h e i r e f f e c t s on conduction, a c t i o n p o t e n t i a l d u r a t i o n and responsiveness. Drugs such as q u i n i d i n e , procainamide and p r o p r a n o l o l , which depress conduction and responsiveness, were placed i n one c l a s s and agents such as l i d o c a i n e and diphenylhydantoin, which have no depressant e f f e c t but might improve conduction and responsiveness, were placed i n another. However, the above c l a s s i f i c a t i o n by Hoffman and Bigger i s not g e n e r a l l y accepted. Vaughan Williams (49), u s i n g d i f f e r e n t experimental techniques and c r i t e r i a , has proposed four classes of antiarrhythmic drugs. This c l a s s i f i c a t i o n has grouped drugs according to d i r e c t e l e c t r o p h y s i o l o g i c a l a c t i o n s , sympathetic blockade, p r o l o n g a t i o n of the a c t i o n p o t e n t i a l , and m o d i f i c a t i o n of the slow inward calcium c u r r e n t s . I t i s very l i k e l y that f u r t h e r experimentation w i l l produce ever i n c r e a s i n g refinements i n the c l a s s i f i c a t i o n of an t i a r r h y t h m i c drugs and i d e n t i f y the molecular b a s i s of t h e i r a ntiarrhythmic a c t i o n s . The purpose of the foregoing m a t e r i a l was to place the general problems of studying antiarrhythmic drug a c t i o n i n t o some k i n d of persp e c t i v e and to introduce the concept that membrane i n t e r a c t i o n s of a v a r i e t y of types are l i k e l y to be important i n the a c t i o n of antiarrhythmic drugs. The aim of the work to be described here was to approach t h i s problem at a very b a s i c l e v e l , by i n v e s t i g a t i n g antiarrhythmic drug-induced s t r u c t u r a l and f u n c t i o n a l perturbations i n model membrane systems. I t was hoped that such studies might provide patterns of molecular changes as a b a s i s f o r understanding the d i v e r s e Page 11 electrophysiological actions of antiarrhythmics in excitable tissues. Ultimately such information may indicate the molecular factors governing the effectiveness of those agents in particular c l i n i c a l situations. In order to understand the membrane molecular mechanisms by which antiarrhythmic drugs alter the electrophysiological properties of myocardial tissues, i t may be important to obtain basic information regarding the nature of structural and functional perturbations induced in l i p i d and protein components of membranes by antiarrhythmic drugs. Membrane structural pertur-bations induced by drugs can be analyzed either in terms of alterations in the nature and extent of the incorporation of group-specific chemical probes into membrane structural components (50-51) as has been described recently for propranolol (52), or in terms of changes produced in the functional properties of membrane-associated enzymes (53). It should be emphasized that, in the studies described, effects of drugs on membrane enzyme activities were examined in conjunction with experiments using chemical probes in order to learn something of the correlations between group availability and enzyme changes of drug-membrane interaction. This is not to imply that the same, or analogous, enzymes in the membranes of excitable tissues represent pharmaco-logical sites of antiarrhythmic drug action. Our i n i t i a l assumption has been, therefore, that information obtained from studies in a simple well-defined membrane system, such as that of the human erythrocyte, might provide a basis for analyzing the molecular characteristics of antiarrhythmic interaction with structurally and functionally more complex membrane systems derived from excitable tissues. Page 12 Our choice of the erythrocyte as a simple membrane system was dictated not only by the relative ease of preparation of erythrocyte membranes in large quantity and in highly homogeneous form, but by the suggestion that the molecular features governing the antihemolytic or membrane stabilizing prop-erties of antiarrhythmics in red cells might be analogous to those determining electrical stabilization in excitable tissues (54). Thus, Seeman has shown that a l l lipid-soluble local and general anaesthetics protect erythrocytes from hypotonic hemolysis (54) and the concentrations of these substances producing 50% antihemolysis in erythrocytes and blockade of peripheral nerves are virtually identical (55-57). Originally the term 'membrane stabilization 1 was used to describe the a b i l i t y of local anaesthetics to abolish the propagation of action potentials in excitable tissues (58-59). Shanes (60-61) used the term 'electrical stabilizer' to describe the action of local anaesthetics because these agents blocked membrane action potentials without appreciably altering resting mem-brane potential. Subsequent studies indicated that a wide variety of l i p i d -soluble compounds including tranquilizers (62), barbiturates (63-64) and detergents (65) also may be shown to possess such 'electrical stabilizing' properties. The idea that the stabilization of excitable tissues by drugs might represent one manifestation of a more general phenomenon of drug-induced membrane structural alterations emerged from studies of the effects of lipid-soluble substances on non-excitable membrane systems. Among the key observations here were the findings that phenothiazines such as promethazine Page 13 and chlorpromazine were able to inhibit mitochondrial swelling induced by a variety of agents (66-67), to inhibit the release of acid phosphatase from rat liver lysosomes (68-70), and to stabilize erythrocytes against hypotonic lysis (71-73). These findings suggested that the interaction of local anaesthetic drugs with ce l l membranes in general may give rise to alterations in membrane permeability characteristics, the functional consequences of which would depend upon the particular tissue or subcellular organelle involved. The similar concentration dependence of such actions in diverse membrane systems (56) suggested the possibility that the structural basis of these effects might be fundamentally similar in excitable and non-excitable membranes. The fact that high concentrations of membrane stabilizers can irreversibly damage both excitable and non-excitable membranes (56); the similarity i n membrane/buffer partition coefficients for local and general anaesthetics in erythrocyte and synaptosomal membranes (74); the observation that local and general anaesthetics cause expansion of both nerve and erythrocyte membranes (54,57); and the finding that the cationic form of amine anaesthetics is active on both erythrocyte and nerve membrane (56) supported the possibility that the structural basis of 'membrane stabilization' might be similar in excitable and non-excitable membranes. It was therefore f e l t that information on the structural and functional consequences of the interaction of anti-arrhythmics with erythrocyte membranes might provide some insight into the molecular basis of antiarrhythmic drug action at the level of excitable tissue membranes, especially of the myocardium. Page 14 A v a r i e t y of l i p i d - s o l u b l e substances have been shown to pr o t e c t the erythrocyte membrane from osmotic, mechanical or a c i d l y s i s (54). These agents, which i n c l u d e s t e r o i d s (75), t r a n q u i l i z e r s (71), and anti-inflammatory compounds (76) cause an expansion of the membrane (74,77-78) r e s u l t i n g i n an increased r e s i s t a n c e of erythrocytes to hypotonic hemolysis. While the molecular basis of t h i s membrane phenomenon has yet to be f u l l y e l u c i d a t e d , a number of recent observations have shed l i g h t on i t . The expansion of erythrocyte membranes by drugs appears to in v o l v e c o n f i g u r a t i o n a l changes i n both p r o t e i n and phospholipid components of the membrane (54). In a d e t a i l e d study by Godin et a l (52), p r o p r a n o l o l , at concentrations e x e r t i n g a n t i -hemolytic e f f e c t s , was shown to modify the i n c o r p o r a t i o n of a chemical probe ( t r i n i t r o b e n z e n e s u l f o n i c acid) i n t o both phospholipid and p r o t e i n components of red blood c e l l ghosts. F u r t h e r , propranolol-induced antihemolysis of i n t a c t erythrocytes c o r r e l a t e s b e t t e r w i t h the r a t i o of drug-stimulated t r i n i t r o b e n z e n e s u l f o n i c a c i d i n c o r p o r a t i o n i n t o phospholipids r e l a t i v e to proteins than w i t h the drug-stimulated l a b e l l i n g of e i t h e r component i n d i v i d u a l l y . In a d d i t i o n , the high degree of c o r r e l a t i o n between the drug-induced l a b e l l i n g of membrane pho s p h o l i p i d by t r i n i t r o b e n z e n e s u l f o n i c a c i d and i n h i b i t i o n of two enzymatic processes a s s o c i a t e d w i t h the membrane suggested that the enzyme i n h i b i t i o n f o l lows from an a l t e r a t i o n by p r o p r a n o l o l i n the s t r u c t u r a l s t a t e of membrane phospholipids which would s e c o n d a r i l y t r i g g e r c o n f i g u r a t i o n a l changes i n c a t a l y t i c a l l y a c t i v e membrane p r o t e i n s . Page 15 F u n c t i o n a l changes r e s u l t i n g from drug-membrane i n t e r a c t i o n have been analyzed i n membrane systems other than the e r y t h r o c y t e . Suko et a l (79) u s i n g sarcoplasmic r e t i c u l u m v e s i c l e s prepared from r a b b i t s k e l e t a l muscle, proposed that the mechanism of a c t i o n of l o c a l anaesthetics i n the r e d u c t i o n of calcium e f f l u x mediated by the calcium t r a n s l o c a t i n g ATPase may be explained by a d i r e c t drug i n t e r a c t i o n w i t h the p r o t e i n components and/or by drug-induced a l t e r a t i o n s i n p r o t e i n - l i p i d i n t e r a c t i o n s i n membrane. In another study, Harrow and D h a l l a (80) showed that both sarcolemmal Mg + -ATPase and Ca + +-ATPase, but not Na +-K + ATPase, a c t i v i t i e s were increased by q u i n i d i n e , procainamide and l i d o c a i n e while only q u i n i d i n e decreased the adenylate cyclase of the r a t myocardium. None of these agents had any e f f e c t on the m y o f i b r i l l a r Mg + +-ATPase or C a + + - s t i m u l a t e d ATPase a c t i v i t i e s . The r e s u l t s suggested some d i f f e r e n c e s i n the a c t i o n of these drugs w i t h i n the myocardial membranes and showed not a l l membrane enzymes are a f f e c t e d i n the same way by these antiarrhythmic drugs. Membrane i n t e g r i t y i s important i n determining p e r m e a b i l i t y of membranes to ions (54). L o c a l anaesthetics and phenothiazines are able to cause a f l u i d i z a t i o n of membrane components (81-82). Such changes have been a t t r i b u t e d to an i n t e r a c t i o n between drug and phospholipids which i n t u r n would a l t e r the o r g a n i z a t i o n of membrane s t r u c t u r a l components. Among the p o s s i b l e consequences of such drug-induced membrane s t r u c t u r a l p e r t u r b a t i o n s are a l t e r e d membrane p e r m e a b i l i t y to sodium and potassium ions and d i s p l a c e -ment of membrane bound calcium (54,83-85). With regard to the r o l e of Page 16 divalent cations in influencing membrane structure, both protein and phospho-l i p i d components would appear to be involved, with the relative contribution of each of these structural components depending upon the particular divalent cation in question (50-51). Thus, the interaction of alkaline earth cations with the membrane involved an important contribution from membrane phospho-li p i d s , while another group of divalent cations (zinc, cadmium, nickel) inter-acted directly with membrane protein sulfhydryl groups in three different membrane systems. Further, besides grouping these cations according to their ion perturbational mechanisms, they may also be separated into similar groups based on their functional properties in excitable tissues. Alkaline earth elements are shown to be able to carry the slow inward current i n mammalian ventricular myocardium while another group (nickel, cobalt, manganese) is found to exert blocking actions on this current (86). The relevance of divalent cation-membrane interactions to mechanism of action of anti-arrhythmics is not certain. However, a recent study of Porzig (87) showed that propranolol was able to increase the efflux of potassium from erythro-cytes, an effect apparently mediated by calcium displaced from the membrane as the result of drug-membrane interaction. This effect of propranolol was produced at concentrations previously shown to alter the configurational state of erythrocyte membrane components (50,52). It can be proposed that i f a comparable mechanism were operative in the myocardial membrane, i t would provide a plausible molecular basis for a propranolol-induced decrease in automaticity in terms of altered potassium conductance. Another example of Page 17 the m o d i f i c a t i o n of membrane-cation i n t e r a c t i o n s by antiarrhythmics i s provided by verapamil, and i t s methoxy d e r i v a t i v e , D-600, which were reported to block the slow inward calcium current i n cardiac f i b e r s (45). More recent i n v e s t i g a t i o n s have pointed out that verapamil also i n h i b i t s slow channels where the current (88) i s c a r r i e d by sodium. Hence, the primary a c t i o n of these drugs may be on the slow channels i n general and not on calcium move-ments s p e c i f i c a l l y . The importance of both potassium and calcium i n myo-cardium has been long recognized (1,89-90). However, the complete d e t a i l s of the r o l e s these ions play i n determining normal and abnormal f u n c t i o n a l p r o p e r t i e s of the myocardium have yet to be understood. I t i s reasonable, t h e r e f o r e , to b e l i e v e that i n f o r m a t i o n generated from the studies of a n t i -arrhythmic drugs on membrane-associated enzymes that regulate potassium and calcium fluxes across erythrocyte membrane has c e r t a i n values and may be rele v a n t to heart t i s s u e s . C u r r e n t l y a v a i l a b l e information concerning the membrane actions of antiarrhythmics i s rat h e r l i m i t e d and incomplete. Therefore, the aim of the present study was to i n v e s t i g a t e the e f f e c t s of various classes of a n t i -arrhythmics at the molecular l e v e l on b i o l o g i c a l membranes. I n i t i a l l y , membrane p e r t u r b a t i o n a l e f f e c t s of antiarrhythmics would be examined i n a model membrane system (namely, the human erythrocyte membrane) which bears a number of analogies to e x c i t a b l e membranes, such as drug-induced s t a b i l i z a t i o n and s i m i l a r mechanisms governing a c t i v e c a t i o n t r a n s p o r t . In t h i s work, one of the main o b j e c t i v e s was to i n v e s t i g a t e a n t i h e m o l y t i c e f f e c t s of v a r i o u s Page 18 antiarrhythmic drugs: l o c a l anaesthetics and c e r t a i n quaternary analogues, beta-blockers ( p r a c t o l o l , p r o p r a n o l o l and i t s quaternary analogue, pranolium), a calcium blocker (D-600), and a nerve b l o c k i n g agent ( b r e t y l i u m ) . Attempts were then made to c o r r e l a t e a n t i h e m o l y t i c e f f e c t s w i t h membrane s t r u c t u r a l p e r t u r b a t i o n s induced by these agents i n erythrocyte membranes using two chemical probes, t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS), a reagent with consider-able s p e c i f i c i t y f o r primary amino groups (91), and 5 , 5 1 - d i t h i o - b i s -( 2 - n i t r o b e n z o i c acid) (DTNB). which i s s e l e c t i v e l y incorporated i n t o s u l f h y d r y l groups (92). Drug e f f e c t s on f u n c t i o n a l p r o p e r t i e s of membrane were i n v e s t i g a t e d by examining v a r i o u s membrane associated enzymes e s p e c i a l l y those i n v o l v e d i n i o n + + . ++ movements, such as Na , K - s t i m u l a t e d Mg -dependent ATPase, b a s a l and K + - s t i m u l a t e d p-nitrophenyl phosphatase, and C a + + - s t i m u l a t e d Mg + +-dependent ATPase. Since the enzyme a c e t y l c h o l i n e s t e r a s e i s present at the outer surface of erythrocytes (93) and can t h e r e f o r e be assayed i n both i n t a c t c e l l s and i s o l a t e d membranes, e f f e c t s of antiarrhythmics on the a c t i v i t y of t h i s enzyme can be studied i n i n t a c t erythrocytes and h y p o t o n i c a l l y l y s e d erythrocyte ghosts. Such studies w i l l provide an opportunity to l e a r n whether or not drug e f f e c t s i n an i s o l a t e d membrane system are r e l e v a n t to the s i t u a t i o n where membranes of the i n t a c t c e l l are i n v o l v e d and a l s o provide a means of d e r i v i n g information on the environment or s t r u c t u r a l d i s p o s i t i o n of membrane enzymes as w e l l as g i v i n g more d e t a i l e d i n s i g h t i n t o d i v e r s e f u n c t i o n a l consequences of drug-membrane i n t e r a c t i o n . Page 19 Finally, these experiments were extended to excitable tissues using brain synaptosomes as a model in order to determine the extent to which drug-induced perturbations studied in erythrocytes approximate those in electrically excit-able cells. Cardiac myocardial membranes were also utilized to study the effects of these drugs in order to gain more specific information which might be relevant to the molecular basis of their antiarrhythmic actions. It is hoped that the results of the studies presented in this thesis may not only help to provide safer and more effective therapy with these valuable but potentially dangerous agents but also enable these agents to be used as probes to investigate the molecular details of myocardial function and dysfunction. Page 20 MATERIALS The a n t i a r r h y t h m i c drugs used i n these studies were q u i n i d i n e hydro-c h l o r i d e and s u l f a t e (K & K L a b o r a t o r i e s ) , pranolium c h l o r i d e (SC-27761) (G.D. Searle & Co), d l - p r o p r a n o l o l hydrochloride (Sigma Chemical Co), p r a c t o l o l , f r e e base (Ayerst L a b o r a t o r i e s ) , b r e t y l i u m t o s y l a t e (Burrough Wellcome L t d ) , QX-572 hydrochloride ( A s t r a Pharmaceutical Product), D-600 hyd r o c h l o r i d e ( K n o l l A G Chemische F a b r i k e n ) , l i d o c a i n e , f r e e base (K & K L a b o r a t o r i e s ) . The f o l l o w i n g chemicals were obtained from Sigma Chemical Company: a c e t y l -t h i o c h o l i n e c h l o r i d e , t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS), sodium dodecyl s u l f a t e (SDS), t r i s (hydroxymethyl) aminomethane (Trizma base), 5 , 5 ' - d i t h i o -b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB), ouabain (Strophanthin G), d l - d i t h i o t h r e i t o l , disodium adenosine-5'-triphosphate (ATP), p-nitrophenylphosphate (104 phosphatase s u b s t r a t e ) , imidazole (grade 111), N-acetylneuraminic a c i d (type 111 from egg), c h o l e s t e r o l standard, phosphorus standard (20y grams i n o r g a n i c P/ml as KH^PO^)), p-nitrophenol standard s o l u t i o n (10u moles/ml) . Sucrose ( s p e c i a l enzyme grade) was obtained from Schwarz/Mann. Bovine serum albumin ( f r a c t i o n V) was purchased from Armour Pharmaceutical Company. S i l i c o t u n g s t i c a c i d was supplied by F i s h e r S c i e n t i f i c Company and p e r c h l o r i c a c i d from Baker & Adamson Chemical. Ninhydrin ("Baker TLC Reagent") was obtained from Baker Chemical Company. Sephadex G-200 was purchased from Pharmacia. A l l other chemicals, solvents and reagents were a n a l y t i c a l reagent grade. They were used without f u r t h e r p u r i f i c a t i o n . Page 21 METHODS MEMBRANE PREPARATIONS a) Erythrocyte Membranes Erythrocyte membranes were prepared from outdated human blood ( 0 + ) stored i n a c i d - c i t r a t e - d e x t r o s e by a m o d i f i c a t i o n of the method of S c h r i e r (94) as described p r e v i o u s l y (95). I t involved a step-wise hypotonic hemolysis procedure. A u n i t of blood (approximately 500 ml) was d i l u t e d to 1200 ml w i t h i s o t o n i c s a l i n e and ce n t r i f u g e d at 650 x g f o r 5 minutes to remove the plasma and buf f y coat. This washing was repeated once. Approx-imately 100 ml packed red blood c e l l s were then processed f u r t h e r by d i l u t i o n to 1200 ml w i t h 0.08M NaCl. This mixture was s t i r r e d at 4°C for 10 min. and ce n t r i f u g e d at 16,000 x g f o r 5 minutes. The supernatant was removed and the procedure repeated w i t h 0.06M, 0.04M, 0.02M, and 0.009M NaCl. At the l a s t two NaCl concentrations, the pH was adjusted to 7.4 with T r i s . The f i n a l step involved treatment w i t h lOmM T r i s (pH 7.4) followed by c e n t r i f u g a t i o n . The erythrocyte membranes were d i l u t e d to a p r o t e i n concentration of 3-5 mg/ml, quick frozen using dry ice/acetone and stored at -20°C. b) Cardiac Plasma Membrane Enriched F r a c t i o n Guinea p i g cardiac plasma membranes were prepared by combining the methods of Hui et a l (96) and Sulakhe et a l (97). Guinea pigs of e i t h e r sex (250-400 g) were k i l l e d by a quick d i s l o c a t i o n of t h e i r necks. Hearts were removed and Page 22 placed i n i c e - c o l d 10 mM T r i s - H C l (pH 7.5) con t a i n i n g 2 mM d i t h i o t h r e i t o l (T-D b u f f e r ) , and were gently compressed a few times to express r e s i d u a l blood. A l l the f o l l o w i n g operations were performed at 4°C. V e n t r i c u l a r t i s s u e was freed from f a t and large v e s s e l s , b l o t t e d , weighed and minced w i t h s c i s s o r s . The amount of v e n t r i c u l a r t i s s u e used for each pre p a r a t i o n was 6-7 grams. The minced t i s s u e was suspended i n 5 volumes (v/w) of T-D b u f f e r (based on t i s s u e wet weight), and homogenized for 15 seconds at a s e t t i n g of 3 i n a p o l y t r o n PT 10 homogenizer followed by 2 seconds at maximum speed. The homogenate was d i l u t e d w i t h 5 volumes of T-D b u f f e r , passed through a 250 ym mesh under m i l d s u c t i o n and c e n t r i f u g e d at 620 x g f o r 10 minutes. The supernatant was removed w i t h a Pasteur pipet and discarded. A d i s t i n c t l a y e r of l o o s e l y packed mi t o c h o n d r i a l and c a p i l l a r y m a t e r i a l (as determined by phase contrast microscopy) sedimenting on the surface of the p e l l e t s was loosened by adding 1 ml of T-D buffe r and gently r o t a t i n g the tube i n a r o c k i n g motion. The s l u r r y was then removed w i t h a Pasteur p i p e t . The p e l l e t was thoroughly dispersed i n 10 volumes of T-D bu f f e r (based on o r i g i n a l t i s s u e wet weight). The suspension was homogenized i n P o l y t r o n PT 10 homogenizer at maximum speed f o r two 5 second i n t e r v a l s a l l o w i n g a 30 second c o o l i n g i n t e r v a l between each homogenization. To t h i s homogenate was added (dropwise) an equal volume of 2.5M KC1 i n T-D b u f f e r . The suspension was s t i r r e d g e n t l y f o r 10 minutes and c e n t r i f u g e d at 9,000 x g for 20 minutes. The p e l l e t was resuspended i n 10 volumes (based on o r i g i n a l t i s s u e weight) of T-D bu f f e r c o n t a i n i n g 1.25M KC1, kept on i c e f o r 10 minutes, and c e n t r i f u g e d at 4,000 x g f o r 20 minutes. The Page 23 p e l l e t was washed twice by c e n t r i f u g a t i o n at 3,000 x g f o r 20 minutes. The washed ext r a c t e d p a r t i c l e s were suspended i n 10% sucrose (w/v) i n T-D b u f f e r and homogenized i n a Pyrex ground glass homogenizer u n t i l the suspension appeared completely homogeneous. This suspension was then f r a c t i o n a t e d using discontinuous sucrose gradient c e n t r i f u g a t i o n . The gradient c o n s i s t e d of 3 ml of 65% sucrose, 2.5 ml each of 60%, 55%, 50% sucrose (w/v) i n T-D b u f f e r , pH 8.2, and 1.5 ml of the membrane m a t e r i a l layered on top of each gradient. C e n t r i f u g a t i o n was c a r r i e d out i n a Beckman L2 u l t r a c e n t r i f u g e (SW 40 r o t o r ) f o r 1 hour at 40,000 x g. Four d i s t i n c t bands were l o c a t e d i n the f o l l o w i n g regions: 10%-50% sucrose i n t e r f a c e ( F l ) , 50%-55% sucrose i n t e r f a c e (F2), 55%-60% sucrose i n t e r f a c e (F3) , 60%-65% sucrose i n t e r f a c e (F4). The p a r t i c l e s i n each region were c o l l e c t e d w i t h Pasteur p i p e t s , d i l u t e d three f o l d w i t h T—D b u f f e r and c e n t r i f u g e d at 11,000 x g f o r 30 minutes. The p e l l e t s were homogenized i n T-D b u f f e r c o n t a i n i n g 10% sucrose (w/v), pH 7.5, i n Pyrex ground glass homogenizer. The suspensions were quick f r o z e n i n dry ice/acetone and stored at -20°C. c) B r a i n Synaptosomal Plasma Membranes Guinea p i g b r a i n synaptic plasma membranes were prepared by the method of Jones and Matus (98). The enriched f r a c t i o n of synaptosomal plasma membranes was recovered from the i n t e r f a c e of a sucrose d e n s i t y gradient on which a h y p o t o n i c a l l y l y s e d crude membrane f r a c t i o n from b r a i n had been separated by Page 24 simultaneous sedimentation and f l o t a t i o n c e n t r i f u g a t i o n . The membranes harvested were washed three times w i t h 0.15 M KC1 and suspended i n the same bu f f e r and quick frozen using dry ice/acetone and stored at -20°C. COMPOSITIONAL ASSAYS OF MEMBRANES P r o t e i n contents of e r y t h r o c y t e , cardiac and b r a i n synaptosomal plasma membranes were determined by the method of Lowry et a l (99) using bovine serum albumin (1 mg/ml) as the standard. Phos p h o l i p i d f or a l l membranes was estimated by B a r l e t t ' s m o d i f i c a t i o n of the Fiske-SubbaRow phosphate a n a l y s i s (100). Membrane c h o l e s t e r o l content was determined by the method of Zak et a l (101). S i a l i c a c i d was estimated by h y d r o l y z i n g the samples with 0.1 N H^SO^ at 80°C for 30 minutes before assaying according to the method of Warren (102). P r i o r to the ph o s p h o l i p i d , c h o l e s t e r o l and s i a l i c a c i d assays, the cardiac membrane pr e p a r a t i o n was washed three times w i t h T-D b u f f e r , pH 7.5 to remove the sucrose i n the o r i g i n a l suspending b u f f e r . This process was necessary as the sucrose was found to i n t e r f e r e w i t h the co l o r r e a c t i o n of the assays. THIN LAYER CHROMATOGRAPHIC ANALYSIS OF MEMBRANE PHOSPHOLIPIDS Ery t h r o c y t e membrane p e l l e t s e q uivalent to 1.5-2.0 mg p r o t e i n were obtained by c e n t r i f u g a t i o n and were e x t r a c t e d twice w i t h 2 ml of a 2:1 (v/v) chloroform:methanol mixture. The e x t r a c t was washed three times w i t h 1 ml 0.75% (w/v) NaCl s o l u t i o n ; the chloroform phases were pooled, and the aqueous Page 25 phases discarded. The pooled chloroform phases were evaporated to dryness and q u a n t i t i v e l y spotted onto an a c t i v a t e d (30 minutes at 110°C) s i l i c a g e l F-254 p l a t e (0.25 mm t h i c k n e s s , Brinkmann). The p l a t e was run i n a solvent mixture c o n t a i n i n g chloroform:methanol:ammonia (14:6:1, v/v/v). P h o s p h o l i p i d spots were i d e n t i f i e d by t h e i r v a l u e s . Aminophospholipids were v i s u a l i z e d by spraying the p l a t e w i t h n i n h y d r i n reagent while the remaining phospholipids were located using iodine vapour. The v a r i o u s phospholipid components were q u a n t i f i e d by e x t r a c t i o n from the s i l i c a g e l using methanol, evaporation of the e x t r a c t to dryness and a n a l y s i s of the residue f or inorganic phosphorous as described p r e v i o u s l y (100). The above procedure was a l s o used to analyze the ph o s p h o l i p i d components of b r a i n synaptosomal membranes. ENZYMATIC ASSAYS a) S u c c i n i c Dehydrogenase Preparations of cardiac plasma membranes and b r a i n synaptosomal membranes were assayed f o r s u c c i n i c dehydrogenase a c t i v i t y i n order to gain some estimate of the degree of m i t o c h o n d r i a l contamination present. The method used i s that described by S l a t e r and Bonner (103). The assay was c a r r i e d out at room temperature, and the r e a c t i o n mixture contained the f o l l o w i n g i n a f i n a l volume of 3 ml: 0.3 ml n e u t r a l i z e d KCN (0.1 M), 0.3 ml K_Fe(CN), 3 o (0.01 M),0.2 ml sodium succinate (0.2 M), 1.5 ml 0.2 M phosphate b u f f e r , pH 7.2, and 100 ug of membrane p r o t e i n . Reactions were i n i t i a t e d by the a d d i t i o n Page 26 of 0.2 ml membrane pr e p a r a t i o n and the o p t i c a l d ensity changes at 400 nm were followed for 15 minutes. The a c t i v i t y of s u c c i n i c dehydrogenase was expressed as the absorbance decrease during the f i r s t 15 minutes of the r e a c t i o n per mg membrane p r o t e i n . b) Adenosine-5 1 Triphosphatases (ATPases) ( i ) Mg + +-dependent and Mg + +-dependent Na +, K + - s t i m u l a t e d ATPase Mg + +-dependent ATPase a c t i v i t y of the erythrocyte membrane was det e r -mined by incu b a t i n g membrane p r o t e i n (0.6-0.8 mg) i n a f i n a l volume of 3 ml co n t a i n i n g : 1.0 ml 165 mM T r i s - H C l b u f f e r , pH 7.4, 0.36 ml ATP (25 mM), 0.1 ml MgCl 2 (90 mM), 0.1 ml ethyleneglycol-bis-Cg-aminoethylether) N,N-tetraacetic a c i d (EGTA) (3 mM). Mg + +-dependent N a + + , K + - s t i m u l a t e d ATPase a c t i v i t y was measured by the i n c l u s i o n of 0.1 ml KC1 (0.6 M) and 0.1 ml NaCl (2.4 M). The d i f f e r e n c e i n a c t i v i t y of Mg + +-dependent ATPase and Mg + +-dependent Na +, K + - s t i m u l a t e d ATPase was taken as the Na +, K +- st i m u l a t e d ATPase ac t i v i t y . The r e a c t i o n s were i n i t i a t e d w i t h membrane p r o t e i n , incubated f o r one hour at 37+ 0.5°C and terminated by the a d d i t i o n of 1 ml i c e c o l d 20% (w/v) t r i c h l o r o a c e t i c a c i d (TCA). The mixture was c e n t r i f u g e d (40,000 x g, 5 min.) and a 3.0 ml a l i q u o t of supernatant was assayed for in o r g a n i c phosphate by the method of Fiske-SubbaRow (100). A c t i v i t y of Mg + + and Mg + +, Na +-K + ATPase of cardiac plasma membranes and b r a i n synaptosomal membranes was assayed as above but using d i f f e r e n t amounts of p r o t e i n and a shorter incubation time. For cardiac Page 27 membranes, 100-150 yg p r o t e i n and i n c u b a t i o n of 10 minutes were used while synaptosomal membrane assay mixtures contained 60-80 yg membrane p r o t e i n and an i n c u b a t i o n time of 5 minutes was used. In the case of the car d i a c membrane prep a r a t i o n , o u a b a i n - s e n s i t i v e Na +-K + ATPase was also assayed by incub-a t i n g the r e a c t i o n mixture w i t h ouabain (30 mM). The d i f f e r e n c e between the ATPase a c t i v i t y i n the presence and absence of ouabain i s taken as the ouabai n - s e n s i t i v e Na +-K + ATPase. S p e c i f i c a c t i v i t i e s of the enzyme were expressed as moles of inorganic phosphate l i b e r a t e d per mg p r o t e i n per hour. ( i i ) Mg + +-dependent C a + + - s t i m u l a t e d ATPase. The r e a c t i o n mixture for determining the C a + + - s t i m u l a t e d Mg + +-dependent ATPase inc l u d e d : 1.0 ml 165 mM T r i s - H C l b u f f e r , pH 7.4, 0.1 ml MgCl 2 (192 mM), 0.24 ml ATP (25 mM), 0.1 ml EGTA (3mM), 0.1 ml C a C l 2 (0.09 mM and 0.2 mM) (corresponding to f i n a l f r e e C a + + c o n c e n t r a t i o n of 1.0 and 90 yM r e s p e c t i v e l y ) * , 0.6-0.8 mg erythrocyte membrane p r o t e i n , and 1 ml each of various concentrations of t e s t drugs, a l l i n a f i n a l volume of 3 ml. M g + + - s t i m u l a t e d ATPase a c t i v i t y was determined i n the same r e a c t i o n mixture without calcium. * Free C a + + concentrations i n the EGTA-Ca + + b u f f e r system were determined by a computer programme provided by Dr. R. R o u f o g a l i s , F a c u l t y of Pharma-c e u t i c a l Science, U.B.C. Page 28 Reactions were terminated a f t e r 1 hour w i t h 1 ml i c e c o l d 8% s i l c o t u n g s t i c a c i d i n 8% p e r c h l o r i c a c i d , a mixture which allowed the removal of drugs from s o l u t i o n by c e n t r i f u g a t i o n (40,000 x g, 5 min.) and thereby prevented i n t e r -ference w i t h the assay f o r i n o r g a n i c phosphate as p r e v i o u s l y described (100). C a + + - s t i m u l a t e d ATPase a c t i v i t y was obtained as the d i f f e r e n c e between a c t i v i t y measured i n the presence of calcium and that measured i n i t s absence. c) A c e t y l c h o l i n e s t e r a s e ( i ) E f f e c t of Antiarrhythmics on the A c t i v i t y of A c e t y l c h o l i n e s t e r a s e Fresh human blood obtained by venous puncture was h e p a r i n i z e d and c e n t r i -fuged. The plasma was removed and the erythrocytes were washed twice w i t h i s o t o n i c NaCl, followed by one wash w i t h 0.1 M phosphate b u f f e r , pH 8.0 and c e n t r i f u g a t i o n . S i x t y m i c r o l i t e r s of the packed erythrocytes were resuspended i n 12 ml of 0.1 M phosphate b u f f e r , pH 8.0 to o b t a i n a d i l u t i o n of 1:200 f o r the enzyme assays with various a n t i a r r h y t h m i c s . The a c e t y l c h o l i n e s t e r a s e a c t i v i t y was assayed s p e c t r o p h o t o m e t r i c a l l y at room temperature. The r e a c t i o n mixture included 1.0 ml phosphate b u f f e r (0.2 M), pH 8.0, 0.6-0.8 ml phosphate b u f f e r (0.1 M), pH 8.0, 0.1 ml DTNB (10 mM) ( i n 0.1 M phosphate b u f f e r , pH 8.0), 1.0 ml each of various concentrations of drugs to be tested and 0.2 ml erythrocyte suspension. The r e a c t i o n was s t a r t e d w i t h 0.1 ml 30 mM a c e t y l t h i o c h o l i n e c h l o r i d e immediately a f t e r an i n i t i a l absorbance reading was recorded f o l l o w i n g the a d d i t i o n of the erythrocyte suspension. The r e a c t i o n was allowed to proceed Page 29 for a period of five minutes and absorbance readings at 412 nm were recorded every 30 seconds. Non-enzymatic hydrolysis of the substrate was corrected for in a l l assays. The activities of the enzyme in the absence and presence of drug were calculated from the absorbance increase at 412 nm during the f i r s t 5 minutes of the reaction. Erythrocyte ghosts and brain synaptosomal membranes were also subjected to acetylcholinesterase assay with and without drugs. They were washed as described above in order to follow as closely as possible the conditions used in the assay of intact erythrocyte acetylcholinesterase. Dilutions of 1:200 and 1:8 were used for erythrocyte ghost membranes and brain synaptosomal membranes respectively. ( i i ) Kinetic Analysis of the Effects of Antiarrhythmics on the Enzyme The effect of various drugs on the substrate kinetics of acetyl-cholinesterase was further studied with intact human erythrocytes or erythro-cyte ghosts using each drug at a concentration producing a 35% inhibition of enzyme activity at a saturating concentration of substrate. A l l experimental procedures and reaction materials used were as described in the above section except that 5 different substrate concentrations of acetylthiocholine chloride (0.15, 0.3, 0.6, 1.5, 3.0 mM) were used. Correc-tion was made for the non-enzymatic hydrolysis of the substrate in each case. Page 30 The k i n e t i c parameters K and V f o r each drug r e a c t i o n were deter-m max ° mined from the slope and y - i n t e r c e p t of the best f i t l i n e computed f o r the experimental data expressed i n the form of an Eadie p l o t . ( i i i ) E f f e c t of Transmembrane C h l o r i d e Gradient on Drug I n h i b i t i o n of the Enzyme In t a c t human erythrocytes were used i n the experiments i n which the i n f l u e n c e of the transmembrane c h l o r i d e gradient on drug i n h i b i t i o n of a c e t y l -c h o l i n e s t e r a s e was i n v e s t i g a t e d . Erythrocytes were prepared as described on page 28 except that the two i s o t o n i c s a l i n e washes were omitted and i n s t e a d the c e l l s were washed three times w i t h 0.1 M phosphate b u f f e r , pH 8.0. The f i n a l r e a c t i o n mixture f o r the a c e t y l c h o l i n e s t e r a s e assay was as described on page 28 w i t h the exception that the 1.0 ml phosphate b u f f e r (0.2 M), pH 8.0, contained e i t h e r 0.45 M NaCl or 0.3 M Na2S0^ to give f i n a l s a l t concentrations of 150 mM and 100 mM r e s p e c t i v e l y as described by Livne and Barr-Yaakov (104). d) p-Nitrophenylphosphatase (NPPase) Basal (no Mg ), Mg -dependent and Mg -dependent K - s t i m u l a t e d NPPase a c t i v i t i e s were studied on erythrocyte ghosts and cardiac plasma membranes i n the absence and presence of a n t i a r r h y t h m i c drugs. + ++ The r e a c t i o n mixture for assaying K - s t i m u l a t e d Mg -dependent NPPase of erythrocyte ghosts c o n s i s t e d of the f o l l o w i n g : 1.0 ml 0.15 M imidazole-HCl b u f f e r , pH 7.4, 0.1 ml p-nitrophenyl phosphate (90 mM), 0.1 ml MgCl- (90 Page 31 mM), 0.1 ml KC1 (0.9 M), 0.6-0.8 mg membrane p r o t e i n and 1.0 ml each of various concentrations of t e s t drugs, a l l made up to a f i n a l volume of 3 ml w i t h water. The Mg + +-dependent NPPase was determined as above except that KC1 was omitted from the r e a c t i o n mixture. Basal NPPase was measured as above but i n the absence of both KC1 and MgC^. NPPase r e a c t i o n s were i n i t i a t e d w i t h membrane p r o t e i n , incubated f o r 1 hour at 37+0.5°C and terminated w i t h 1 ml ice c o l d 20% (w/v) TCA. Mixtures were c e n t r i f u g e d (40,000 x g, 5 min.) to remove membrane m a t e r i a l . An a l i q u o t (3.0 ml) of supernatant was added to 1 ml of 1.5 M T r i s s o l u t i o n . The amount of p-nitrophenol i n the a l k a l i n i z e d supernatant was q u a n t i f i e d spectrophoto-m e t r i c a l l y by measuring the absorbance of the s o l u t i o n at 412 nm. S p e c i f i c a c t i v i t y of membrane NPPase was expressed as umoles p-nitrophenol l i b e r a t e d per hour per mg p r o t e i n . A l l three NPPase a c t i v i t i e s of cardiac plasma membranes were determined by the same procedure but 0.3-0.5 mg cardiac membrane p r o t e i n and i n c u b a t i o n times of 10 minutes were used. In a l l cases c o r r e c t i o n was made f o r the spontaneous (non-enzymatic) h y d r o l y s i s of the s u b s t r a t e . CHEMICAL PROBE STUDIES a) Gel F i l t r a t i o n of Membranes l a b e l l e d w i t h T r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) Two m i l l i l i t e r s of erythrocyte membranes were l a b e l l e d w i t h TNBS i n a r e a c t i o n mixture containing 7.0 ml 20 mM T r i s , pH 8.0, 0.7 ml 10 mM TNBS, pH 8.0, 0.25-0.5 ml of each t e s t drug at various c o n c e n t r a t i o n s , and water to Page 32 make a f i n a l volume of 20 ml. Samples were incubated at 37+0.5 C for 1 hour and r e a c t i o n s stopped by a d d i t i o n of 1 M HC1. Samples were c e n t r i f u g e d at 40,000 x g for 10 minutes and washed once w i t h 20 mM T r i s , pH 8.0 by c e n t r i -f u g a t i o n . P e l l e t s were resuspended i n 1.5 ml water, q u a n t i t a t i v e l y t r a n s -f e r r e d to d i a l y s i s sacs and d i a l y z e d against 5 mM EDTA-5 mM 2-mercaptoethanol, pH 7.5, at 4 C for 70 ho urs. The d i a l y z e d membranes were s o l u b i l i z e d by 0.4 ml 10% SDS and b o i l e d f o r 10 minutes. A one ml a l i q u o t was a p p l i e d to a 16 x 100 mm column of Sephadex G-200 and el u t e d w i t h a s o l u t i o n c o n t a i n i n g 1% SDS-0.02% NaN3-0.05 M NH 4HC0 3 as described by Lenard (105). 50 drop f r a c t i o n s (approx. 1 ml) were c o l l e c t e d at a flow r a t e of 15-20 seconds per drop and the absorbance at 335 nm of each f r a c t i o n was measured. b) I n c o r p o r a t i o n of 5 , 5 1 - d i t h i o b i s - ( 2 - n i t r o b e n z o i c acid) (DTNB) The e f f e c t of antiarrhythmics on the membrane surface s u l f h y d r y l group r e a c t i v i t y was analyzed by studying the i n c o r p o r a t i o n of DTNB. Each sample contained 1.0 ml 0.15 M imidazole b u f f e r , pH 7.5, 0.1 ml 3mM DTNB ( i n imidazole b u f f e r ) , 1.0 ml of s o l u t i o n s c o n t a i n i n g d i f f e r e n t concentrations of te s t drug, 0.6-0.8 mg of membrane p r o t e i n and water i n a f i n a l volume 3 ml. In c o n t r o l s , water was s u b s t i t u t e d f o r the drugs. The samples were incubated at 37+0.5°C for 30 minutes, c e n t r i f u g e d at 40,000 x g f o r 10 minutes and the absorbance of the supernatant at 412 nm determined. The number of a c c e s s i b l e s u l f h y d r y l groups r e l a t i v e to the c o n t r o l was c a l c u l a t e d using a molar e x t i n c -Page 33 t i o n c o e f f i c i e n t of 1.36 x 10 (92). T o t a l s u l f h y d r y l group t i t e r of the membranes was determined by d i s r u p t i n g the membranes w i t h SDS at a f i n a l c o ncentration of 1% (w/v) i n the absence of t e s t drugs. HEMOLYSIS STUDIES ON INTACT ERYTHROCYTES The procedure used here was a m o d i f i c a t i o n of that described by Machleidt et a l (106). A l l glasswares and t e s t tubes used i n the assay were t r e a t e d w i t h chromic a c i d i n order to remove any trace of detergent which would i n f l u e n c e the hemolysis of red c e l l s . Fresh human blood was withdrawn i n t o a h e p a r i n i z e d syringe and the blood was c e n t r i f u g e d at top speed f o r 5 minutes using a c l i n i c a l c e n t r i f u g e . Erythrocytes were washed three times i n 0.9% NaCl/15 mM T r i s HCL, pH 7.0, usi n g four volumes of b u f f e r per volume of packed er y t h r o -cytes. A f t e r the f i n a l wash, 2 ml of packed c e l l s were suspended i n 32 ml of the i s o t o n i c b u f f e r . The hemolysis t e s t i n v o l v e d an i n i t i a l i n c u b a t i o n at room temperature of 0.2 ml erythrocyte suspension i n i s o t o n i c b u f f e r (0.9% NaCl/15 mM T r i s - H C l , pH 7.0) co n t a i n i n g various concentrations of t e s t drugs. This was followed by a hypotonic challenge w i t h 15 mM T r i s - H C l , pH 7.0 b u f f e r c o n t a i n i n g the same concentration of t e s t drug as was present i n the previous i n c u b a t i o n . F o l l o w i n g a 10 minute i n c u b a t i o n at room temperature, samples were c e n t r i f u g e d at 13,000 x g f o r 1 minute. The absorbance of supernatant at 540 nm was determined and t h i s value was expressed as a percent of t o t a l hemolysis of 0.2 ml erythrocytes i n d i s t i l l e d water. Each experiment was performed i n t r i p l i c a t e and r e s u l t s were expressed as mean + S.E.M. Page 34 RESULTS The o b j e c t i v e of t h i s study was to describe the membrane p e r t u r b a t i o n a l e f f e c t s of sev e r a l d i f f e r e n t pharmacological agents using the human e r y t h r o -cyte membrane as a model system and u t i l i s i n g a v a r i e t y of physiochemical and biochemical techniques. ANTIARRHYTHMIC AGENTS INVESTIGATED The membrane p e r t u r b a t i o n a l p r o p e r t i e s of conventional a n t i a r r h y t h m i c drugs such as q u i n i d i n e , l i d o c a i n e and propanolol were compared to those of p r a c t o l o l , a 3"blocker which, u n l i k e p r o p r a n o l o l , appears to l a c k d i r e c t cardiodepressant p r o p e r t i e s (107); pranolium, a quaternary analogue of pro p r a n o l o l possessing a n t i a r r h y t h m i c p r o p e r t i e s but devoid of g-adrenergic b l o c k i n g p r o p e r t i e s (108); D-600, a d e r i v a t i v e of verapamil which may exert a n t i a r r h y t h m i c e f f e c t s , at l e a s t i n p a r t , by v i r t u e of i t s a b i l i t y to block slow C a + + channels a c t i v a t e d during the plateau phase of the cardiac a c t i o n p o t e n t i a l (109); b r e t y l i u m , a quaternary adrenergic neurone blocker (42), and f i n a l l y , QX 572, a quaternary a n t i a r r h y t h m i c r e l a t e d to l i d o c a i n e (110). ANTIHEMOLYSIS STUDIES The s t a b i l i z a t i o n of erythrocytes against hypotonic hemolysis i n the presence of various antiarrhythmic drugs i s shown i n Figures 1 and 2. A l l the drugs examined produced some degree of s t a b i l i z a t i o n which v a r i e d w i t h each Page 35 FIGURE 1. An t i h e m o l y t i c e f f e c t s of q u i n i d i n e , D-600, l i d o c a i n e and p r a c t o l o l . These experiments were performed using chromic acid-washed glassware e x a c t l y as described p r e v i o u s l y (52). Each point represents the mean of t r i p l i c a t e experiments performed on a minimum of three d i f f e r e n t blood samples obtained by venipuncture from healthy v o l u n t e e r s . Page 36 Page 37 FIGURE 2. A n t i h e m o l y t i c e f f e c t s of propranol, pranolium, QX 572, and b r e t y l i u m . See legend to Figure 1. Page 38 Page 39 individual agent. Four general groups could be distinguished on the basis of the various maxima of stabilization obtained. Quinidine, D-600, lidocaine and QX 572 a l l showed approximately the same degree of protection of 55% while propranolol and pranolium had a relatively higher degree of stabilization of 65% and bretylium displayed a much lower maximum of 40%. Practolol was the only drug that seemed to produce an increasing degree of protection of intact erythrocytes from lysis in hypotonic media. The preliminary grouping of these antiarrhythmics on the basis of their antihemolytic characteristics led to the following questions (i) are the molecular characteristics of erythrocyte membrane stabilization identical for a l l the agents studied; and ( i i ) for each agent exhibiting a biphasic effect, are the molecular characteristics governing the stabilization phase identical to those underlying the l y t i c phase. Our attempts to obtain information bearing on these questions have involved a study of the influence of antiarrhythmics on the incorporation of group-specific chemical probes into erythrocyte membrane structural compon-ents and on the activity of membrane-associated enzymes. Before the studies with various chemical probes are presented, i t is necessary to comment on the homogeneity of membrane preparations employed in these experiments. ANALYSIS OF MITOCHONDRIAL CONTAMINATION AND COMPOSITIONAL CHARACTERISTICS Succinic dehydrogenase was used as an indication of mitochondrial contam-ination in the cardiac membrane preparation from guinea pig hearts. Enzyme Page 40 a c t i v i t y i n various sucrose gradient f r a c t i o n s obtained using two d i f f e r e n t methods of membrane prep a r a t i o n i s reported i n Table 1. A progressive decrease i n the s p e c i f i c a c t i v i t y of s u c c i n i c dehydrogenase i n bands to F^ was observed i n f r a c t i o n s obtained by e i t h e r method. However, the sucrose gradient f r a c t i o n s obtained by the procedure described i n the Methods (that i s , the combined methods of Hui et a l (96) and Sulakhe et a l (97)) e x h i b i t e d a r e l a t i v e l y lower a c t i v i t y of the enzyme as compared to the c o r r e s -ponding f r a c t i o n s obtained by the method of Sulakhe et a l i n d i c a t i n g a l e s s e r degree of m i t o c h o n d r i a l contamination when membranes were prepared by the former combined method. The a c t i v i t y of Mg + +-dependent Na +-K + ATPase, a plasma membrane marker enzyme of a l l f r a c t i o n s was a l s o assayed (Table I I ) . The combined F^ and F^ f r a c t i o n was found to have higher Na +-K + ATPase a c t i v i t y than F^ or f r a c t i o n and i t was found to be ouabain s e n s i t i v e (to the extent of approximately 20%). The s p e c i f i c a c t i v i t y of ouabain s e n s i t i v e Mg + +-dependent Na +-K + ATPase found i n the present membrane preparation (combined F^ and F^ f r a c t i o n s ) ( 9 . 2 ymoles Pi/mg/hr) was very comparable to that reported e a r l i e r by Sulakhe et a l (111) f o r the same f r a c t i o n s which were termed "plasma membrane enriched f r a c t i o n s " . Guinea p i g b r a i n synaptosomal membranes were al s o assayed f o r s u c c i n i c dehydrogenase a c t i v i t y . I t was found that the synaptosomal membranes were v i r t u a l l y devoid of measurable m i t o c h o n d r i a l contamination and thus c o n s t i -tuted a more homogeneous membrane prep a r a t i o n than that obtained from myocar-d i a l t i s s u e - at l e a s t as judged by m i t o c h o n d r i a l enzyme a c t i v i t y . The Page 41 Table I Comparison of the a c t i v i t y of s u c c i n i c dehydrogenase i n guinea p i g myocardial membrane f r a c t i o n s prepared by various procedures A c t i v i t y (A absorbance/mg/15 min.) Membrane F r a c t i o n Procedure 1* Procedure 2* Crude E x t r a c t 0.339 + 0.019 0.251 + 0.020 Band F-^  0.753 + 0.070 0.424 + 0.036 Band F 2 0.529 + 0.057 0.269 + 0.040 Band F3 0.384 + 0.045 0.197 + 0.011 Band F4 0.280 + 0.046 0.132 + 0.028 *Data expressed as mean + SEM was obtained from f i v e d i f f e r e n t preparations i n the case of procedure 1, using the method described by Sulakhe et a l (97), and from seven d i f f e r e n t preparations employing procedure 2, which represents our m o d i f i c a t i o n of the methods of Hui et a l (96) and Sulakhe et a l (97). Note that the crude f r a c t i o n obtained from procedure 2 had a lower a c t i v i t y than procedure 1. Page 42 Table I I Ouabain-sensitive Mg -dependent Na , K -st i m u l a t e d ATPase a c t i v i t y of guinea p i g myocardial membrane f r a c t i o n s prepared by the combined procedures of Hui et a l (96) and Sulakhe et a l (97) F r a c t i o n Mg + +(Na +-K +) ATPase a c t i v i t y ( y moles Pi/mg/hr) Crude 2.2 + 0.2 F-L 4.6 + 0.6 F 2 5 . 7 + 0 . 9 F 3 + F 4 9 . 2 + 2 . 1 F r a c t i o n s were i s o l a t e d and assayed as described i n the Methods. Results are the mean values +_ S.E.M. f o r a minimum of three d i f f e r e n t preparations. Page 43 compositional c h a r a c t e r i s t i c s of b r a i n s y n a p t i c membranes were therefore analyzed and compared w i t h those of human erythrocyte membrane. Table I I I shows the values of t o t a l p hospholipid, c h o l e s t e r o l and s i a l i c a c i d i n both erythrocyte and synaptosomal membrane. In erythrocyte membranes, as i n most plasma membranes, phospholipid and c h o l e s t e r o l are present i n approximately equimolar amounts. In synaptic membranes, both p h o s p h o l i p i d and c h o l e s t e r o l were present i n greater absolute q u a n t i t i e s on a per mg p r o t e i n basis than i n the erythrocyte membrane and the molar r a t i o of p h o s p h o l i p i d / c h o l e s t e r o l i s greater as w e l l . The amount of s i a l i c a c i d i n both preparations was the same. These p r e l i m i n a r y compositional analyses suggested that membranes ex t r a c t e d from e x c i t a b l e t i s s u e d i f f e r c o m p o s i t i o n a l l y from those of e r y t h r o c y t e s . Phospholipids from both membranes were f u r t h e r separated and examined by means of t h i n l a y e r chromatography (TLC). Four d i f f e r e n t phospho-l i p i d components were apparent on the TLC of erythrocytes w h i l e f i v e compon-ents were seen w i t h synaptosomes. I n d i v i d u a l phosphlipids were i d e n t i f i e d on the basis of t h e i r known values i n the solvent system employed. The R^ values f o r a l l the components are presented i n Table IV. Four phospholipids (phosphatidylethanolamine, p h o s p h a t i d y l c h o l i n e , sphingomyelin and phospha-t i d y l s e r i n e ) were common to both erythrocyte and synaptosomal membranes. In synaptosomal membrane, the f i f t h spot which appeared on top of the phospha-tidylethanolamine on the TLC was probably cerebroside (a g a l a c t o l i p i d found only i n b r a i n t i s s u e s ) on the basis of i t s r e l a t i v e l y high R value. Page 44 Table I I I Compositional data of human erythrocyte and guinea p i g b r a i n synaptosomal membranes. Membrane T o t a l Phospholipids C h o l e s t e r o l P L i p i d r a t j . 0 S i a l i c A c i d (u moles/mg) (u moles/mg) Choi (n moles/mg) Erythrocy t e 0.748 + 0.020 0.606 + 0.016 1.26 + 0.04 112.2 + 3.2 Synaptosome 1.273 + 0.072 0.822 + 0.070 1.60 + 0.09 114.1 + 10.8 Results were obtained from a minimum of s i x t e e n d i f f e r e n t preparations f o r human erythrocyte and s i x f o r b r a i n synaptosomal membranes. Various compositional analyses are described i n the s e c t i o n of Methods. Data are expressed as mean + S.E.M. Page 45 Table IV Values* and relative proportion of various phospholipids from membranes of human erythrocyte and guinea pig brain synaptosome. Phospolipid Erythrocyte Rf % Synaptosome Rf % Phosphatidyl- 0.138+0.004 15.7+0.8 0.135+0.012 14.8+1.3 serine Sphingomyelin 0.205+0.006 9.5+1.5 0.201+0.018 2.6+1.1 Phosphatidyl- 0.339 + 0.009 29.4 + 0.5 0.307 + 0.014 36.2 + 2.8 choline Phosphatidyl- 0.468+0.008 43.8+1.8 0.447+0.018 46.5+2.0 ethanolamine Cerebroside 0.578 +0.017 0.8+0.3 *Rf values by thin layer chromatography on S i l i c a gel plates using chloroform-methanol-ammonia (16:4:1, v/v/v) as solvent. Results presented are averages from sixteen different preparations of human erythrocyte and four of synaptosomal membranes. Data are expressed as mean +_ S.E.M. Page 46 R e l a t i v e amounts of each i n d i v i d u a l phospholipid are a l s o reported (Table IV). In both erythrocytes and synaptosomes, phosphatidylethanolamine and phosph a t i d y l c h o l i n e were the predominant phospholipids present. However, b r a i n synaptosomes possessed r e l a t i v e l y greater amounts of these two phospho-l i p i d s than e r y t h r o c y t e s . The f i n d i n g that b r a i n membrane has higher q u a n t i t i e s of these two phospholipids i s i n accordance w i t h the r e s u l t s of other workers (112-113). The minute q u a n t i t y of cerebrosides present i n the membrane i s also c o n s i s t e n t w i t h the f i n d i n g s of the above workers (112-113). MEMBRANE PERTURBATIONAL PROPERTIES OF THE ANTIARRHYTHMIC AGENTS a) E f f e c t s of Antiarrhythmics on L a b e l l i n g of Membranes by DTNB The p r o t e i n p e r t u r b a t i o n a l e f f e c t s of the antiarrhythmics were monitored i n terms of a l t e r a t i o n s produced i n the r e a c t i o n of membrane s u l f h y d r y l groups with 5 , 5 ' - d i t h i b - b i s - ( 2 - n i t r o b e n z o i c acid)(DTNB). I t has been reported that the i n t e g r i t y of membrane-protein s u l f h y d r y l groups i s a c r i t i c a l f a c t o r i n determining the hemolytic behaviour of erythrocytes (114). Godin et a l (52) have shown that a n t i h e m o l y t i c concentrations of p r o p r a n o l o l cause a progres-s i v e i n c r e a s e i n the r e a c t i v i t y of erythrocyte membrane s u l f h y d r y l groups towards DTNB. This approach i s now f u r t h e r extended to analyze the membrane p e r t u r b a t i o n a l p r o p e r t i e s of other antiarrhythmic agents. The r e s u l t s of these experiments are presented i n Figures 3 and 4. With most drugs, a progressive increase i n the a c c e s s i b i l i t y and/or r e a c t i v i t y of membrane s u l f h y d r y l groups towards DTNB i s observed i n the range of drug Page 47 FIGURE 3. M o d i f i c a t i o n of erythrocyte membrane s u l f h y d r y l groups by 5 , 5 1 - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) i n the presence of ant i a r r h y t h m i c s . Each point represents the mean of experiments performed on a minimum of four d i f f e r e n t membrane preparations. Results are expressed as the incremental increase i n s u l f h y d r y l m o d i f i c a t i o n above the c o n t r o l l e v e l i n the absence of drug. Mean c o n t r o l value +_ S.E.M. was 23.6 + 0.2 umoles SH/mg p r o t e i n . Page 48 Page 49 FIGURE 4. M o d i f i c a t i o n of erythrocyte membrane s u l f h y d r y l groups by 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) i n the presence of ant i a r r h y t h m i c s . See legend to Figure 3. Page 50 Page 51 concentrations s t a b i l i z i n g i n t a c t erythrocytes against hypotonic hemolysis. However, q u a n t i t a t i v e d i f f e r e n c e s i n the a b i l i t i e s of drugs to perturb the -3 membrane are apparent. At a concentration of 10 M, QX 572, propanolol and pranolium produce a higher degree of DTNB i n c o r p o r a t i o n than q u i n i d i n e and D-600. The two exceptions to t h i s general increase of DTNB i n c o r p o r a t i o n are p r a c t o l o l ( F i g u r e 3) and b r e t y l i u m (Figure 4 ) . Th e i r i n t e r a c t i o n w i t h the erythrocyte membrane at ant i h e m o l y t i c drug concentrations does not appreciably a l t e r the m o d i f i c a t i o n of membrane s u l f h y d r y l s by DTNB. To i n v e s t i g a t e the in f l u e n c e of p r o t e i n components on the s t a b i l i z a t i o n and d e s t a b i l i z a t i o n of i n t a c t e r y t h r o c y t e s , increases i n DTNB l a b e l l i n g produced by antiarrhythmics were expressed r e l a t i v e to the corresponding a n t i h e m o l y t i c e f f e c t s of these agents. When such r e l a t i o n s h i p s were explored (Figure 5 ) , evidence was found suggesting a common molecular p e r t u r b a t i o n a l mechanism f o r q u i n i d i n e , D-600, l i d o c a i n e and QX 572 i n the s t a b i l i z i n g range of drug concentrations but d i f f e r i n g p e r t u r b a t i o n a l mechanisms i n the d e s t a b i l i z i n g range. P r o p r a n o l o l and i t s quaternary d e r i v a t i v e , pranolium, (Figure 6) e x h i b i t e d p r o p e r t i e s q u a l i t a t i v e l y s i m i l a r to the i n i t i a l s t a b i l i z i n g component of other a n t i -arrhythmics examined, although q u a n t i t a t i v e d i f f e r e n c e s between these two r e l a t e d compounds were apparent. In p a r t i c u l a r , i t appeared that f o r a given degree of s u l f h y d r y l p e r t u r b a t i o n , the quaternary p r o p r a n o l o l analogue pro-duced a l e s s e r degree of antihemolysis than p r o p r a n o l o l . The above r e s u l t s suggest that there i s an a s s o c i a t i o n between membrane p r o t e i n s t r u c t u r a l p e rturbations and the antihemolysis induced by various a n t i a r r h y t h m i c agents Page 52 FIGURE 5. Relationship between the concentration-dependence of antihemolysis and of the incremental increases in DTNB modification of erythrocyte membranes produced by antiarrhythmics. Original data in Figures 1-4. Solid lines and dotted lines represent drug concentrations that stabilize and destabilize intact erythrocytes from hypotonic l y s i s , respectively. Page 53 Ul >• J • UJ I h Z < 6 a 4 0 S O ••„. Q X - 5 7 E L I D O C A I N E o cr-° • Q U I N I D I N E • D - B O O • 5 4 6 8 IIMCREIVIEIMTAL S U L F H Y D R Y L MODIFICATION (ju M O L E S / mg PROTEIN) Page 54 FIGURE 6. R e l a t i o n s h i p between the concentration-dependence of antihemolysis and of the incremental increases i n DTNB m o d i f i c a t i o n of erythrocyte membranes produced by pr o p r a n o l o l and pranolium. O r i g i n a l data i n Figures 2 and 4. Page 55 1 1 I I i a 4 B 8 IO IS I N C R E M E N T A L S U L F H Y D R Y L MODIFICATION ( A J M O L E S / m g PROTEIN) Page 56 and may f u r t h e r suggest that the decrease i n antihemolysis at high drug concentrations involves progressive membrane d i s r u p t i o n i n v o l v i n g a f u r t h e r exposure of s u l f h y d r y l s i t e s . E f f e c t s of the antia r r h y t h m i c drugs on DTNB i n c o r p o r a t i o n i n t o membrane s u l f h y d r y l groups were a l s o studied i n a synapt i c membrane pr e p a r a t i o n obtained from guinea p i g b r a i n and the r e s u l t s are shown i n Figure 7. With the exception of pranolium, most agents (QX 572, q u i n i d i n e , D-600, l i d o c a i n e and p r o p r a n o l o l ) e x h i b i t e d s i m i l a r DTNB i n c o r p o r a t i o n patterns as were observed i n erythrocyte ghost membranes. Pranolium d i f f e r e d from others i n that the agent produced e f f e c t s on DTNB i n c o r p o r a t i o n which e x h i b i t e d _3 s a t u r a t i o n at 10 M. The d i f f e r e n c e observed i n the DTNB i n c o r p o r a t i o n of pranolium may be due to i t s l e s s e r a b i l i t y to permeate synaptosomal membranes or to i t s l e s s e r a b i l i t y to perturb the environment surrounding membrane s u l f h y d r y l s i t e s , or some combination of both. b) E f f e c t s of Antiarrhythmics on L a b e l l i n g of the Erythrocyte Membrane by TNBS The foregoing experiments u t i l i z i n g DTNB focussed e n t i r e l y on the p e r t u r -b a t i o n a l e f f e c t s of antiarrhythmics at the l e v e l of membrane p r o t e i n s . I t i s known, however, that membrane phospholipids are even more s u s c e p t i b l e to the modifying i n f l u e n c e of l i p i d - s o l u b l e anaesthetic agents (50-51). Previous work of Godin and co-workers had shown that a n t i h e m o l y t i c p r o p e r t i e s of pro-p r a n o l o l c o r r e l a t e w e l l w i t h i t s a b i l i t y to increase the i n c o r p o r a t i o n of the amino g r o u p - s p e c i f i c probe, t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) i n t o e r y t h r o -Page 57 FIGURE 7. M o d i f i c a t i o n of guinea p i g b r a i n synaptosomal membrane s u l f h y d r y l groups by 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) i n the presence of antia r r h y t h m i c s . Each point represents the mean of experiments performed on a minimum of three d i f f e r e n t membrane preparations. Results are expressed as the incremental increase i n s u l f h y d r y l m o d i f i c a t i o n above the c o n t r o l l e v e l i n the absence of drug. Mean c o n t r o l value + S.E.M. was 26.8 + 2.3 umoles SH/mg P r o t e i n . Page 58 Page 59 cyte membrane s t r u c t u r a l components (52). Therefore, we undertook to analyze the molecular p e r t u r b a t i o n a l c h a r a c t e r i s t i c s of other antiarrhythmics i n terms of t h e i r i n f l u e n c e on the i n c o r p o r a t i o n of TNBS i n t o e r y t hrocyte membrane p r o t e i n and phospholipid amino groups. Membranes were l a b e l l e d w i t h TNBS i n the presence or absence of a n t i -arrhythmic agents at various concentrations, s o l u b i l i z e d i n sodium dodecyl s u l f a t e , and the p r o t e i n and phospholipid components resolved by g e l f i l t r a -t i o n using Sephadex G-200. This r e s u l t e d i n complete separation of the p r o t e i n components, which e l u t e d i n a complex p a t t e r n beginning at the v o i d volume, from the phospholipid components, which e l u t e d much l a t e r , thus enabling the e f f e c t s of antiarrhythmic agents on p r o t e i n and phospholipid components to be viewed independently. The data from these experiments i n d i c a t e d that antiarrhythmic agents increase TNBS i n c o r p o r a t i o n i n t o both proteins and phospholipids and that these agents d i f f e r a p p r e c i a b l y i n t h e i r r e l a t i v e p e r t u r b a t i o n a l actions on the membrane phospholipid and p r o t e i n components (Figure 8). These e f f e c t s were most conveniently expressed i n terms of the slope of the l i n e a r r e l a t i o n -s h i p c h a r a c t e r i z i n g drug-stimulated i n c o r p o r a t i o n of TNBS i n t o phospholipids as a f u n c t i o n of corresponding i n c o r p o r a t i o n of TNBS i n t o p r o t e i n s at concen-t r a t i o n s of antiarrhythmics causing s t a b i l i z a t i o n of i n t a c t erythrocytes (Table V). The f a c t that a l l slopes were greater than one r e f l e c t s the greater p e r t u r b a t i o n a l a c t i o n s of these pharmacological agents on membrane phospholipids as compared with membrane p r o t e i n s . The r e l a t i o n s h i p s between Page 60 FIGURE 8. R e l a t i v e increases i n the i n c o r p o r a t i o n of t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) i n t o phospholipid and p r o t e i n components of erythrocyte membranes i n the presence of i n c r e a s i n g concentrations of ant i a r r h y t h m i c s . Membranes were l a b e l l e d w i t h TNBS i n the presence of antiarrhythmics and the extent of t r i n i t r o p h e n y l a t i o n of phospholipid and p r o t e i n components resolved by g e l f i l t r a t i o n using Sephadex G-200 was determined s p e c t r o p h o t o m e t r i c a l l y (52). Each point represents the mean of experiments performed on two d i f f e r e n t membrane preparations. Page 61 RELATIVE PROTEIN LABELLING Page 62 Table V E f f e c t s of antiarrhythmics on the slope of the l i n e a r r e l a t i o n s h i p c h a r a c t e r i z i n g membrane phospholipid l a b e l l i n g by TNBS as a f u n c t i o n of membrane p r o t e i n l a b e l l i n g Drug Concentration Range C o r r e l a t i o n s l o p e * (mean + S.E.M.) Lidocaine 2.0-10 mM 2.6 + 0.6 Quinidine 0.1-0.8 mM 3.6 + 0.3 P r o p r a n o l o l 0.05-1.00 mM 5.3 + 0.9 D-600 0.05-0.60 mM 6.6 + 1.2 QX-572 0.01-0.60 mM 9.0 + 0.9 Pranolium 0.02-1.00 mM 12.1 + 2.4 * L a b e l l i n g experiments were performed on a minimum of two d i f f e r e n t membrane pre p a r a t i o n s . Slopes of the r e g r e s s i o n l i n e s d e s c r i b i n g the r e l a t i o n s h i p between drug-induced increases i n phospholipid l a b e l l i n g as a f u n c t i o n of p r o t e i n l a b e l l i n g were evaluated using a Compucorp ( S t a t i s t i c i a n ) c a l c u l a t o r . Page 63 antih e m o l y t i c p r o p e r t i e s of the drugs examined and t h e i r membrane perturb-a t i o n a l actions were assessed by expressing the maximal TNBS i n c o r p o r a t i o n i n t o p r o t e i n and phospholipid components (as determined by absorbance at 335 nm) at each drug concentration as a f u n c t i o n of erythrocyte hemolytic s t a b i l -i z a t i o n produced at the same concentration (Figure 9). These analyses revealed that q u i n i d i n e , QX 572, p r o p r a n o l o l and pranolium a l l produce i d e n t i c a l e f f e c t s on the i n c o r p o r a t i o n of TNBS i n t o p r o t e i n s ( l e f t panel, Figure 9), w h i l e l i d o c a i n e and D-600 deviate from t h i s p a t t e r n , w i t h the former e x h i b i t i n g the greatest p r o t e i n p e r t u r b a t i o n a l a c t i o n of a l l the agents and the l a t t e r a c t u a l l y causing an i n i t i a l decrease i n TNBS i n c o r p o r a t i o n r e l a t i v e to that i n the absence of added drug. When the drug-induced increases i n TNBS l a b e l l i n g of erythrocyte membrane phospholipids were analyzed, a completely d i f f e r e n t spectrum of l a b e l l i n g p r o f i l e s was obtained ( r i g h t panel, Figure 9). The two quaternary agents, pranolium and QX 572, produced equivalent e f f e c t s which, on the b a s i s of t h e i r sharp increase with drug c o n c e n t r a t i o n , appeared cooperative i n nature. Quinidine and D-600 showed e q u i v a l e n t l i p i d p e r t u r b a t i o n a l p r o p e r t i e s which d i f f e r e d from those of l i d o c a i n e , w i t h p r o p r a n o l o l occupying an intermediate p o s i t i o n between these two groups. The small e f f e c t s of b r e t y l i u m and p r a c t o l o l on TNBS in c o r p o r -a t i o n were only observed at rather high drug concentrations although both drugs, e s p e c i a l l y p r a c t o l o l , have p r e v i o u s l y been shown to possess a n t i -hemolytic p r o p e r t i e s (Figures 1 and 2). This apparent l a c k of p e r t u r b a t i o n a l actions on membrane s t r u c t u r a l components by these two d i f f e r e n t c l a s s e s of Page 64 FIGURE 9. E f f e c t s of antiarrhythmics on the l a b e l l i n g of p r o t e i n and phospholipid components of erythrocyte membranes. The extent of t r i n i t r o p h e n y l a t i o n was assessed as described i n Figure 8. Each point i s the mean of experiments performed on two d i f f e r e n t membrane preparations. Page 65 7 /o A N T I H E M O L Y S I S Page 66 antiarrhythmic agents might suggest that t h e i r a n t i h e m o l y t i c p r o p e r t i e s are produced by a molecular mechanism which d i f f e r s from that of the other substances examined. The s t r u c t u r a l b a s i s of the d e s t a b i l i z i n g or l y t i c phase of a n t i -arrhythmics-erythrocyte i n t e r a c t i o n was examined using two drugs w i t h pronounced l y t i c actions at high c o n c e n t r a t i o n s , namely q u i n i d i n e and D-600 (see F i g u r e 1). In both cases, a sharp increase i n phospholipid l a b e l l i n g was observed at l y t i c drug concentrations. A corresponding increase i n p r o t e i n l a b e l l i n g was al s o seen at l y t i c concentrations of q u i n i d i n e but not D-600. I t would appear, t h e r e f o r e , t h a t , as the experiments using DTNB had suggested (Fig u r e 5 ) , the molecular c h a r a c t e r i s t i c s of drug-induced l y s i s of ery t h r o -cytes by q u i n i d i n e and D-600 are not i d e n t i c a l . EFFECTS OF ANTIARRHYTHMICS ON ACTIVITY OF MEMBRANE-ASSOCIATED ENZYMES Results from the foregoing experiments have provided evidence that a n t i -arrhythmics d i f f e r considerably i n t h e i r p e r t u r b a t i o n a l c h a r a c t e r i s t i c s i n erythrocyte membranes and that these d i f f e r i n g patterns of s t r u c t u r a l a l t e r -a t i o n s may be relevant to the antihemolytic p r o p e r t i e s of these agents i n i n t a c t e r y t h r o c y t e s . Another approach to analyze the f u n c t i o n a l consequences of these molecular i n t e r a c t i o n s i n v o l v e d a study of the e f f e c t s of i n c r e a s i n g concentrations of antiarrhythmics on the a c t i v i t y of se v e r a l membrane-associated enzymatic processes. When the e f f e c t s of antiarrhythmic agents on the a c t i v i t i e s of Page 67 Mg -independent (or basal) and Mg - s t i m u l a t e d NPPases of erythrocyte ghosts were s t u d i e d , most drugs showed a small i n h i b i t o r y e f f e c t on both b a s a l and M g + + - s t i m u l a t e d NPPase a c t i v i t i e s . The i n h i b i t i o n e x h i b i t e d a weak dependence on concentration of drug, so t h a t , f o r convenience, only an average value f o r i n h i b i t i o n over the whole range of drug concentrations i s reported (Table V I ) . I t i s c l e a r from these data that the i n h i b i t o r y p r o p e r t i e s of these drugs are v i r t u a l l y equivalent i n the s t a b i l i z i n g range of concen-t r a t i o n s w i t h the b a s a l NPPase a c t i v i t y showing approximately two-fold greater i n h i b i t i o n than the M g + + - s t i m u l a t e d NPPase. At concentrations producing l y s i s of i n t a c t e r y t h r o c y t e s , D-600 and q u i n i d i n e both e x h i b i t e d somewhat enhanced i n h i b i t i o n . The i n h i b i t o r y actions of p r a c t o l o l (Figure 10) were very much greater and showed a concentration dependency compared to other agents examined (Table VI) but were q u a l i t a t i v e l y s i m i l a r , i n that the basal NPPase a c t i v i t y was approximately twice as s u s c e p t i b l e to i n a c t i v a t i o n as the Mg + + - s t i m u l a t e d NPPase a c t i v i t y . In the small i n h i b i t o r y e f f e c t s of p r o p r a n o l o l , another 3 - b l o c k e r , t h i s p a t t e r n appeared to be reversed so that basal a c t i v i t y was l e s s s u s c e p t i l e to i n h i b i t i o n (Figure 10). The a c t i v i t y of the ouabain-sensitive K + - s t i m u l a t e d component of Mg + +-dependent NPPase (K +-NPPase) was very much more s u s c e p t i b l e to progressive i n h i b i t i o n by i n c r e a s i n g drug concentrations. The agents shown i n Figure 11 appeared to share an i n i t i a l common i n h i b i t o r y component which was followed at higher concentrations by i n h i b i t o r y patterns c h a r a c t e r i s t i c f o r each agent. The e f f e c t s of p r o p r a n o l o l on the a c t i v i t y of K +-NPPase ( r i g h t Page 68 Table VI E f f e c t s of antiarrhythmics on a c t i v i t y of b a s a l (Mg -independent) and M g + + - s t i m u l a t e d erythrocyte membrane p-nitrophenylphosphatase Average I n h i b i t i o n * * Drug* b a s a l M g + + - s t i m u l a t e d B r e t y l i u m 8.0 + 1. .3 3.5 + 1.1 Lidocaine 11.1 + 1 .7 6.7 + 1.0 Quinidine s t a b i l i z i n g range 13.7 + 3. .4 6.6 + 2.1 d e s t a b i l i z i n g range 37.1 + 3 .0 14.9 + 1.2 D-600 s t a b i l i z i n g range 13.2 + 1. .3 6.5 + 0.8 d e s t a b i l i z i n g range 15.7 + 3 .2 11.5 + 1.5 Pranolium 10.2 + 1. .0 5.6 + 2.1 Pr o p r a n o l o l 4.1 + 0. .9 9.2 + 0.8 *A11 drugs were tested i n the range of concentrations producing s t a b i l i z a t i o n of i n t a c t erythrocytes (see F i g s . 1 and 2) unless otherwise i n d i c a t e d . **Results are expressed as mean + S.E.M. of experiments using three d i f f e r e n t membrane preparations. Page 69 FIGURE 10. I n h i b i t i o n of bas a l (Mg + +-independent) and Mg + +-dependent p-nitrophenylphosphatase (NPPase) by p r a c t o l o l and pro p r a n o l o l i n r e l a t i o n to the an t i h e m o l y t i c e f f e c t s of these agents. Each point represents the mean of experiments performed using three d i f f e r e n t membrane preparations. Page 70 % A N T I H E M O L Y S I S Page 71 FIGURE 11. I n h i b i t i o n of the K + - s t i m u l a t e d component of Mg + +-dependent p-nitrophenyphosphatase (K +-NPPase) by antiarrhythmics i n r e l a t i o n to the ant i h e m o l y t i c e f f e c t s of these agents. Each point represents the mean of experiments performed using three d i f f e r e n t membrane preparations. Page 72 % A N T I H E M O L Y S I S Page 73 panel, Figure 11) was q u a l i t a t i v e l y s i m i l a r but q u a n t i t i v e l y d i f f e r e n t from those shown on the l e f t panel of the same f i g u r e (Figure 11). Both b r e t y l i u m and p r a c t o l o l again e x h i b i t e d d i f f e r e n t i n h i b i t o r y p r o p e r t i e s which were u n l i k e those of the other pharmacological agents t e s t e d , w i t h the l a t t e r showing minimal i n h i b i t o r y potency and the former e x h i b i t i n g e f f e c t s which were v i r t u a l l y independent of concentration i n the range stud i e d ( r i g h t panel, Figure 11). Since K + - s t i m u l a t e d p-nitrophenylphosphatase i s a wi d e l y d i s t r i b u t e d plasma membrane enzyme, a p r e l i m i n a r y i n v e s t i g a t i o n was undertaken to compare the i n h i b i t o r y e f f e c t s of antiarrhythmics on the enzyme from human erythrocyte membranes w i t h these i n p a r t i a l l y p u r i f i e d sarcolemmal membranes derived from guinea p i g heart (96-97). The myocardial enzyme was shown to be i n h i b i t e d by four of the f i v e drugs t e s t e d , although to a l e s s e r degree than the erythro-cyte enzyme. Further, i n most cases, a s t i m u l a t o r y component was noted at lowest drug concentrations w i t h the myocardial K +-NPPase (Figure 12). Again, d i s t i n c t groupings of drugs emerged with q u i n i d i n e and D-600 apparently sharing a common r e l a t i o n s h i p which d i f f e r e d from that f o r e i t h e r p r o p r a n o l o l or pranolium, and l i d o c a i n e e x h i b i t i n g s t i m u l a t o r y e f f e c t s on the myocardial enzyme over the e n t i r e range of concentrations t e s t e d . These p r e l i m i n a r y studies on myocardial sarcolemmal membranes were again i n d i c a t i v e of perturb-a t i o n a l actions of antiarrhythmics which d i f f e r e d w i t h the pharmacological agent i n question and the i n h i b i t o r y patterns i n the two systems stud i e d Page 74 FIGURE 12. R e l a t i o n s h i p between erythrocyte and myocardial membrane K +-NPPase i n h i b i t i o n by various a n t i a r r h y t h m i c s . The erythrocyte data are those presented i n F i g . 11 and the myocardial data were obtained using four d i f f e r e n t sarcolemmal preparations (see Methods). Enzyme a c t i v i t i e s were determined i n e x a c t l y the same manner for both preparations (52) except that r e a c t i o n times of 60 minutes and 10 minutes were used f o r the erythrocyte and cardiac preparations r e s p e c t i v e l y . Page 75 % INHIBITION E R Y T H R O C Y T E K + -NPPase Page 76 suggested that the s t r u c t u r a l consequences of drug-membrane i n t e r a c t i o n are not equivalent i n the erythrocyte membrane and the plasma membrane of the myocardium. ++ ++ The f i n a l membrane enzyme system studied was Mg -dependent Ca stimulated ATPase which, i n the erythrocyte membrane, c o n s i s t s of the two ++ -3 d i f f e r e n t components, one stimulated by Ca at concentrations of 10 M (low a f f i n i t y Ca + +-ATPase) and a second component a c t i v a t e d by concen-t r a t i o n s of C a + + i n the range of 10 ^ M (high a f f i n i t y Ca + +-ATPase) (52,115). The extent to which each of these two a c t i v i t i e s i s i n v o l v e d i n the e n e r g i z a t i o n of a c t i v e C a + + e x t r u s i o n from erythrocytes as the r e s u l t of adenosine triphosphate (ATP) h y d r o l y s i s has not as yet been f i r m l y e s t a b l i s h e d (115-116). The i n h i b i t o r y p r o p e r t i e s of ant i a r r h y t h m i c agents on these two components were not i d e n t i c a l (Figure 13). With p r o p r a n o l o l and pranolium (both of which show s i m i l a r e f f e c t s ) and l i d o c a i n e , the component stimulated by low concentrations of C a + + (termed the high a f f i n i t y a c t i v i t y ) was more r e a d i l y i n h i b i t e d than the low a f f i n i t y a c t i v i t y . In c o n t r a s t , q u i n i d i n e and D-600 i n h i b i t e d both a c t i v i t e s to about the same extent (Figure 13). Results with b r e t y l i u m and p r a c t o l o l are not shown as both of them d i d not appreciably modify the a c t i v i t y of e i t h e r component. EFFECTS OF ANTIARRHYTHMICS ON ACTIVITY OF ACETYLCHOLINESTERASE As an extension of the a n a l y s i s of drug-induced p r o t e i n s t r u c t u r a l a l t e r -a t i o n i n terms of the f u n c t i o n a l consequences of drug-membrane i n t e r a c t i o n , Page 77 FIGURE 13. R e l a t i v e i n h i b i t i o n of high and low a f f i n i t y C a + + - s t i m u l a t e d Mg + +-dependent ATPase (Ca + +-ATPase) from erythrocyte membranes by various a n t i a r r h y t h m i c s . The assays f o r high and low a f f i n i t y a c t i v i t i e s were i d e n t i c a l (52) except that a Ca + +-EGTA b u f f e r system was used to achieve concentrations of free C a + + i n the media of 1 and 90 M r e s p e c t i v e l y . Each point represents the mean of experiments performed using three d i f f e r e n t membrane preparations. Page 78 Page 79 the influence of antiarrhythmics on the activity of membrane-associated acetylcholinesterase was studied. This enzyme has been well characterized in erythrocytes and is known to be located on the external surface of the eryth-rocyte membrane (93). Thus, the activity of this enzyme can be measured in both intact erythrocytes and in hemoglobin-free erythrocyte membranes using the method described by Ellman (117) wherein acetylthiocholine is employed as substrate and the rate of formation of thiocholine is measured colori-metrically using 5,5 1-dithio-bis-(2-nitrobenzoic acid). These experiments enabled us to determine the extent to which the membrane actions of anti-arrhythmics in isolated erythrocyte membranes are relevant to those in the membrane of the intact cells. Furthermore, the presence of acetylcholin-esterase in plasma membrane fractions derived from guinea pig brain tissue has enabled a direct comparison of the membrane perturbations produced by the antiarrhythmic agents in excitable membranes with those in the membrane of the erythrocyte. Such a comparison was prompted by the correlation which appears to exist between the antihemolytic properties of a number of lipid-soluble anaesthetic molecules and the a b i l i t y of these substances to exert electrical stabilization in excitable tissues (54). Antiarrhythmics were shown to cause progressive inhibition of erythrocyte acetylcholinesterase at concentrations producing stabilization of these cells against hypotonic hemolysis. When inhibitory effects at each drug concen-tration were related to corresponding degree of erythrocyte antihemolysis, the various antiarrhythmics examined f e l l into a number of distinct categories Page 80 (Figur e 14). Again, q u i n i d i n e , D-600 and QX 572 a l l emerged as one c l a s s w i t h b r e t y l i u m , p r a c t o l o l and p r o p r a n o l o l e x i s t i n g as a separate group. The exception was l i d o c a i n e which apparently d i d not belong to e i t h e r one of the above c l a s s e s . This f i n d i n g suggested the p o s s i b i l i t y that d i f f e r e n c e s may e x i s t i n the molecular b a s i s of enzyme i n h i b i t i o n induced by these various agents. Pranolium could not be stud i e d as i t was found to cause hemolysis of i n t a c t erythrocytes under the experimental conditions of the enzyme assay. The i n h i b i t o r y e f f e c t s of antiarrhythmics on the a c e t y l c h o l i n e s t e r a s e of i n t a c t e r y t h r o c y t e s , erythrocyte ghost membranes and guinea p i g b r a i n s y n a p t i c membranes were compared using H i l l p l o t a n a l y s i s , as i l l u s t r a t e d f o r b r e t y l i u m , p r o p r a n o l o l and l i d o c a i n e i n Figure 15. The slopes of these l i n e a r r e l a t i o n s h i p s provide a measure of the c o o p e r a t i v i t y and/or the st o i c h i o m e t r y c h a r a c t e r i z i n g the i n h i b i t o r y process (118) and the a n t i l o g of the x - i n t e r c e p t represents the K ^ value of the i n h i b i t o r ; that i s , the concentration required to produce 50% i n h i b i t i o n of a c e t y l c h o l i n e s t e r a s e a c t i v i t y . The r e s u l t s of these studies are summarized i n Table V I I . No marked d i f f e r e n c e s between the actions of these agents i n the i n t a c t erythrocyte and i t s i s o l a t e d membrane were apparent, although for a few drugs, namely, l i d o c a i n e and p r a c t o l o l , K values f or enzyme i n h i b i t i o n were somewhat greater f o r the membranes than f o r the i n t a c t c e l l s . Considerably greater d i f f e r e n c e s i n K ^ values were noted when erythrocyte membranes and b r a i n s y n a p t i c membranes were compared (Table V I I ) . This was p a r t i c u l a r l y so i n the case of q u i n i d i n e , l i d o c a i n e , p r a c t o l o l and QX 572, suggesting that the membrane Page 81 FIGURE 14. Inhibition of intact erythrocyte acetylcholinesterase activity by various antiarrhythmics relative to their antihemolytic properties. Each point represents the mean + S.E.M. of experiments using three different samples of blood. Page 82 Page 83 FIGURE 15. H i l l p l o t a n a l y s i s of the i n h i b i t i o n of erythrocyte membrane and synaptic membrane a c e t y l c h o l i n e s t e r a s e by a n t i a r r h y t h m i c s . Each poi n t represents the mean + S.E.M. of experiments using three d i f f e r e n t erythrocyte membrane preparations and two d i f f e r e n t synaptic membrane preparations. Page 84 ERYTHROCYTE M E M B R A N E BRETYLIUM P R O P R A N O L O L ,6 LIDOCAINE SYNAPTIC M E M B R A N E BRETYLIUM P R O P R A N O L O L ° LIDOCAINI • 1 -S L O G D R U G COIMC. Page 85 Table V I I I n h i b i t o r y e f f e c t s of antiarrhythmics on the a c e t y l c h o l i n e s t e r a s e a c t i v i t y of i n t a c t e r y t h r o c y t e s , e r y t h r o c y t e membranes and b r a i n s y n a p t i c membranes* Drug Ery t h r o c y t e E r y t h r o c y t e membranes Synaptic Membranes K 5 H i l l K.5 H i l l K.5 H i l l (mM) Slope (mM) Slope (mM) Slope B r e t y l i u m 0.22+.03 1. 19+.13 0.21+.0 0.92+0 0.28+.01 0.87+.02 Quinidine 1.84+.28 0. 92+.06 1.84+.09 0.97+0 5.00+.31 0.96+.01 Lidocaine 6.05+.40 1. 15+.04 8.50+.04 1.02+.09 22.7+0 0.95+.12 D-600 0.90+.07 0. 72+.03 0.61+0 1.00+.03 0.67+.02 1.24+.04 P r a c t o l o l 0. 70+.03 0. 84+.03 0.99+.05 1.03+.03 1.58+.03 1.01+.19 Pr o p r a n o l o l 1.10+.11 1. 06+.17 1.40+.08 1.13+.01 1.14+.06 1.54+.04 QX-572 0.73+.06 1. 11+.13 0.76+.03 0.87+.10 1.73+.06 1.63+.13 * The data f o r erythrocyte and synapt i c membranes were obtained using a minimum of two d i f f e r e n t membrane preparations and the experiments w i t h i n t a c t erythrocytes were performed i n t r i p l i c a t e on a minimum of three d i f f e r e n t blood samples. In each case, i n h i b i t i o n data were p l o t t e d as shown i n F i g . 15 and i n h i b i t o r y constants evaluated from r e g r e s s i o n l i n e s determined using a Compucorp (140) s t a t i s t i c a l c a l c u l a t o r . Data are expressed as mean + S.E.M. Page 86 environments where these drugs i n t e r a c t are not i d e n t i c a l i n the two systems s t u d i e d . Examination of H i l l p l o t slopes for the i n h i b i t i o n revealed an apparent l a c k of c o o p e r a t i v i t y or of stoichiometry greater than one i n the i n h i b i t o r y e f f e c t s of most antiarrhythmics so that H i l l c o e f f i c i e n t s of u n i t y were observed i n most cases (118). However, two exceptions were observed f o r propr a n o l o l and QX 572 whose i n t e r a c t i o n w i t h s y n a p t i c membranes (but not erythrocyte membranes) was c h a r a c t e r i z e d by a slope s i g n i f i c a n t l y greater than one. In order to determine the nature of the i n h i b i t i o n exerted by the a n t i -arrhythmics on a c e t y l c h o l i n e s t e r a s e , enzyme k i n e t i c studies were performed using drug concentrations producing the same degree of a c e t y l c h o l i n e s t e r a s e i n h i b i t i o n . A value of 35% was a r b i t r a r i l y chosen as a convenient l e v e l of i n h i b i t i o n . Since the i n f l u e n c e of drugs on the a c t i v i t y of a c e t y l c h o l i n -esterase i n i n t a c t c e l l s was not found to d i f f e r from that of the ghost membranes, only i n t a c t erythrocytes were used for the k i n e t i c study. The values of K (Michaelis-Menten constant, an i n d i c a t i o n of substrate m concentration which gives h a l f the numerical value of maximal v e l o c i t y ) and V (the maximal rat e of enzymatic r e a c t i o n at high substrate concen-max 1 & t r a t i o n ) f o r a c e t y l c h o l i n e s t e r a s e i n the absence and presence of drug were obtained by Eadie p l o t a n a l y s i s , as i l l u s t r a t e d f o r the c o n t r o l , p r a c t o l o l and D-600 i n Figure 16 where the slope and the y - i n t e r c e p t of the l i n e a r r e l a t i o n -ship give the value of K and V r e s p e c t i v e l y . The r e s u l t s are summar-r & m max r 3 i z e d i n Table V I I I . A l l drugs except p r a c t o l o l showed an increased f o r Page 87 FIGURE 16. Eadie p l o t a n a l y s i s of the nature of the i n h i b i t i o n of i n t a c t erythrocyte a c e t y l c h o l i n e s t e r a s e a c t i v i t y by a n t i a r r h y t h m i c s . C o n t r o l values were obtained from experiments performed i n the absence of any added drug. Each point represents the means _+ S.E.M. of experiments using two d i f f e r e n t samples of blood. Page 88 Page 89 Table VIII Influence of antiarrhythmics on the enzyme-substrate kinetics of intact erythrocytes* Drug KmCmM) Control .083 + .005 .088 + .004 Practolol .086 + .001 .058 + .005 Bretylium .224 + .013 .054 + .002 Propranolol .143 + .002 .050 + .001 Quinidine .280 + .103 .070 + .001 D-600 .195 + .008 .054 + .006 Lidocaine .161 + .033 .058 + 0 QX-571 .411 + .130 .065 + .004 *The data for intact erythrocytes were obtained using a minimum of two different blood samples. Experimental details were described in the Methods section. In each case, data were plotted as shown in Fig. 16 and kinetic constants were derived from Eadie plots and evaluated from regression lines determined using a Compucorp (140) sta t i s t i c a l calculator. Data are expressed as mean + S.E.M. Page 90 the substrate but a decrease V of the enzyme. These observations implied max J that the inhibition exerted by these drugs on the activity of acetylcholin-esterase of intact erythrocytes was of a complex or mixed type. Practolol was the only drug that showed a non-competitive type of inhibiton of the enzyme as characterized by a parallel shift from the control in the Eadie plot (Figure 16) . which gave a decreased V of the acetylcholinesterase but an iden-max J t i c a l K for the substrate, m Finally, the recent observations of Livne and Bar-Yaakov (104) indicating that the inhibitory effects of the antihemolytic substance linolenoyl sorbitol on erythrocyte acetylcholinesterase are modulated by the magnitude of the transmembrane chloride gradient have suggested a comparable approach with antiarrhythmics to study potential dependent properties of these pharmacological agents i n the erythrocyte model system. Acetylcholinesterase activity of intact erythrocytes in the presence of antiarrhythmics was assayed in a high chloride medium or in a low chloride medium (with sulfate ion replacing chloride). The inhibition of the enzyme by most of the drugs tested was independent of the nature of the medium (Figure 17) . However, the inhibitory properties of both quinidine and propranolol were enhanced in a low chloride medium (Figure 17). These latter findings paralleled the results of Livne and Bar-Yaakov (104) who demonstrated that inhibition of erythrocyte acetylcholinesterase by linolenoyl sorbitol was enhanced as the magnitude of the transmembrane chloride gradient increased. Page 91 FIGURE 17. E f f e c t of transmembrane c h l o r i d e gradient on the i n h i b i t i o n of i n t a c t erythrocyte a c e t y l c h o l i n e s t e r a s e a c t i v i t y by ant i a r r h y t h m i c s . Each point represents the means + S.E.M. of experiments using a minimum of two d i f f e r e n t samples of blood. Page 92 11 (fl < a LU h tf) 11 Z - i • I U J > 11 a < Z • h E i z R 90 • 11 7 Q 1 2 11 • 50 a • i u 3 ° i (D I , a • BRETYLIUM / o LIDOCAINE j\ a D-600 e o a a e a BO IOO PROPRANOLOL SO 40 EO BO IOO 7 10 INHIBITION A C E T Y L C H O L I N E S T E R A S E (LOW CHLORIDE MEDIUM) Page 93 The fact that the membrane actions of antiarrhythmics in the present model system vary in their dependence on transmembrane potential may point to possible differences in the molecular basis by which these compounds modify the functional properties of electrically excitable tissues. Page 94 DISCUSSION A large number of studies e x i s t which have been concerned with the e f f e c t s of drugs on the p r o p e r t i e s of membranes or membrane-associated processes. By and l a r g e , these studies have been of a d e s c r i p t i v e nature w i t h l i t t l e d e t a i l e d i n f o r m a t i o n of the molecular mechanism governing the i n t e r a c t i o n . This s i t u a t i o n i s p a r t i c u l a r l y true i n the case of a n t i a r r h y t h m i c s , where studies have focused e i t h e r on t h e i r e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s or on t h e i r c l i n i c a l e f f e c t i v e n e s s i n a v a r i e t y of s i t u a t i o n s , w h i l e the studies of molecular aspects governing the actions of these drugs have been very much neglected. The experiments reported i n t h i s study provide ample evidence for d i f f e r e n c e s i n the s t r u c t u r a l and f u n c t i o n a l consequences of the i n t e r a c t i o n of pharmacologically d i v e r s e antiarrhythmic agents w i t h a simple model membrane system, namely that of the human eryth r o c y t e . I t i s suggested that these observations may u l t i m a t e l y allow a b e t t e r understanding of the molecular b a s i s by which these substances modify myocardial f u n c t i o n . I t has g e n e r a l l y been assumed that the a b i l i t y of c e r t a i n l i p i d - s o l u b l e amphipathic molecules to produce d i r e c t myocardial depression, sometimes r e f e r r e d to as a q u i n i d i n e - l i k e a c t i o n , i s synonymous w i t h the s t a b i l i z a t i o n by these compounds of erythrocytes against hypotonic l y s i s (54). A l l of the a n t i a r r h y t h m i c agents examined here, i n c l u d i n g the permanently charged quaternary d e r i v a t i v e s pranolium, b r e t y l i u m and QX 572, d i d produce a considerable degree of antihemolysis (Figures 1 and 2) as expected from t h e i r Page 95 known cardiodepressant actions. An exception was the $-adrenergic antagonist practolol, which produced a high degree of erythrocyte stabilization (Figure 1) but unlike propranolol, a related g-adrenergic antagonist, practolol is virtually devoid of direct cardiodepressant activity (119-120). For several other agents (lidocaine, quinidine, QX 572 and D-600) a typical pattern of hypotonic stabilization at low concentrations followed by c e l l lysis at higher concentrations was observed. Such biphasic actions of l i p i d soluble anaes-thetics on red cel l s t a b i l i t y have been reviewed in detail by Seeman (54,56). The mechanism governing the stabilization of erythrocytes by anaesthetics is believed to be related to their surface-active properties at low concen-trations (54,56). Thus, i t seems safe to assume that protection of red cells from lysis is the result of a physical interaction of the anaesthetic agents within the membrane of the erythrocyte. Such membrane interactions are related to the l i p i d solubilities of the anaesthetic molecules and an excellent correlation exists between hydrophobicity and anaesthetic potency as well as with the ab i l i t y of general and local anaesthetics to fluidize membrane lipids (54). However, i t is not known i f both types of anaesthetics interact with a common hydrophobic site in neuronal membranes. The investi-gations of Kendig and Cohen (121) on pressure reversal of anaesthesia have provided evidence to show that both general and local anaesthetics have similar sites of action in biological membranes. Seeman suggested that proteins figure prominently in the expansion of biological membranes produced by anaesthetic molecules (122). The fact that there is an approximately Page 96 t e n - f o l d discrepancy between the volume of occupation and the extent of membrane expansion produced by membrane s t a b i l i z e r s i n erythrocyte and synaptosome membranes but not i n p r o t e i n - f r e e pure l i p i d systems i n d i c a t e s that proteins are important i n the process of membrane expansion (54,56). There i s much evidence showing that drug-membrane i n t e r a c t i o n causes p e r t u r -bations i n membrane s t r u c t u r a l components with both p r o t e i n and phospholipid components being i n v o l v e d (54). Experiments using DTNB to detect drug-induced p r o t e i n perturbations i n erythrocyte membranes showed that most of the drugs i n v e s t i g a t e d here produced an increase i n DTNB i n c o r p o r a t i o n i n t o membrane p r o t e i n s u l f h y d r y l groups (Figures 3 and 4). The only drugs that d i d not e x h i b i t any enhancement of DTNB l a b e l l i n g of erythrocyte membrane p r o t e i n components were p r a c t o l o l and b r e t y l i u m . In con t r a s t to the marked s t i m u l -atory e f f e c t s of p r o p r a n o l o l (Figure 4), p r a c t o l o l a c t u a l l y caused a decrease i n the i n c o r p o r a t i o n of DTNB i n t o erythrocyte membranes (Figure 3). The absence of d i r e c t cardiodepressant e f f e c t s of p r a c t o l o l might have some bearing on i t s i n a b i l i t y to enchance DTNB l a b e l l i n g of p r o t e i n components i n erythrocyte membranes. However, the observed membrane s t a b i l i z i n g a ctions of p r a c t o l o l (Figure 1) may be relevant to the a b i l i t y of t h i s molecule to antagonize ouabain-induced arrhythmias (120,123) p o s s i b l y as the r e s u l t of a depressant a c t i o n at the l e v e l of adrenergic nerves (124). I t i s s i g n i f i c a n t that b r e t y l i u m , which i s al s o b e l i e v e d to act at l e a s t i n part by depressing cardiac adrenergic nerve f u n c t i o n (42), a l s o exerted a n t i h e m o l y t i c e f f e c t s i n i n t a c t erythrocytes which were not associated with an increased i n c o r p o r a t i o n Page 97 of DTNB (Figu r e 4) i n t o erythrocyte membranes. Our f i n d i n g s suggest that the molecular mechanisms by which these two antia r r h y t h m i c agents exert a n t i -hemolytic e f f e c t s i n i n t a c t erythrocytes may d i f f e r from those by which the other antiarrhythmics studied s t a b i l i z e red c e l l s . As for the group of agents that exerted a b i p h a s i c a c t i o n on the s t a b i l i z a t i o n of erythrocyte from hypo-t o n i c l y s i s (namely D-600, q u i n i d i n e , l i d o c a i n e and QX 572), an i d e n t i c a l p a t t e r n of DTNB i n c o r p o r a t i o n i n t o erythrocyte membrane s u l f h y d r y l s was common to a l l of them at concentrations i n the s t a b i l i z a t i o n range (Figure 5) but, at concentrations of drug producing l y s i s , the above mentioned drugs d i f f e r e d from each other i n the r e l a t i o n s h i p between p r o t e i n p e r t u r b a t i o n (as r e f l e c t e d i n DTNB i n c o r p o r a t i o n ) and hemolysis (Figure 5). The f a c t that l i d o c a i n e and QX 572 d i d not appear to d i f f e r i n t h e i r d e s t a b i l i z a t i o n phases i s i n t e r e s t i n g i n the l i g h t of the cl o s e s t r u c t u r a l resemblance between these two molecules and t h e i r comparable anti a r r h y t h m i c actions (125). S i m i l a r i t i e s i n membrane p r o t e i n p e r t u r b a t i o n a l c h a r a c t e r i s t i c s were observed with p r o p r a n o l o l and i t s quaternary d e r i v a t i v e , pranolium (Figure 6). However, at a given degree of antihemolysis, the quaternary d e r i v a t i v e produced a greater degree of membrane s u l f h y d r y l p e r t u r b a t i o n . Despite the q u a n t i t a t i v e d i f f e r e n c e i n the p e r t u r -b a t i o n a l a c t i v i t y , the s i m i l a r i t i e s between pranolium and p r o p r a n o l o l are cons i s t e n t w i t h the s i m i l a r spectra of antiarrhythmic a c t i v i t y observed when these agents are used to antagonize ouabain-induced arrhythmias, an a c t i o n b e l i e v e d to be governed s o l e l y by the membrane-stabilizing p r o p e r t i e s of these molecules (126) Page 98 When p r o t e i n p e r t u r b a t i o n was studied i n guinea p i g b r a i n membranes using the same approach, q u i n i d i n e , D-600 and QX 572 a l l produced an equivalent enhancement of s u l f h y d r y l l a b e l l i n g when these agents were stud i e d i n the same concentration range as had been used i n the erythrocyte s t u d i e s . U n l i k e the pat t e r n observed i n the er y t h r o c y t e , p r o p r a n o l o l d i f f e r e d from pranolium i n that the quaternary d e r i v a t i v e produced a l e s s e r increase i n DTNB in c o r p o r -a t i o n than p r o p r a n o l o l . Lidocaine produced p r a c t i c a l l y no s t i m u l a t i o n of DTNB i n t o the s u l f h y d r y l groups of b r a i n synaptosomal membranes i n the concen-t r a t i o n range that s t a b i l i z e d i n t a c t e r y t h r o c y t e s . This d i f f e r e n c e cannot be a t t r i b u t e d to d i f f e r i n g absolute amounts of s u l f h y d r y l groups i n synaptosomal membranes as compared with erythrocytes because the t o t a l number of s u l f h y d r y l groups i n both preparations (expressed on a per mg p r o t e i n b a s i s ) was the same ( r e s u l t s not shown). Rather, i t would seem that d i f f e r e n c e s e x i s t i n the s t r u c t u r a l consequences of lidocaine-membrane i n t e r a c t i o n i n the two systems. This i n tu r n would suggest the p o s s i b i l i t y that fundamental d i f f e r e n c e s may e x i s t i n the molecular basis of membrane s t a b i l i z a t i o n by antiarrhythmics i n e x c i t a b l e and non-excitable t i s s u e s . Experiments r e l a t i n g the ant i h e m o l y t i c p r o p e r t i e s of antiarrhythmics to t h e i r e f f e c t s on the m o d i f i c a t i o n of membrane s u l f h y d r y l groups by DTNB (Figure 3-6) and on the a c t i v i t y of membrane enzymes (Figure 11, Table VI) suggested that a l l drugs, with the p r e v i o u s l y noted exception of b r e t y l i u m and p r a c t o l o l , produce a comparable degree of membrane p r o t e i n p e r t u r b a t i o n f or a given l e v e l of antihemolysis at concentrations i n the s t a b i l i z i n g range. Page 99 However, when drug-induced membrane a l t e r a t i o n s were viewed i n terms of both phospholipid and p r o t e i n p e r t u r b a t i o n a l e f f e c t s using the amino group t r i n i t r o p h e n y l a t i n g reagent TNBS (52), d i f f e r e n c e s i n drug-induced s t r u c t u r a l a l t e r a t i o n s at the l e v e l of both membrane proteins and phospholipids became apparent (Figure 8 and 9, Table V). The l a c k of obvious p a r a l l e l i s m between drug e f f e c t s on p r o t e i n and phospholipid l a b e l l i n g i n d i c a t e d that a consi d e r -able degree of s t r u c t u r a l independence e x i s t s between membrane p r o t e i n and phospholipid s t r u c t u r a l components. The f a c t that DTNB (Figure 5) and TNBS (Figure 9) y i e l d d i f f e r i n g i n f o r m a t i o n on the s t r u c t u r a l c h a r a c t e r i s t i c s of drug-induced p r o t e i n m o d i f i c a t i o n suggested that the e f f e c t s of a n t i a r r y t h m i c s are g e n e r a l i z e d and that these two reagents do not probe i d e n t i c a l p r o t e i n domains i n the membrane. The foregoing experimental r e s u l t s have shown that antiarrhythmics are able to exert widespread and s t r u c t u r a l l y d i v e r s e e f f e c t s on the c o n f i g u r -a t i o n a l s t a t e of membrane p r o t e i n and l i p i d components. In order to o b t a i n more d e t a i l e d i n f o r m a t i o n regarding the s t r u c t u r a l consequences of membrane-drug i n t e r a c t i o n , the i n f l u e n c e of s e v e r a l antiarrhythmics on the K +-sti m u l a t e d , o u a b a i n - s e n s i t i v e component of p-nitrophenylphosphatase (K +-NPPase)(127) and on the C a + + - s t i m u l a t e d component of Mg + +-dependent ATPase (Ca + +-ATPase)(116) - two enzyme systems whose r o l e i n a c t i v e i o n transport i s c r i t i c a l l y dependent on i n t e r a c t i o n s w i t h membrane phospholipid -was examined. Page 100 With the exception of p r a c t o l o l and b r e t y l i u m (Figure 11, Table V I ) , whose membrane ac t i o n s were g e n e r a l l y a t y p i c a l , a l l a n t i a r r h y t h m i c s were consider-ably more i n h i b i t o r y towards the K + - s t i m u l a t e d component of Mg + +-dependent p-nitrophenylphosphatase as compared w i t h the a c t i v i t i e s i n the presence of Mg + + alone or the basal a c t i v i t y i n the absence of added Mg + + (Figure 10, Table V I ) . This presumably r e f l e c t s the greater dependence of the ouabain-s e n s i t i v e K + - s t i m u l a t e d component on the s t r u c t u r a l i n t e g r i t y of the membrane. The more marked i n h i b i t i o n of K +-NPPase al s o suggests that these agents may exert t h e i r p e r t u r b a t i o n a l e f f e c t s on the outer surface of the erythrocyte membrane where the K + - s t i m u l a t e d component of the enzyme complex i s l o c a t e d (128). I n h i b i t o r y actions of antiarrhythmics have p r e v i o u s l y been demonstrated - f o r example by Lowry et a l (129) who found that q u i n i d i n e i n h i b i t e d the K +-NPPase i n r a t b r a i n t i s s u e s , hemoglobin-free human e r y t h r o -cyte and beef heart membrane. The experiments with K +-NPPase allowed a d i r e c t comparison of the p e r t u r b a t i o n a l c h a r a c t e r i s t i c s of various antiarrhythmics on erythrocyte membranes and cardiac sarcolemmal membranes (Figure 12). With the exception of l i d o c a i n e , high concentrations of antiarrhythmics were i n h i b i t o r y , a l b e i t to v a r y i n g degrees, i n both systems. In myocardial membranes, however, the e f f e c t s of l i d o c a i n e were s t i m u l a t o r y , a property shared by other a n t i -arrhythmics at low concentrations. The common pr o p e r t i e s of q u i n i d i n e and D-600 and the d i f f e r i n g a c t i o n s of pr o p r a n o l o l and pranolium when compared i n these two systems tended to p a r a l l e l the ph o s p h o l i p i d p e r t u r b a t i o n a l e f f e c t Page 101 seen w i t h TNBS i n erythrocyte membrane (Figure 9) while the p r o p e r t i e s of l i d o c a i n e may be governed by i t s d i s t i n c t i v e balance between ph o s p h o l i p i d and p r o t e i n p e r t u r b i n g a c t i o n s (Figure 9, Table V). The foregoing observations were important i n emphasizing that the consequences of antiarrhythmic-membrane i n t e r a c t i o n s are governed not only by the s t r u c t u r e of the drug i n question but also by the nature of the membrane. Antiarrhythmic substances have been shown to modify Ca + +-dependent processes i n a v a r i e t y of membrane systems (87,130-132) and these e f f e c t s may w e l l be important i n determining some of t h e i r c a r d i o v a s c u l a r actions (87). This i s p a r t i c u l a r l y so i n the case of D-600, a verapamil d e r i v a t i v e whose antiarrhythmic p r o p e r t i e s are b e l i e v e d to in v o l v e the blockade of slow C a + + channels a c t i v a t e d during the plateau phase of the car d i a c a c t i o n p o t e n t i a l (109,133). The C a + + - s t i m u l a t e d ATPase system of erythrocyte membranes provided a convenient model with which to i n v e s t i g a t e the i n f l u e n c e of various antiarrhythmics on f u n c t i o n a l l y s i g n i f i c a n t Ca + +-membrane i n t e r a c t i o n s . The f a c t that antiarrhythmics e x h i b i t e d marked d i f f e r e n c e s i n t h e i r a b i l i t y to i n h i b i t the high and low a f f i n i t y components ( a c t i v a t e d by 10 ^ and 10 4 M concentrations of free C a + + r e s p e c t i v e l y ) suggested that these a c t i v i t i e s do not a r i s e from i d e n t i c a l molecular species w i t h i n the membrane and these drugs may provide an experimental means of assessing the r o l e of each component i n determining a c t i v e C a + + e x t r u s i o n from the erythrocyte (115-116). More ge n e r a l l y , however, the groupings of drugs observed l i k e l y r e f l e c t d i f f e r i n g patterns of membrane p r o t e i n and phospholipid perturbations which vary i n Page 102 terms of t h e i r i n f l u e n c e on membrane-Ca i n t e r a c t i o n . No d e f i n i t i v e evidence could be found i n any of our experiments to i n d i c a t e that the mem-brane p e r t u r b a t i o n a l actions of D-600 studi e d here have any d i r e c t relevance to i t s a c t i o n i n b l o c k i n g C a + + channels i n the heart. However, the c a p a c i t y of t h i s molecule to i n t e r a c t with and perturb membrane s t r u c t u r a l components may w e l l account for i t s c o n c e n t r a t i o n - and time-dependent e f f e c t s on the k i n e t i c s of the C a + + - c a r r y i n g system and on the steady s t a t e outward current i n cat p a p i l l a r y muscle (133) and i t s a b i l i t y to modify K + fluxes i n canine P u r k i n j e f i b e r s (134). Moreover, i t has r e c e n t l y been shown that l o c a l anaesthetics are able to i n f l u e n c e slow C a + + - c u r r e n t s at concentrations comparable to those causing blockade of f a s t Na +-currents (135). The suggestion of the authors that these r e s u l t s may r e f l e c t p e r t u r b a t i o n by these agents of a sarcolemmal l i p o p r o t e i n m a t r i x common to both channels (135) i s e n t i r e l y c o n s i s t e n t w i t h the f i n d i n g s described here. A l t e r a t i o n s i n the a u t o m a t i c i t y of myocardial t i s s u e s produced by a n t i -arrhythmics probably represent the most fundamental mechanism whereby these substances produce t h e i r t h e r a p e u t i c a c t i o n s . Although the i o n i c b a s i s of a u t o m a t i c i t y i s as yet incompletely understood and l i k e l y i s not i d e n t i c a l i n a l l regions of the h e a r t , the a b i l i t y of antiarrhythmics such as l i d o c a i n e (136) and p r o p r a n o l o l (87,137) to modify transmembrane fl u x e s of K + has been i m p l i c a t e d i n t h e i r a c t i o n s on cardiac a u t o m a t i c i t y . Model st u d i e s i n erythrocytes (87) have demonstrated that p r o p r a n o l o l , at concentrations causing s t a b i l i z a t i o n of erythrocytes (52) and an enhancement of K + e f f l u x Page 103 from canine P u r k i n j e f i b e r s (137), produces an increase i n K" e f f l u x from these c e l l s , presumably as the r e s u l t of drug-induced membrane s t r u c t u r a l perturbations leading to the displacement of membrane-bound C a + + (87). Lidocaine does not share t h i s property and q u i n i d i n e has been shown to i n h i b i t the enhancement of K + e f f l u x from erythrocytes caused by increased i n t r a -c e l l u l a r l e v e l s of C a + + (132). These f i n d i n g s are c e r t a i n l y i n accord w i t h the d i f f e r i n g p r o p e r t i e s of these three agents as revealed i n the present study of erythrocyte membranes and suggest, by analogy, that the molecular mechanisms by which these three antiarrhythmics a l t e r a u t o m a t i c i t y i n myocardial t i s s u e s may be d i f f e r e n t . This i n t u r n may have a d i r e c t bearing on the di v e r s e patterns of antiarrhythmic a c t i v i t y e x h i b i t e d by these substances when used c l i n i c a l l y (136,138-139). These considerations serve to emphasize the inappropriateness of the general term q u i n i d i n e - l i k e a c t i o n when used to describe the d i r e c t membrane e f f e c t s of antiarrhythmics other than q u i n i d i n e on the heart. Furthermore, i f a p a r a l l e l e x i s t s between the b i p h a s i c a c t i o n of antiarrhythmics i n erythrocytes (Figures 1 and 2) and t h e i r c h a r a c t e r i s t i c p a t t e r n of antiarrhythmogenesis followed by cardiodepression at high doses when these agents are employed c l i n i c a l l y , our p r e l i m i n a r y a n a l y s i s of the molecular b a s i s of the s t a b i l i z a t i o n and l y t i c phases i n erythrocytes (see R e s u l t s ) suggests the p o s s i b i l i t y that these two phases of anti a r r h y t h m i c drug a c t i o n i n v i v o may not n e c e s s a r i l y represent a continuum but might be to some extent m e c h a n i s t i c a l l y d i s t i n c t . Page 104 The erythrocyte membrane has proven to be convenient model system with which to investigate the molecular consequences of drug-membrane interaction. The fact that substances such as propranolol induce alterations in the structural and functional characteristics of erythrocyte membranes (52) at concentrations producing hypotonic stabilization of intact erythrocytes (52,54) and electrical stabilization of nerves (56,140) suggests that an analysis of drug-induced perturbations in isolated erythrocyte membranes might provide meaningful information regarding the mechanism by which pharmaco-logical agents alter the functional properties of excitable tissues. It therefore becomes important to ascertain whether or not molecular aspects of drug action studies in isolated membranes are relevant to the situation in the intact c e l l . In this regard, Aloni and Livne (141) have recently demonstrated that the interaction of the antihemolytic substance, linolenoyl sorbitol with the membrane of the intact erythrocyte causes a concentration-dependent inhibition of the acetylcholinesterase which is not observed in isolated erythrocyte membranes or in solubilized preparations of the enzyme. It has been reported previously that membranes prepared by hypotonic hemolysis do not necessarily possess the same composition, structure, or s t a b i l i t y as the original erythrocyte membrane (142-144). Retention of aldolase and glycer-aldehyde phosphate dehydrogenase by human erythrocyte ghosts has been shown to be dependent upon pH and osmotic strength of the washing buffer (142). Mitchell and Hanahan (143) reported that human erythrocyte ghosts prepared from hypotonic hemolysis were less stable than intact cells in terms of the Page 105 l o s s of membrane l i p i d s and p r o t e i n s i n the presence of hypertonic s a l i n e s o l u t i o n . Burger et a l (144) al s o demonstrated that bovine erythrocyte ghosts l o s t most of t h e i r g l y c o l y t i c enzymes and a c e r t a i n amount of the l i p o p r o t e i n s of the i n t a c t c e l l by s o l u b i l i z a t i o n during hypotonic hemolysis. Information obtained from chemical l a b e l l i n g experiments have f u r t h e r provided evidence to show that membranes of i n t a c t erythrocytes and ghosts d i f f e r from each other i n terms of membrane r e a c t i v i t y and s t r u c t u r e s . Both phosphatidylethanolamine and phosphatidylserine reacted e q u a l l y w i t h d i n i t r o f l u o r o b e n z e n e i n ghost membranes but only the phosphatidylethanolamine of the i n t a c t e r y t h rocyte membranes was l a b e l l e d by the agent (145), suggesting that the ghost membranes possessed higher a c c e s s i b i l i t y to d i n i t r o f l u o r o b e n z e n e . In a double l a b e l l i n g study w i t h a c e t i c anhydride, the higher l i p i d r e a c t i v i t y observed i n the ghost membranes but not i n i n t a c t c e l l s was b e l i e v e d to r e s u l t from an a l t e r a t i o n i n the l i p i d - l i p i d or l i p i d - p r o t e i n i n t e r a c t i o n s r e s u l t i n g from the prep a r a t i o n of the ghost membranes (146). Landsberger et a l (147) found that the bovine serum albumin-spin l a b e l complex was t i g h t l y bound to the ghost membranes but not to i n t a c t c e l l s . They suggested that such d i f f e r e n c e i n b i n d i n g may be due to the a c c e s s i b i l i t y of the inner surface of the ghost membrane to large molecules and/or an a l t e r a t i o n of the ghost membrane surface. Therefore, i s o l a t e d erythrocyte membranes may be considered to be c l o s e d e r i v a t i v e s of the membrane of i n t a c t c e l l s , r e t a i n i n g many of i t s s t r u c t u r a l components and f u n c t i o n a l p r o p e r t i e s , e.g. l i p i d s , enzymes mediating i o n t r a n s p o r t , e t c . However, i n a d d i t i o n to r e t e n t i o n of i t s osmotic p r o p e r t i e s and l i p i d content, Page 106 human hemoglobin-free erythrocyte membranes prepared by hemolysis and repeated washings with hypotonic s a l t s o l u t i o n s , s t i l l r e t a i n the a c e t y l c h o l i n e s t e r a s e a c t i v i t y of the o r i g i n a l i n t a c t erythrocyte (93, 142-143). Thus, a c e t y l -c h o l i n e s t e r a s e i s b e l i e v e d to be an i n t e g r a l membrane component (142-143). The l o c a l i z a t i o n of t h i s enzyme on the outer surface of the membrane (93) allows measurement of i t s a c t i v i t y i n i n t a c t c e l l s as w e l l as i n i s o l a t e d membranes and t h i s property has provided one means of d i r e c t l y comparing the p e r t u r b a t i o n a l actions of antiarrhythmics on both the i n t a c t and i s o l a t e d c e l l membrane of the e r y t h r o c y t e . The experiments i n the present studies have shown that the dependence of a c e t y l c h o l i n e s t e r a s e i n a c t i v a t i o n by a n t i h e m o l y t i c agents on c e l l u l a r i n t e g r i t y as described for l i n o l e n o y l s o r b i t o l (141) i s not a general phenomenon (Figu r e 15, Table V I I ) . Thus, the i n h i b i t o r y e f f e c t s of b r e t y l i u m , q u i n i d i n e , and QX 572 on erythrocyte a c e t y l c h o l i n e s t e r a s e of i n t a c t c e l l s were v i r t u a l l y i d e n t i c a l to those i n the i s o l a t e d membrane. For other drugs, namely l i d o c a i n e , D-600 and p r a c t o l o l , small but s i g n i f i c a n t d i f f e r e n c e s i n the concentration of drug producing 50% i n h i b i t i o n (K value) i n membranes versus i n t a c t c e l l s were noted. These d i f f e r e n c e s may a r i s e from a l t e r a t i o n s i n membrane a r c h i t e c t u r e produced during hypotonic l y s i s (148-149). Expression of the enzyme i n h i b i t i o n by antiarrhythmics r e l a t i v e to t h e i r corresponding a n t i h e m o l y t i c e f f e c t s (Figure 14) i l l u s t r a t e d that for a given degree of membrane occupation by drug (54), the s t r u c t u r a l consequence of t h i s i n t e r a c t i o n vary w i t h the s t r u c t u r e of the agent i n question. The a c e t y l -Page 107 c h o l i n e s t e r a s e of erythrocyte membrane has been shown to be a l i p o p r o t e i n (143) whose pr o p e r t i e s are i n f l u e n c e d by changes i n the l i p i d environment of the membrane (150-151). The studies w i t h TNBS presented here as w e l l as work by Godin et a l (52) have shown that antiarrhythmics d i f f e r markedly i n t h e i r a b i l i t y to perturb erythrocyte membrane s t r u c t u r e , as r e f l e c t e d i n the e f f e c t s of these agents on the t r i n i t r o p h e n y l a t i o n of p r o t e i n and phospholipid amino groups by the amino-group probe, TNBS. Br e t y l i u m was minimally d i s r u p t i v e i n t h i s regard and t h i s f i n d i n g , when taken w i t h the pronounced and v i r t u a l l y i d e n t i c a l i n h i b i t o r y e f f e c t s of t h i s compound on the membrane a c e t y l -c h o l i n e s t e r a s e of both erythrocytes and synaptosomes (Table VII) may i n d i c a t e a r e l a t i v e l y s p e c i f i c a c t i o n of t h i s quaternary molecule on the enzyme, pos-s i b l e at the a n i o n i c s i t e normally i n v o l v e d i n the b i n d i n g of the c a t i o n i c n i t r o g e n of the a c e t y l c h o l i n e molecule. I t would seem l i k e l y that the d i v e r s e e f f e c t s of the other antiarrhythmics on membrane a c e t y l c h o l i n e s t e r a s e a c t i v i t y are governed by the d i f f e r i n g a b i l i t i e s of these agents to i n t e r a c t with and modify p r o t e i n and phospholipid s t r u c t u r a l components of the membrane. Fur-ther experimentation w i l l be required before a d e t a i l e d a n a l y s i s of the molecular basis of these i n h i b i t o r y e f f e c t s i s p o s s i b l e . Our experiments are important i n emphasizing that l i p i d - s o l u b l e amphiphatic substances, which have g e n e r a l l y been assumed to protect erythrocytes against hypotonic l y s i s and s t a b i l i z e e l e c t r i c a l l y e x c i t a b l e membranes by a comparable mechanism (54), do not exert i d e n t i c a l p e r t u r b a t i o n a l actions on the e r y t h r o c y t e membrane. While these f i n d i n g s i n erythrocytes suggest a corresponding degree of complexity i n Page 108 the d i r e c t membrane actions of antiarrhythmics on myocardial t i s s u e s , e x t r a p -o l a t i o n of r e s u l t s obtained w i t h erythrocytes to the s i t u a t i o n i n more complex e x c i t a b l e membrane systems must be undertaken with considerable caution. This was apparent from the d i f f e r i n g i n h i b i t o r y e f f e c t s of antiarrhythmics on the K +-NPPase a c t i v i t y of erythrocyte and heart membranes (Figure 11) and a l s o from the d i f f e r e n c e s i n K r values and H i l l c o e f f i c i e n t s f o r c h o l i n e s t e r a s e i n h i b i t i o n noted f o r some drugs when erythrocyte and synapt i c membrane prepar-ations were compared d i r e c t l y (Table V I I ) . Since membrane p a r t i t i o n c o e f f i c -i e n t s f o r drugs i n erythrocytes and synaptosomal membranes are g e n e r a l l y equivalent (74) and the molecular p r o p e r t i e s of c h o l i n e s t e r a s e i n erythrocytes comparable to those of the enzyme derived from e x c i t a b l e t i s s u e s (152), these d i f f e r e n c e s point to the importance of membrane s t r u c t u r a l o r g a n i z a t i o n i n determining the f u n c t i o n a l consequences of drug i n t e r a c t i o n i n a p a r t i c u l a r membrane system. When the b a s i c mode of i n h i b i t i o n of a c e t y l c h o l i n e s t e r a s e exerted by the antiarrhythmics was explored, a type of mixed i n h i b i t i o n , wherein both enzyme-substrate i n t e r a c t i o n (as r e f l e c t e d i n K ) and c a t a l y t i c c a p a c i t y (V ) are a f f e c t e d , was observed. This probably r e s u l t s from a l t e r a t i o n s i n enzyme environment produced by m u l t i p l e types of drug-induced membrane s t r u c t u r a l p e rturbations as were apparent i n our experiments i n v o l v i n g TNBS and DTNB. P r a c t o l o l was the only drug which showed a non-competitive type of i n h i b i t i o n . I t i s p o s s i b l e that t h i s d i s t i n c t i v e i n h i b i t o r y p a t t e r n may be Page 109 governed by s i m i l a r s t r u c t u r a l features which determine i t s unique 8 - b l o c k i n g a c t i o n s which are associated w i t h minimal d i r e c t cardiodepressant p r o p e r t i e s and i t s c h a r a c t e r i s t i c e f f e c t s on erythrocyte membranes described p r e v i o u s l y . Erythrocytes have been shown to possess s i g n i f i c a n t d i f f u s i o n p o t e n t i a l s using f l u o r e s c e n t cyanine dyes whose d i s t r i b u t i o n across the membrane i s p o t e n t i a l - s e n s i t i v e (153) or by means of d i r e c t microelectrode measurements i n the case of salamander giant red c e l l s (154). In human erythrocytes the magnitude of t h i s p o t e n t i a l , which i s l a r g e l y determined by the d i s t r i b u t i o n of c h l o r i d e i o n and i s approximately -9 mV (153), has been shown to govern the i n h i b i t i o n of erythrocyte a c e t y l c h o l i n e s t e r a s e by l i n o l e n o y l s o r b i t o l (104). The present studies have shown that enzyme i n h i b i t i o n by b r e t y l i u m , D-600 and l i d o c a i n e i s independent of the c h l o r i d e gradient across the ery t h r o c y t e membrane (F i g u r e 17). On the other hand, the i n h i b i t o r y e f f e c t s of q u i n i d i n e and p r o p r a n o l o l were enhanced i n the presence of an increased transmembrane c h l o r i d e gradient. Although i t i s known that the i n t e r a c t i o n of propr a n o l o l w i t h i n t a c t erythrocytes causes an increase i n the membrane p e r m e a b i l i t y to K + (87), an a c t i o n which might f u r t h e r magnify the p o t e n t i a l d i f f e r e n c e s across the membrane of erythrocytes suspended i n a low c h l o r i d e medium, t h i s a c t i o n does not seem to be shared by q u i n i d i n e which, r a t h e r , tends to decrease membrane p e r m e a b i l i t y to K + (132,155). The greater i n h i b i t o r y e f f e c t s o f p r o p r a n o l o l and q u i n i d i n e i n lower c h l o r i d e media might be the r e s u l t of a l t e r a t i o n s i n c o n f i g u r a t i o n of membrane s t r u c t u r a l components induced by the augmented transmembrane p o t e n t i a l g r a d i e n t , which a l t e r a t i o n s Page 110 could modify the a b i l i t y of quinidine and propranolol to interact with and/or perturb the structure of the erythrocyte membrane. Further experiments w i l l attempt to elucidate the molecular basis of this proposed potential-dependent membrane configurational change using covalent bond-forming group-specific chromophoric probes as described earlier (52). It is hoped that eventually these studies w i l l be extended to include an invest-igation of membrane structural changes accompanying depolarization of excit-able tissues using isolated synaptosomes, which undergo depolarization in  vitro in the presence of veratridine or high potassium concentrations (87, 154), as a model. This system might also enable a detailed analysis of the effects of membrane-active drugs on potential-dependent membrane phenomena. Page 111 BIBLIOGRAPHY 1. Singh, B.N. Pharmac. Ther. (C) 2, 125-150 (1977). 2. Rosen, M.R., Hoffman, B.F., and Wit, A.L. Amer. Heart J. 89, 115-122 (1975). 3. Josephson, M.E., Seides, S.F., Batsford, W.P., Weisfogel, G.M., Akhtar, M., Caracta, A.R., Lau, S.H., and Damato, A.N. Amer. Heart J. 8_7, 55-64 (1974) . 4. Josephson, M.E., Caracta, A.R., R i c c i u t t i , M.A., Lau, S.H., and Damato, A.N. Amer. J. Cardiol. 33, 596-603 (1974). 5. Seides, S.F., Josephson, M.E., Batsford, W.P., Weisfogel, G.M., Lau, S.H., and Damato, A.N. Amer. 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