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Membrane actions of antiarrhythmic drugs Au, Tony Long Sang 1978

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MEMBRANE ACTIONS OF ANTIARRHYTHMIC DRUGS by  B.Sc,  TONY LONG SANG AU U n i v e r s i t y o f 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 in THE FACULTY OF GRADUATE STUDIES Department o f Pharmacology  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1978  © Tony Long Sang Au, 1978  In p r e s e n t i n g t h i s an  advanced degree at  the L i b r a r y I  further  for  this  in p a r t i a l  freely  available  for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e  representatives. thesis for  Department of  of  the  requirements  Columbia,  I agree  r e f e r e n c e and copying o f  this  It  financial  i s understood that gain s h a l l  Pharmacology  The U n i v e r s i t y of B r i t i s h  2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5  27th April, 1978  Columbia  not  copying or  for  that  study. thesis  purposes may be granted by the Head of my Department  written permission.  Date  fulfilment  the U n i v e r s i t y of B r i t i s h  s h a l l make it  scholarly  by h i s of  thesis  or  publication  be allowed without my  ii ABSTRACT  The structural and functional consequences of the interaction of various antiarrhythmics with human erythrocyte membranes, guinea pig brain synaptosomes and myocardial sarcolemmal membranes were studied at drug concentrations affecting the s t a b i l i t y of intact erythrocytes to hypotonic l y s i s . It was assumed that such stabilization might bear some molecular resemblance to the e l e c t r i c a l s t a b i l i z i n g properties of these drugs i n excitable tissues. Membrane perturbational actions of these drugs were measured i n terms of the specific incorporation of the chromophoric probes, 5,5 -dithio-bis1  (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 quaternary analogues QX 572 and pranolium, exhibited a concentration-dependent stimulation of DTNB and TNBS incorporation.  At drug concentrations producing  erythrocyte s t a b i l i z a t i o n , the protein perturbational properties of quinidine, lidocaine, D-600 and QX 572 as viewed i n terms of DTNB labelling were equivalent while differences were apparent with quinidine, D-600 and lidocaine at high concentrations  i n the destabilizing range. Most agents, with the excep-  tion of pranolium, showed a similar pattern of DTNB incorporation i n brain synaptic membranes as i n erythrocytes.  Studies of the incorporation of TNBS  iii  i n t o e r y t h r o c y t e membranes i n d i c a t e d t h a t a n t i a r r h y t h m i c s induce g r e a t e r s t r u c t u r a l a l t e r a t i o n s i n membrane p h o s p h o l i p i d s proteins.  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 m i n i m a l 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 i n c o r p o r a t i o n s i n t o  e r y t h r o c y t e membranes, a l t h o u g h  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 , t h a t the membrane  p e r t u r b a t i o n a l a c t i o n s of a n t i a r r h y t h m i c s g r o u p - s p e c i f i c chemical  as analyzed  here by means of  probes are a b e t t e r i n d e x of t h e i r d i r e c t m y o c a r d i a l  membrane a c t i o n s than e r y t h r o c y t e The  as compared w i t h membrane  stabilization.  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 a n t i a r r h y t h m i c s were shown to be c r i t i c a l l y dependent on the drug and membrane i n q u e s t i o n . e r y t h r o c y t e membrane o u a b a i n - s e n s i t i v e K - s t i m u l a t e d +  phosphatase (K -NPPase) was +  The  a c t i v i t y of  p-nitrophenyl-  more r e a d i l y i n h i b i t e d than t h a t of  M g - i n d e p e n d e n t and M g - s t i m u l a t e d NPPase by most drugs examined. ++  myocardial  In  + +  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 a c t i o n s o n l y at the lowest concentrations.  The  C a - A T P a s e system i n the e r y t h r o c y t e membrane was ++  i n h i b i t e d by a n t i a r r h y t h m i c s w i t h p r o p r a n o l o l , p r a n o l i u m a r e l a t i v e l y h i g h e r degree of i n h i b i t i o n of the h i g h C a  and + +  drug  also  l i d o c a i n e showing  a f f i n i t y component  w h i l e q u i n i d i n e and D-600 e x e r t e d e q u a l i n h i b i t o r y a c t i o n s on b o t h h i g h  and  iv low C a  ++  a f f i n i t y components of the enzyme. A comparison of the  perturbational actions of antiarrhythmics i n isolated erythrocyte membranes, in the membranes of the intact erythrocyte and i n brain synaptic membranes was made by analyzing the effects of drugs on the a c t i v i t y of the membrane acetylcholinesterase present i n these preparations.  Inhibitory actions of a l l  drugs tested were comparable i n both intact and isolated erythrocyte membranes but differed i n 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 noncompetitively.  The transmembrane chloride gradient had no influence on the  inhibition by bretylium, lidocaine and D-600 of the acetylcholinesterase a c t i v i t y of the intact cells but the inhibition produced by quinidine and propranolol was enhanced when erythrocytes were suspended i n a low chloride medium. The foregoing results, therefore, indicate that the membrane perturbational actions of antiarrhythmics vary with the agent i n question and with the particular membrane system.  It i s suggested that the molecular mechanisms  by which these drugs alter cardiac automaticity may not be identical and  may  d i f f e r i n various regions of the myocardium. This i n turn may underlie the differing spectra of c l i n i c a l effectiveness exhibited by these agents.  pharmacological  V  TABLE OF CONTENTS Page INTRODUCTION  1  MATERIALS  20  METHODS  21  Membrane Preparations Compositional Assays of Membranes Thin Layer Chromatographic Analysis of Membrane Phospholipids Enzyme Assays Chemical Probe Studies Hemolysis Studies RESULTS Antiarrhythmic Agents Investigated Antihemolytic Studies Analysis of Mitochondrial Contamination and Compositional Characteristics Membrane Perturbational Properties of the Antiarrhythmic Agents Effects of Antiarrhythmics on A c t i v i t y of Membrane-Associated Enzymes Effects of Antiarrhythmics on A c t i v i t y of Acetylcholinesterase DISCUSSION BIBLIOGRAPHY  21 24 24 25 31 33 34 34 34 39 46 66 76 94 111  vi  LIST OF TABLES  No.  Title  Page  I.  Comparison o f the a c t i v i t y o f s u c c i n i c dehydrogenase i n guinea p i g m y o c a r d i a l membrane f r a c t i o n s prepared by v a r i o u s procedures.  41  II.  O u a b a i n - s e n s i t i v e Mg -dependent N a , K - s t i m u l a t e d ATPase a c t i v i t y o f guinea p i g m y o c a r d i a l membrane f r a c t i o n s prepared by the combined procedures o f H u i e t a l and Sulakhe et a l .  42  III.  C o m p o s i t i o n a l data o f human e r y t h r o c y t e and guinea p i g b r a i n synaptosomal membranes.  44  Rf v a l u e s and r e l a t i v e p r o p o r t i o n o f v a r i o u s p h o s p h o l i p i d s from membranes o f human e r y t h r o c y t e and guinea p i g b r a i n synaptosome.  45  E f f e c t s o f a n t i a r r h y t h m i c s on the s l o p e o f 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 p h o s p h o l i p i d l a b e l l i n g by TNBS as a f u n c t i o n o f membrane p r o t e i n l a b e l l i n g .  62  E f f e c t s o f a n t i a r r h y t h m i c s on a c t i v i t y o f b a s a l ( M g - i n d e p e n d e n t ) and M g - s t i m u l a t e d e r y t h r o c y t e membrane p-nitrophenylphosphatase.  68  I n h i b i t o r y e f f e c t s o f a n t i a r r h y t h m i c s on the a c e t y l cholinesterase a c t i v i t y of intact erythrocytes, erythrocyte membranes and b r a i n s y n a p t i c membranes.  85  Influence of antiarrhythmics of i n t a c t e r y t h r o c y t e s .  89  IV.  V.  VI.  ++  ++  VII.  VIII.  +  +  + +  on the enzyme-substrate k i n e t i c s  Vll  LIST OF FIGURES No.  Title  1.  A n t i h e m o l y t i c e f f e c t s o f q u i n i d i n e , D-600, l i d o c a i n e and practolol.  2.  Antihemolytic e f f e c t s of p r o p r a n o l o l , pranolium, bretylium.  3.  M o d i f i c a t i o n o f e r y t h r o c y t e 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 t h e presence o f antiarrhythmics.  4.  M o d i f i c a t i o n o f e r y t h r o c y t e 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 t h e presence o f ant i a r r h y thmi c s.  5.  R e l a t i o n s h i p between t h e c o n c e n t r a t i o n - d e p e n d e n c e o f a n t i h e m o l y s i s and o f t h e i n c r e m e n t a l i n c r e a s e s i n DTNB m o d i f i c a t i o n o f e r y t h r o c y t e 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 t h e c o n c e n t r a t i o n - d e p e n d e n c e o f a n t i h e m o l y s i s and o f the i n c r e m e n t a l i n c r e a s e s i n DTNB m o d i f i c a t i o n o f e r y t h r o c y t e membranes produced by p r o p r a n o l o l and pranolium.  7.  M o d i f i c a t i o n o f 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 t h e presence o f a n 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 e r y t h r o c y t e membranes i n t h e presence o f i n c r e a s i n g concentrations of antiarrhythmics.  9.  E f f e c t s o f a n t i a r r h y t h m i c s on t h e l a b e l l i n g o f p r o t e i n and p h o s p h o l i p i d components o f e r y t h r o c y t e membranes.  QX 572, and  10.  I n h i b i t i o n o f b a s a l ( M g - i n d e p e n d e n t ) and M g - d e p e n d e n t p - n i t r o p h e n y l p h o s p h a t a s e (NPPase) by p r a c t o l o l and p r o p r a n o l o l i n r e l a t i o n t o t h e a n t i h e m o l y t i c e f f e c t s o f these a g e n t s .  11.  I n h i b i t i o n o f the K - s t i m u l a t e d component o f M g - d e p e n d e n t p - n i t r o p h e n y l p h o s p h a t a s e (K+-NPPase) by a n t i a r r h y t h m i c s i n r e l a t i o n t o t h e a n t i h e m o l y t i c e f f e c t s o f these a g e n t s .  ++  +  ++  ++  viii  No.  Title  Page  12.  R e l a t i o n s h i p between e r y t h r o c y t e and m y o c a r d i a l membrane K NPPase i n h i b i t i o n by v a r i o u s a n t i a r r h y t h m i c s .  74  13.  R e l a t i v e i n h i b i t i o n o f 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 ( C a - A T P a s e ) from e r y t h r o c y t e membranes by v a r i o u s a n t i a r r h y t h m i c s .  77  14.  I n h i b i t i o n of intact erythrocyte acetylcholinesterase a c t i v i t y by v a r i o u s a n t i a r r h y t h m i c s r e l a t i v e t o t h e i r a n t i h e m o l y t i c properties.  81  15.  H i l l p l o t a n a l y s i s o f the i n h i b i t i o n o f e r y t h r o c y t e membrane and s y n a p t i c 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 .  83  16.  E a d i e p l o t a n a l y s i s o f the nature o f the i n h i b i t i o n o f i n t a c t e r y t h r o c y t e 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 .  87  17.  E f f e c t o f transmembrane c h l o r i d e g r a d i e n t on the i n h i b i t i o n o f 91 i n t a c t e r y t h r o c y t e 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 .  +  + +  ++  ++  ix  ACKNOWLEDGEMENT  I would l i k e to express my gratitude to Dr. David V. Godin for his resourceful and valuable supervision i n the preparation of this thesis and to Dr. Michael J.A. Walker for introducing me to antiarrhythmic I wish also to thank Mrs.  research.  T.W. Ng and Miss M.E. Garnett for their  technical assistance and Mr. Glenn C o l l i n s for photographic work.  Page 1  INTRODUCTION  Although a considerable  amount of i n f o r m a t i o n  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 a n t i a r r h y t h m i c s  i s available regarding  the  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 m o l e c u l a r mechanisms governing antiarrhythmic the use o f a n t i a r r h y t h m i c disorders  drug a c t i o n have y e t to be f u l l y e l u c i d a t e d . drugs c l i n i c a l l y  Thus,  i n the treatment of c a r d i a c rhythm  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.  quinidine 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 a r r h y t h m i a s (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 ness of q u i n i d i n e and  l i d o c a i n e i n t h e s e two  regions  of the h e a r t  (2).  The e f f e c t i v e -  d i f f e r e n t c o n d i t i o n s may  be r e l a t e d t o d i f f e r e n c e s i n the mechanism u n d e r l y i n g i n t h e s e two  (1).  the rhythm  However, i t may  i n part  abnormality  a l s o be t h a t 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 a c c u m u l a t i o n 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 .  these two  p r o p e r t i e s but at p r e s e n t ,  hindered  of the m e c h a n i s t i c  for this  drug a c t i o n i s a v a i l a b l e .  a s p e c t s of a n t i a r r h y t h m i c  g r e a t l y by a number of e x p e r i m e n t a l  difficulties  w e l l i n v o l v e some c o m b i n a t i o n of  no s a t i s f a c t o r y e x p l a n a t i o n  r e l a t i v e s e l e c t i v i t y of antiarrhythmic Studies  The o v e r a l l  1  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  drug a c t i o n have been  difficulties.  Some of  the  a r i s e from the complex t i m e and voltage-dependent b e h a v i o r  e x c i t a b l e t i s s u e s i n g e n e r a l , w h i l e others s t r u c t u r a l and  may  are p e c u l i a r to the  f u c t i o n a l p r o p e r t i e s of the h e a r t .  of  unique  A normal c o n t r a c t i o n o f  the  Page 2  h e a r t i n v o l v e s the p r o p a g a t i o n of an impulse from the s i n u s node t h r o u g h  the  s p e c i a l i z e d c o n d u c t i o n 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 . a c t i o n s of a n t i a r r h y t h m i c s  I t i s important,  t h e r e f o r e , t o study  i n the f u n c t i o n i n g i n t a c t h e a r t .  s i t u a t i o n here i s c o m p l i c a t e d  However, the  by the f a c t t h a t a n t i a r r h y t h m i c s  produce 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 a c t i o n s i n one h e a r t as compared w i t h another.  the  frequently  r e g i o n of  For example, b o t h q u i n i d i n e and  the  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 p e r i o d of the a t r i o v e n t r i c u l a r node but i n c r e a s e t h a t of the H i s - P u r k i n j e system ( 3 - 4 ) , w h i l e p r o p r a n o l o l has e f f e c t on the H i s - P u r k i n j e system of normal or d i s e a s e d  subjects  o t h e r 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 a n t i a r r h y t h m i c may  little  (5-7).  such as  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 a c t i o n s and  In  quinidine alter-  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 - b o t h o f which may  d i f f e r considerably  i n various regions  of the h e a r t  Another c o m p l i c a t i o n i n the study of a n t i a r r h y t h m i c the f a c t t h a t i s c h e m i c  or h y p o x i c m y o c a r d i a l  (8-10).  drug a c t i o n r e l a t e s to  tissues i n s i t u often exhibit  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 c a r d i a c t i s s u e s (11).  I t has  been shown, f o r example, t h a t i s c h e m i c  t i s s u e s are c o n s i d e r a b l y more s u s c e p t i b l e to the m o d i f y i n g antiarrhythmic  drugs than normal t i s s u e s .  myocardial  e f f e c t s of c e r t a i n  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 myocardium than i n a d e q u a t e l y oxygenated m y o c a r d i a l  t i s s u e s (12).  of propranolol i n slowing  refractoriness, i n  conduction,  i n prolonging  The  canine effects  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 p e r i o d of the ischemic zones o f the h e a r t p r o b a b l y e x p l a i n p a r t o f i t s a n t i a r r h y t h m i c a c t i o n s i n acute m y o c a r d i a l  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 o f b e t a - a d r e n e r g i c combination  of both.  p o t e n t i a l amplitude  b l o c k a d e or l o c a l a n a e s t h e t i c a c t i o n o r some  Hondeghem e t a l (13) found t h a t e x c i t a b i l i t y , a c t i o n and maximum r a t e o f r i s e were a l l decreased t o a g r e a t e r  e x t e n t by q u i n i d i n e , l i d o c a i n e , p r o c a i n a m i d e and d i p h e n y l h y d a n t o i n guinea  i n hypoxic  p i g p a p i l l a r y f i b e r s as compared w i t h n o r m a l l y oxygenated c e l l s .  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 i m p o r t a n t  a n t i a r r h y t h m i c drug a c t i o n i n man.  Such  i n determining  T h e r e f o r e , e x t r a p o l a t i o n o f the r e s u l t s o f  s t u d i e s u s i n g normal i s o l a t e d m y o c a r d i a l  t i s s u e t o 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 c o n s i d e r a b l e c a u t i o n . A final  c o m p l i c a t i o n which s h o u l d be mentioned i s the extreme s e n s i t i v i t y  o f 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 t o the e l e c t r o l y t e c o m p o s i t i o n 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 . diphenylhydantoin potassium.  o f the  The 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 o f  a r e 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 o f  When p o t a s s i u m c o n c e n t r a t i o n i s low (< 3 mM), d i p h e n y l h y d a n t o i n  i n c r e a s e 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 atrial  may  of both  and P u r k i n j e f i b e r s , but when the p o t a s s i u m c o n c e n t r a t i o n o f t h e medium  i s at normal plasma v a l u e s dominantly  depressant  (4-5 mM) or s l i g h t l y h i g h e r , the drug has a p r e -  e f f e c t on the a c t i o n p o t e n t i a l o f b o t h a t r i a l and  P u r k i n j e f i b e r s ( 1 4 ) . S i m i l a r l y , Singh and Vaughan W i l l i a m s (15) showed t h a t a r e d u c t i o n i n e x t e r n a l p o t a s s i u m c o n c e n t r a t i o n from 5.6 mM t o 3 mM caused a  Page 4  decrease i n the a b i l i t y of lidocaine to depress the maximum rate of depolarization and responsiveness i n rabbit a t r i a l and ventricular tissue, such that a ten-fold greater concentration of lidocaine was required to produce a substantial 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 a l (16), hyperkalemic dogs  exhibited an increased susceptibility to quinidine t o x i c i t y , as reflected i n 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.  I t i s proposed that the increased t o x i c i t y 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 i s relevant to their effects on cardiac muscle (17-19). In nerve, these compounds act to cause a rise i n the e l e c t r i c a l threshold of e x c i t a b i l i t y , retardation of impulse propagation and reduction of the rate of rise of action potential and at s u f f i c i e n t l y 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 s e n s i t i v i t y of heart muscle and nerve to  such membrane depressant effects of a given compound is different, e.g. tetro-  Page 5  d o t o x i n b l o c k s nerve c o n d u c t i o n  at a c o n c e n t r a t i o n o f 0.01 mg/1 but concen-  t r a t i o n s up t o a hundred times g r e a t e r have no e f f e c t s on the r a t e o f r i s e or d u r a t i o n o f the c a r d i a c a c t i o n p o t e n t i a l ( 2 5 ) .  I n c o n t r a s t , most l o c a l  a n a e s t h e t i c type a n t i a r r h y t h m i c s depress the r a t e o f r i s e o f the c a r d i a c a c t i o n p o t e n t i a l at c o n c e n t r a t i o n s  10-300 times l e s s than those needed t o  cause a comparable r e d u c t i o n i n the a m p l i t u d e o f 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 o f  l o c a l a n a e s t h e t i c 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 t o t h e i r a b i l i t y t o reduce t h e maximal r a t e o f d e p o l a r i z a t i o n i n c a r d i a c muscle (26-27) because such a r e d u c t i o n by t h e r a p e u t i c c o n c e n t r a t i o n s  o f these drugs has been  found t o be a s s o c i a t e d w i t h ( a ) an i n c r e a s e i n the t h r e s h o l d o f e x c i t a b i l i t y ; (b) a d e p r e s s i o n  i n conduction  v e l o c i t y ; and ( c ) a p r o l o n g a t i o n o f the  e f f e c t i v e refractory period (1).  These a l t e r a t i o n s o c c u r w i t h o u t  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 c a r d i a c muscle may suggest t h a t t h e mechanism o f a c t i o n of l o c a l a n a e s t h e t i c s  i s s i m i l a r i n both t i s s u e s .  I n the case o f m y o c a r d i a l  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 o c c u r e i t h e r by v i r t u e o f the  a b i l i t y o f these agents t o modify autonomic tone or as the r e s u l t o f druginduced 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 t u r n a l t e r i o n f l u x e s the membrane.  W h i l e these two m e c h a n i s t i c  d i s t i n c t , as i n the case o f b e t a - a d r e n e r g i c  p o s s i b i l i t i e s may be s e p a r a t e antagonists  across and  d e v o i d o f 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 a p p r 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 p o s s i b i l i t y . In a study of the experimental antiarrhythmic properties of acebutolol, propranolol and practolol, Basil et a l (28) suggested that membranes t a b i l i z i n g actions of propranolol and acebutolol, rather than the betaadrenoceptor blockade, were the primary determinant of antiarrhythmic properties i n the reversion of ouabain-induced ventricular arrhythmias i n anaesthetized dog by infusion of the drug.  The findings that the infusion of  the d-isomer of propranolol, which is v i r t u a l l y devoid of beta-adrenergic blocking properties, was effective i n abolishing ouabain-induced ventricular tachycardia i n 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 betareceptor blocking drugs against arrhythmias induced by d i 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 i s l i k e l y to be the prime mechanism of antiarrhythmic action i n arrhythmias induced by catecholamines or arrhythmias following myocardial infarction (33). There i s 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 o f p r o p r a n o l o l (40-150 ng/ml) d u r i n g a n t i a r r h y t h m i c therapy  i n man a r e  much lower than the drug c o n c e n t r a t i o n needed i n v i t r o (>3 ug/ml) t o 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 o f normal c a r d i a c (33-34).  cells  C o l t a r t e t a l (35) u s i n g racemic p r o p r a n o l o l have found t h a t w h i l e  plasma l e v e l s o f 40-85 ng/ml may be a n t i a 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 s u p p r e s s i o n i c a n t l y , d-propranolol  of arrhythmias  i n others.  More s i g n i f -  (which i s d e v o i d o f a p p r e c i a b l e b e t a - r e c e p t o r  blocking  p r o p e r t i e s ) a t l e v e l s o f 180-310 ng/ml was i n e f f e c t i v e i n t h e treatment o f chronic s t a b l e v e n t r i c u l a r ectopic beats. of membrane a c t i o n s o f b e t a - r e c e p t o r  Therefore  the q u e s t i o n o f the r o l e  blockers i n determining  antiarrhythmic a c t i o n i s s t i l l unresolved  but the f o r e g o i n g e v i d e n c e would  tend t o suggest t h a t t h i s p r o p e r t y o f c e r t a i n b e t a - a d r e n e r g i c not a major determinant o f a r r h y t h m i c  clinical  antagonists i s  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 .  I n a d d i t i o n t o 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  t o depress c a r d i a c a d r e n e r g i c nerves (36-37) and t o e x e r t c e n t r a l  nervous system e f f e c t s ( 3 8 ) .  I t i s now r e c o g n i z e d  t h a t the c e n t r a l nervous  system may p l a y a c r u c i a l r o l e i n the arrhythmogenic e f f e c t o f c a r d i a c g l y c o s i d e s ( 3 9 ) , and i t may a l s o be important a n t i a r r h y t h m i c drugs (39-40).  i n the a c t i o n s o f c e r t a i n  C o n s e q u e n t l y , the r e c o g n i t i o n o f 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 o f a n t i a r r h y t h m i c drug a c t i o n may p l a c e l i m i t a t i o n s on the importance o f i n f o r m a t i o n d e r i v e d from i n v i t r o s t u d i e s . I t has been suggested t h a t the d i s c r e p a n c y  between animal s t u d i e s and c l i n i c a l  Page 8  usage as regards the relative importance of beta-adrenergic blockade and membrane stabilization i n the antagonism of digitalis-induced arrhythmias by drugs may arise because the level of sympathetic tone i n anaesthetized animals is l i k e l y to be considerably less than would be obtained i n 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 i n d i g i t a l i s toxicity and have instead implicated the peripheral nervous system (ganglia and the peripheral afferent components of the baroreceptor reflex) (41). As regards antiarrhythmic action, peripheral nerves certainly represent a l i k e l y site of action for bretylium. I t i s 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 d i g i 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 hypothalamic 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 drugs may  drug.  The  n e u r a l depressant  p r o p e r t i e s of  w e l l i n v o l v e q u i n i d i n e - l i k e a c t i o n on n e u r o n a l  antiarrhythmic membranes of c e n t r a l  or p e r i p h e r a l f i b e r s which i s somewhat analogous to the proposed s t a b i l i z a t i o n o f the m y o c a r d i a l The  plasma membrane porduced by  these  agents.  above c o n s i d e r a t i o n s have i n t r o d u c e d yet another c o m p l i c a t i o n i n  a n a l y z i n g the p r o p e r t i e s of a n t i a r r h y t h m i c or mechanisms by which a p a r t i c u l a r dependent upon the nature  potential  in atrial  This  and  drugs because the exact mechanism  antiarrhythmic exerts  of the arrhythmogenic s t i m u l u s .  under c o n d i t i o n s of h y p o x i a which may  (44).  electrical  be  i t s effects Arrhythmias  a s s o c i a t e d with a decreased  i s presumed to a r i s e from the f a c t  arising  membrane  P u r k i n j e f i b e r s respond p a r t i c u l a r l y w e l l to  'selectivity'  are  verapamil  that verapamil  is  a b l e to b l o c k the slow inward c a l c i u m c u r r e n t which i s r e s p o n s i b l e f o r the p l a t e a u phase of c a r d i a c a c t i o n p o t e n t i a l ( 4 5 ) . such as a t r i a l  and  atrioventricular  Indeed, v a r i o u s  j u n c t i o n a l arrhythmias  arrhythmias  are b e l i e v e d to  a r i s e from abnormal impulses mediated by such c a l c i u m c u r r e n t s which remain f u n c t i o n a l d e s p i t e the reduced membrane p o t e n t i a l i n d i s e a s e d of the h e a r t  (46-47).  Such e l u c i d a t i o n of m o l e c u l a r  arrhythmogenesis should u l t i m a t e l y p r o v i d e the most a p p r o p r i a t e Due  antiarrhythmic  to the s u c c e s s f u l c l i n i c a l  management of d i f f e r e n t types c l a s s i f y these  mechanisms  areas  underlying  a more r a t i o n a l b a s i s f o r  agent i n a p a r t i c u l a r use  or hypoxic  clinical  of v a r i o u s a n t i a r r h y t h m i c s  choosing  situation. i n the  of rhythm d i s o r d e r s , attempts have been made to  drugs i n t o groups based on s t u d i e s 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 p r o p e r t i e s of the myocardium s t u d i e d both  Page 10  i n v i t r o and i n v i v o .  Hoffman and B i g g e r (48) have c l a s s i f i e d  various  a n t i a r r h y t h m i c s 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 r e s p o n s i v e n e s s .  Drugs such as  q u i n i d i n e , p r o c a i n a m i d e and p r o p r a n o l o l , which depress c o n d u c t i o n and responsiveness,  were p l a c e d i n one c l a s s and agents such as l i d o c a i n e and  diphenylhydantoin, conduction  which have no depressant  and r e s p o n s i v e n e s s ,  e f f e c t b u t might improve  were p l a c e d i n another.  c l a s s i f i c a t i o n by Hoffman and B i g g e r  However, the above  i s not g e n e r a l l y a c c e p t e d .  Williams (49), using d i f f e r e n t experimental  techniques  proposed four c l a s s e s o f a n t i a r r h y t h m i c drugs.  and c r i t e r i a , has  This c l a s s i f i c a t i o n has  grouped drugs a c c o r d i n g t o 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 , blockade, inward  Vaughan  sympathetic  p r o l o n g a t i o n o f 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 o f the slow  calcium currents.  I t i s very l i k e l y that f u r t h e r experimentation  produce ever i n c r e a s i n g r e f i n e m e n t s  will  i n the c l a s s i f i c a t i o n o f a n t i a r r h y t h m i c  drugs and i d e n t i f y t h e m o l e c u l a r b a s i s o f t h e i r a n t i a r r h y t h m i c a c t i o n s . The  purpose o f the f o r e g o i n g m a t e r i a l was t o p l a c e the g e n e r a l problems o f  s t u d y i n g a n t i a r r h y t h m i c drug a c t i o n i n t o some k i n d o f p e r s p e c t i v e and t o i n t r o d u c e the concept t h a t membrane i n t e r a c t i o n s o f a v a r i e t y o f types a r e l i k e l y t o be i m p o r t a n t  i n the a c t i o n o f a n t i a r r h y t h m i c drugs.  The aim o f the  work t o be d e s c r i b e d here was t o approach t h i s problem at a v e r y b a s i c  level,  by i n v e s t i g a t i n g a n t i a r r h y t h m i c drug-induced s t r u c t u r a l and f u n c t i o n a l p e r t u r b a t i o n s i n model membrane systems. provide patterns of molecular  I t was hoped t h a t such s t u d i e s might  changes as a b a s i s f o r u n d e r s t a n d i n g  the d i v e r s e  Page 11  electrophysiological actions of antiarrhythmics i n excitable tissues. Ultimately such information may indicate the molecular factors governing the effectiveness of those agents i n 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 i n l i p i d and protein components of membranes by antiarrhythmic drugs. Membrane structural perturbations induced by drugs can be analyzed either i n terms of alterations i n 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 i n terms of changes produced i n the functional properties of membrane-associated enzymes (53).  I t should be emphasized that, i n the  studies described, effects of drugs on membrane enzyme a c t i v i t i e s were examined i n conjunction with experiments using chemical probes i n order to learn something of the correlations between group a v a i l a b i l i t y and enzyme changes of drug-membrane interaction.  This is not to imply that the same, or  analogous, enzymes i n the membranes of excitable tissues represent pharmacological sites of antiarrhythmic drug action. Our i n i t i a l assumption has been, therefore, that information obtained from studies i n 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 i n large quantity and i n highly homogeneous form, but by the suggestion that the molecular features governing the antihemolytic or membrane s t a b i l i z i n g properties of antiarrhythmics i n red cells might be analogous to those determining e l e c t r i c a l stabilization i n excitable tissues (54). that a l l lipid-soluble  Thus, Seeman has shown  local and general anaesthetics protect erythrocytes  from hypotonic hemolysis (54) and the concentrations of these substances producing 50% antihemolysis i n erythrocytes and blockade of peripheral nerves are v i r t u a l l y identical Originally  (55-57).  the term 'membrane s t a b i l i z a t i o n  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  s t a b i l i z e r ' to describe the action of local anaesthetics because these agents blocked membrane action potentials without appreciably altering resting membrane 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 s t a b i l i z i n g ' 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 i n h i b i t mitochondrial swelling induced by a variety of agents (66-67), to inhibit the release of acid phosphatase from rat l i v e r lysosomes (68-70), and to stabilize erythrocytes against hypotonic l y s i s (71-73).  These findings suggested that the interaction of local anaesthetic  drugs with c e l l membranes i n general may give rise to alterations i n 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 i n diverse membrane systems (56) suggested the p o s s i b i l i t y that the structural basis of these effects might be fundamentally similar i n excitable and non-excitable membranes. The fact that high concentrations of membrane stabilizers can irreversibly damage both excitable and non-excitable membranes (56); the s i m i l a r i t y i n membrane/buffer partition coefficients for local and general anaesthetics i n 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 i s active on both erythrocyte and nerve membrane (56) supported the p o s s i b i l i t y that the structural basis of 'membrane s t a b i l i z a t i o n ' might be similar i n excitable and non-excitable membranes. It was therefore f e l t that information on the structural and functional consequences of the interaction of antiarrhythmics 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 o f l i p i d - s o l u b l e substances have been shown t o p r o t e c t the e r y t h r o c y t e membrane from o s m o t i c , m e c h a n i c a l or a c i d l y s i s  (54).  a g e n t s , which i n c l u d e s t e r o i d s ( 7 5 ) , t r a n q u i l i z e r s ( 7 1 ) , and  These  anti-inflammatory  compounds (76) cause an e x p a n s i o n o f the membrane (74,77-78) r e s u l t i n g i n an increased resistance of erythrocytes  to hypotonic hemolysis.  While the  m o l e c u l a r b a s i s o f t h i s membrane phenomenon has y e t t o be f u l l y e l u c i d a t e d , a number o f r e c e n t o b s e r v a t i o n s  have shed l i g h t on i t .  The e x p a n s i o n o f  e r y t h r o c y t e membranes by drugs appears t o i n v o l v e c o n f i g u r a t i o n a l changes i n b o t h p r o t e i n and p h o s p h o l i p i d  components o f t h e membrane ( 5 4 ) .  study by G o d i n e t a l ( 5 2 ) , p r o p r a n o l o l , a t c o n c e n t r a t i o n s hemolytic  In a detailed  exerting a n t i -  e f f e c t s , was shown t o m o d i f y t h e i n c o r p o r a t i o n o f a c h e m i c a l 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 ) i n t o both p h o s p h o l i p i d of r e d b l o o d  c e l l ghosts.  intact erythrocytes  and p r o t e i n components  Further, propranolol-induced  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 o f  antihemolysis 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 acid incorporation i n t o phospholipids p r o t e i n s than w i t h the d r u g - s t i m u l a t e d individually.  r e l a t i v e to  l a b e l l i n g o f e i t h e r component  I n a d d i t i o n , the h i g h degree o f c o r r e l a t i o n between t h e  drug-induced l a b e l l i n g o f membrane p h o 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 o f two enzymatic p r o c e s s e s a s s o c i a t e d w i t h t h e membrane suggested t h a t the enzyme i n h i b i t i o n f o l l o w s 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 t h e s t r u c t u r a l s t a t e o f membrane p h o s p h o l i p i d s  which would  secondarily  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 .  using sarcoplasmic  r e t i c u l u m v e s i c l e s prepared  Suko e t a l  (79)  from r a b b i t s k e l e t a l m u s c l e ,  proposed t h a t the mechanism of a c t i o n o f l o c a l a n a e s t h e t i c s i n the  reduction  o f c a l c i u m e f f l u x mediated by the c a l c i u m t r a n s l o c a t i n g ATPase may  be  e x p l a i n e d 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. another s t u d y , Harrow and D h a l l a (80)  showed t h a t b o t h sarcolemmal Mg  ATPase and C a - A T P a s e , but not N a - K ++  +  by q u i n i d i n e , p r o c a i n a m i d e and adenylate  In  +  +  -  ATPase, a c t i v i t i e s were i n c r e a s e d  l i d o c a i n e w h i l e o n l y q u i n i d i n e decreased  c y c l a s e o f the r a t myocardium.  None o f these agents had any  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 effect 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 m y o c a r d i a l 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 a n t i a r r h y t h m i c drugs.  Membrane i n t e g r i t y i s important t o i o n s (54).  i n determining  p e r m e a b i l i t y o f membranes  L o c a l a n a e s t h e t i c s and p h e n o t h i a z i n e s  f l u i d i z a t i o n o f membrane components (81-82).  are a b l e to cause a  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 p h o s p h o l i p i d s 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 o f membrane s t r u c t u r a l components. p o s s i b l e consequences o f such drug-induced membrane s t r u c t u r a l  Among the perturbations  are a l t e r e d membrane p e r m e a b i l i t y to sodium and p o t a s s i u m i o n s and d i s p l a c e ment o f membrane bound c a l c i u m (54,83-85).  With r e g a r d to the r o l e o f  Page 16  divalent cations i n influencing membrane structure, both protein and phosphol 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 i n question (50-51).  Thus, the interaction of alkaline earth cations  with the membrane involved an important contribution from membrane phosphol i p i d s , while another group of divalent cations (zinc, cadmium, nickel) interacted directly with membrane protein sulfhydryl groups i n 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 i n 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) i s found to exert blocking actions on this current (86).  The relevance of  divalent cation-membrane interactions to mechanism of action of antiarrhythmics is not certain. However, a recent study of Porzig (87) showed that propranolol was able to increase the efflux of potassium from erythrocytes, 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).  I t can be proposed that i f a  comparable mechanism were operative i n the myocardial membrane, i t would provide a plausible molecular basis for a propranolol-induced automaticity i n terms of altered potassium conductance.  decrease i n  Another example of  Page 17  the m o d i f i c a t i o n o f membrane-cation i n t e r a c t i o n s by a n t i a r r h y t h m i c s i s p r o v i d e d by v e r a p a m i l , and i t s methoxy d e r i v a t i v e , D-600, which were r e p o r t e d to b l o c k t h e slow inward  calcium current i n cardiac f i b e r s (45).  i n v e s t i g a t i o n s have p o i n t e d out t h a t v e r a p a m i l where the c u r r e n t ( 8 8 ) i s c a r r i e d by sodium.  More r e c e n t  a l s o i n h i b i t s slow c h a n n e l s Hence, the p r i m a r y  a c t i o n of  these drugs may be on the slow channels i n g e n e r a l and not on c a l c i u m movements s p e c i f i c a l l y .  The importance o f b o t h p o t a s s i u m and c a l c i u m i n myo-  cardium has been l o n g r e c o g n i z e d  (1,89-90).  the r o l e s these i o n s p l a y i n d e t e r m i n i n g  However, the complete d e t a i l s o f  normal and abnormal f u n c t i o n a l  p r o p e r t i e s o f the myocardium have y e t t o be u n d e r s t o o d .  I t i s reasonable,  t h e r e f o r e , t o b e l i e v e t h a t i n f o r m a t i o n generated from t h e s t u d i e s o f a n t i arrhythmic  drugs on membrane-associated enzymes t h a t r e g u l a t e p o t a s s i u m and  c a l c i u m f l u x e s across e r y t h r o c y t e membrane has c e r t a i n v a l u e s and may be relevant 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 a c t i o n s o f  a n t i a r r h y t h m i c s i s r a t h e r l i m i t e d and i n c o m p l e t e . present  Therefore,  the aim o f t h e  study was t o i n v e s t i g a t e the e f f e c t s o f v a r i o u s c l a s s e s o f a n t i -  arrhythmics  at t h e m o l e c u l a r  l e v e l on b i o l o g i c a l membranes.  Initially,  membrane p e r t u r b a t i o n a l e f f e c t s o f a n t i a r r h y t h m i c s would be examined i n a model membrane system (namely, the human e r y t h r o c y t e membrane) which bears a number o f a n a l o g i e s and  t o e x c i t a b l e membranes, such as drug-induced  s i m i l a r mechanisms g o v e r n i n g  active cation transport.  stabilization  I n t h i s work, one  of the main o b j e c t i v e s was t o 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 o f v a r i o u s  Page 18  a n t i a r r h y t h m i c drugs: beta-blockers  l o c a l a n a e s t h e t i c s and  ( p r a c t o l o l , p r o p r a n o l o l and  c e r t a i n quaternary  i t s quaternary  analogues,  analogue,  pranolium),  a c a l c i u m b l o c k e r (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 e r y t h r o c y t e membranes u s i n g  two  c h e m i c a l p r o b e s , 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 w i t h  consider-  a b l e s p e c i f i c i t y f o r p r i m a r y amino groups ( 9 1 ) , and 5 , 5 - d i t h i o - b i s 1  ( 2 - n i t r o b e n z o i c a c i d ) (DTNB). which i s s e l e c t i v e l y i n c o r p o r a t e d  into  s u l f h y d r y l groups ( 9 2 ) . 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 o f membrane were i n v e s t i g a t e d by examining v a r i o u s membrane a s s o c i a t e d 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 -stimulated +  +  + . ++ , K - s t i m u l a t e d Mg -dependent ATPase, b a s a l  p - n i t r o p h e n y l phosphatase, and C a - s t i m u l a t e d  dependent ATPase.  + +  c e l l s and  Mg + +  S i n c e the enzyme a c e t y l c h o l i n e s t e r a s e i s p r e s e n t  o u t e r s u r f a c e o f e r y t h r o c y t e s (93) and  and  at the  can t h e r e f o r e be assayed i n b o t h i n t a c t  i s o l a t e d membranes, e f f e c t s o f a n t i a r r h y t h m i c s on the a c t i v i t y  of  t h i s enzyme can be s t u d i e d i n i n t a c t e r y t h r o c y t e s and h y p o t o n i c a l l y l y s e d erythrocyte ghosts.  Such s t u d i e s w i l l p r o v i d e an o p p o r t u n i t y t o 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 t o the s i t u a t i o n where membranes o f 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 p r o v i d e  a  means o f d e r i v i n g i n f o r m a t i o n 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 o f 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 i n order to determine the extent to which drug-induced perturbations studied i n erythrocytes approximate those i n e l e c t r i c a l l y excitable c e l l s .  Cardiac myocardial membranes were also u t i l i z e d to study the  effects of these drugs i n 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 i n 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 s t u d i e s were q u i n i d i n e h y d r o 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 ) , p r a n o l i u m c h l o r i d e (SC-27761)  (G.D.  S e a r l e & Co), d l - p r o p r a n o l o l h y d r o c h l o r i d e (Sigma Chemical Co), p r a c t o l o l , f r e e base ( A y e r s t 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 h y d r o c h l o r i d e ( A s t r a P h a r m a c e u t i c a l P r o d u c t ) , D-600 h y d 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 c h e m i c a l s were o b t a i n e d from Sigma Chemical Company:  acetyl-  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 d o d e c y l s u l f a t e (SDS), t r i s  (hydroxymethyl) aminomethane ( T r i z m a b a s e ) , 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), ouabain ( S t r o p h a n t h i n G ) ,  dl-dithiothreitol,  d i s o d i u m a d e n o s i n e - 5 ' - t r i p h o s p h a t e (ATP), p - n i t r o p h e n y l p h o s p h a t e phosphatase  (104  s u b s t r a t e ) , i m i d a z o l e (grade 111), N - a c e t y l n e u r a m i n i c a c i d ( t y p e  111 from egg), c h o l e s t e r o l s t a n d a r d , phosphorus s t a n d a r d (20y grams i n o r g a n i c P/ml  as K H ^ P O ^ ) ) , p - n i t r o p h e n o l s t a n d a r d s o l u t i o n ( 1 0 u moles/ml) .  Sucrose ( s p e c i a l enzyme grade) was o b t a i n e d from Schwarz/Mann. albumin ( f r a c t i o n V) was S i l i c o t u n g s t i c a c i d was  purchased  from Armour P h a r m a c e u t i c a l Company.  s u p p l i e d by F i s h e r S c i e n t i f i c Company and  a c i d from Baker & Adamson C h e m i c a l .  grade.  perchloric  N i n h y d r i n ("Baker TLC Reagent") was  o b t a i n e d from Baker Chemical Company. Pharmacia.  Bovine serum  Sephadex G-200 was  purchased  from  A l l o t h e r c h e m i c a l s , s o l v e n t s and r e a g e n t s were a n a l y t i c a l  They were used w i t h o u t f u r t h e r  purification.  reagent  Page 21  METHODS  MEMBRANE PREPARATIONS a)  E r y t h r o c y t e Membranes E r y t h r o c y t e membranes were prepared  from outdated  human b l o o d ( 0 ) +  s t o r e d 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 o f t h e method o f S c h r i e r (94) as d e s c r i b e d p r e v i o u s l y ( 9 5 ) . hemolysis  procedure.  I t involved a step-wise  A u n i t of blood  (approximately  hypotonic  500 ml) was d i l u t e d t o  1200 ml w i t h i s o t o n i c s a l i n e and c e n t r i f u g e d at 650 x g f o r 5 minutes t o remove the plasma and b u f f y c o a t .  T h i s washing was r e p e a t e d  i m a t e l y 100 ml packed r e d b l o o d c e l l s were then processed to  1200 ml w i t h 0.08M N a C l .  once.  f u r t h e r by d i l u t i o n  This m i x t u r e was s t i r r e d at 4°C f o r 10 min. and  c e n t r i f u g e d at 16,000 x g f o r 5 minutes.  The s u p e r n a t a n t  was removed and the  procedure repeated w i t h 0.06M, 0.04M, 0.02M, and 0.009M N a C l . NaCl c o n c e n t r a t i o n s , the pH was a d j u s t e d t o 7.4 w i t h T r i s . i n v o l v e d treatment  Approx-  A t the l a s t two  The f i n a l  step  w i t h lOmM T r i s (pH 7.4) f o l l o w e d by c e n t r i f u g a t i o n . The  e r y t h r o c y t e membranes were d i l u t e d t o a p r o t e i n c o n c e n t r a t i o n o f 3-5 mg/ml, quick frozen using d r y ice/acetone  b)  and s t o r e d at -20°C.  C a r d i a c Plasma Membrane E n r i c h e d F r a c t i o n Guinea p i g c a r d i a c plasma membranes were prepared  o f H u i e t a l (96) and Sulakhe e t a l ( 9 7 ) .  by combining the methods  Guinea p i g s o f e i t h e r sex (250-400  g) were k i l l e d by a q u i c k d i s l o c a t i o n o f 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) c o n 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 g e n t l y compressed a few times t o express r e s i d u a l b l o o d . All  the f o l l o w i n g o p e r a t i o n s  were performed at 4°C.  V e n t r i c u l a r t i s s u e was  f r e e d from f a t and l a r g e 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 o f v e n t r i c u l a r t i s s u e used f o r each p r e 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) o f T-D b u f f e r (based on t i s s u e wet  weight),  and homogenized f o r 15 seconds at a s e t t i n g o f 3 i n a p o l y t r o n PT  10 homogenizer f o l l o w e d by 2 seconds a t maximum speed.  The homogenate was  d i l u t e d w i t h 5 volumes o f 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 a t 620 x g f o r 10 minutes. removed w i t h a P a s t e u r p i p e t and d i s c a r d e d . packed m i t o c h o n d r i a l  The s u p e r n a t a n t was  A d i s t i n c t layer of loosely  and c a p i l l a r y m a t e r i a l (as determined by phase c o n t r a s t  m i c r o s c o p y ) s e d i m e n t i n g on the s u r f a c e o f the p e l l e t s was l o o s e n e d by a d d i n g 1 ml o f T-D b u f f e r and g e n t l y r o t a t i n g t h e tube i n a r o c k i n g s l u r r y was then removed w i t h a P a s t e u r p i p e t . dispersed The  motion.  The p e l l e t was  The  thoroughly  i n 10 volumes o f T-D b u f f e r (based on o r i g i n a l t i s s u e wet w e i g h t ) .  s u s p e n s i o n 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 ( d r o p w i s e ) an e q u a l volume o f  2.5M KC1 i n T-D b u f f e r .  The s u s p e n s i o n 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 a t 9,000 x g f o r 20 m i n u t e s . 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 w e i g h t ) o f T-D b u 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 m i n u t e s , and c e n t r i f u g e d a t 4,000 x g f o r 20 m i n u t e s .  The  Page 23  p e l l e t was  washed t w i c e by c e n t r i f u g a t i o n at 3,000 x g f o r 20 m i n u t e s .  washed e x t r a c t e d  p a r t i c l e s were suspended i n 10%  s u c r o s e (w/v)  i n T-D  and homogenized i n a P y r e x ground g l a s s homogenizer u n t i l the appeared c o m p l e t e l y homogeneous. discontinuous of 65% 8.2,  sucrose gradient  s u c r o s e , 2.5 ml  and  1.5  centrifugation.  each o f 60%,  55%,  50%  The  then f r a c t i o n a t e d  gradient  s u c r o s e (w/v)  i n T-D  buffer,  10%-50% s u c r o s e i n t e r f a c e ( F l ) , 50%-55% s u c r o s e i n t e r f a c e , 60%-65% s u c r o s e i n t e r f a c e ( F 4 ) .  40  b u f f e r and  centrifuged  p e l l e t s were homogenized i n T-D  buffer containing  i n P y r e x ground g l a s s homogenizer. i c e / a c e t o n e and  c)  at 11,000 x g f o r 30 m i n u t e s .  The  10%  rotor)  following (F2), The  p a r t i c l e s i n each r e g i o n were c o l l e c t e d w i t h P a s t e u r p i p e t s , d i l u t e d f o l d w i t h T—D  pH  gradient.  Four d i s t i n c t bands were l o c a t e d i n the  55%-60% s u c r o s e i n t e r f a c e (F3)  using  c o n s i s t e d of 3 ml  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  f o r 1 hour at 40,000 x g.  buffer  suspension  ml of the membrane m a t e r i a l l a y e r e d on top of each  C e n t r i f u g a t i o n was  regions:  This s u s p e n s i o n was  The  three The  s u c r o s e (w/v), pH  7.5,  s u s p e n s i o n s were q u i c k f r o z e n i n dry  s t o r e d at -20°C.  B r a i n Synaptosomal Plasma Membranes Guinea p i g b r a i n s y n a p t i c plasma membranes were p r e p a r e d by the method of  Jones and Matus ( 9 8 ) . was  The  enriched  f r a c t i o n of synaptosomal plasma membranes  r e c o v e r e d from the i n t e r f a c e of a s u c r o s e d e n s i t y g r a d i e n t  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 s e p a r a t e d  by  Page 24  simultaneous  s e d i m e n t a t i o n 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  h a r v e s t e d were washed t h r e e times w i t h 0.15 M KC1 and suspended i n the same b u f f e r and q u i c k f r o z e n u s i n g d r y i c e / a c e t o n e and s t o r e d a t -20°C.  COMPOSITIONAL ASSAYS OF MEMBRANES P r o t e i n c o n t e n t s o f e r y t h r o c y t e , c a r d i a c and b r a i n synaptosomal plasma membranes were determined by t h e method o f Lowry e t a l ( 9 9 ) u s i n g b o v i n e serum albumin (1 mg/ml) as the s t a n d a r d . P h o s p h o l i p i d f o r a l l membranes was e s t i m a t e d by B a r l e t t ' s m o d i f i c a t i o n o f the Fiske-SubbaRow phosphate a n a l y s i s ( 1 0 0 ) .  Membrane c h o l e s t e r o l content was  determined by the method o f Zak e t a l (101).  S i a l i c a c i d was e s t i m a t e d by  h y d r o l y z i n g the samples w i t h 0.1 N H^SO^ a t 80°C f o r 30 minutes b e f o r e a s s a y i n g a c c o r d i n g t o t h e method o f Warren (102). P r i o r t o the p h 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 a s s a y s , t h e c a r d i a c membrane p r e p a r a t i o n was washed t h r e e times w i t h T-D b u f f e r , pH 7.5 t o remove the s u c r o s e i n the o r i g i n a l suspending  buffer.  T h i s process was n e c e s s a r y as  the s u c r o s e was found t o i n t e r f e r e w i t h the c o l o r r e a c t i o n o f t h e a s s a y s .  THIN LAYER CHROMATOGRAPHIC ANALYSIS OF MEMBRANE PHOSPHOLIPIDS E r y t h r o c y t e membrane p e l l e t s e q u i v a l e n t t o 1.5-2.0 mg p r o t e i n were o b t a i n e d 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 t w i c e w i t h 2 ml o f a 2:1 ( v / v ) chloroform:methanol  mixture.  The e x t r a c t was washed t h r e e times w i t h 1 ml  0.75% (w/v) NaCl s o l u t i o n ; the c h l o r o f o r m phases were p o o l e d , and the aqueous  Page 25  phases d i s c a r d e d .  The pooled  c h l o r o f o r m phases were e v a p o r a t e d to dryness and  q u a n t i t i v e l y s p o t t e d onto an a c t i v a t e d (30 minutes a t 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). mixture  The p l a t e was r u n i n a s o l v e n t  c o n t a i n i n g chloroform:methanol:ammonia (14:6:1, v / v / v ) .  s p o t s were i d e n t i f i e d by t h e i r  values.  Phospholipid  A m i n o p h o s p h o l i p i d s were  v i s u a l i z e d by s p r a y i n g the p l a t e w i t h n i n h y d r i n reagent w h i l e the r e m a i n i n g p h o s p h o l i p i d s were l o c a t e d u s i n g i o d i n e vapour.  The v a r i o u s  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  phospholipid  g e l u s i n g methanol,  e v a p o r a t i o n o f t h e e x t r a c t t o dryness and a n a l y s i s o f t h e r e s i d u e f o r i n o r g a n i c phosphorous as d e s c r i b e d p r e v i o u s l y The  (100).  above procedure was a l s o used t o a n a l y z e the p h o s p h o l i p i d components  o f b r a i n synaptosomal membranes.  ENZYMATIC ASSAYS a)  S u c c i n i c Dehydrogenase P r e p a r a t i o n s o f c a r d i a c 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 o r d e r t o g a i n some e s t i m a t e o f t h e degree o f m i t o c h o n d r i a l c o n t a m i n a t i o n used i s t h a t d e s c r i b e d by S l a t e r and Bonner ( 1 0 3 ) . at room t e m p e r a t u r e , and the r e a c t i o n m i x t u r e f i n a l volume o f 3 m l :  present.  The method  The assay was c a r r i e d out  contained  the f o l l o w i n g i n a  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 s u c c i n a t e (0.2 M), 1.5 ml 0.2 M phosphate b u f f e r , pH 7.2,  and 100 ug o f 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  o f 0.2 ml membrane p r e p a r a t i o n and the o p t i c a l d e n s i t y changes a t 400 nm were f o l l o w e d f o r 15 m i n u t e s .  The a c t i v i t y o f s u c c i n i c dehydrogenase was e x p r e s s e d  as t h e absorbance decrease d u r i n g the f i r s t  15 minutes o f t h e r e a c t i o n p e r mg  membrane p r o t e i n .  b)  Adenosine-5 (i)  1  T r i p h o s p h a t a s e s (ATPases)  Mg -dependent and Mg -dependent N a , K - s t i m u l a t e d ++  ++  +  ATPase  +  Mg -dependent ATPase a c t i v i t y o f t h e e r y t h r o c y t e membrane was d e t e r ++  mined by i n c u 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 o f 3 ml c o 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 m l MgCl  (90 mM), 0.1 ml e t h y l e n e g l y c o l - b i s - C g - a m i n o e t h y l e t h e r )  2  a c i d (EGTA) (3 mM).  N,N-tetraacetic  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 o f 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 o f Mg -dependent ATPase and M g - d e p e n d e n t ++  ++  N a , K - s t i m u l a t e d ATPase was t a k e n as t h e N a , K - s t 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 , i n c u b a t e d  f o r one hour  at 37+ 0.5°C and t e r m i n a t e d by the a d d i t i o n o f 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 m i x t u r e 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 o f s u p e r n a t a n t was assayed f o r i n o r g a n i c phosphate by t h e method o f Fiske-SubbaRow (100). A c t i v i t y of Mg  + +  and M g , N a - K + +  +  +  ATPase o f c a r d i a c plasma  membranes and b r a i n synaptosomal membranes was assayed as above b u t u s i n g d i f f e r e n t amounts o f p r o t e i n and a s h o r t e r i n c u b a t i o n time.  For c a r d i a c  Page 27  membranes, 100-150 y g p r o t e i n and i n c u b a t i o n o f 10 minutes were used w h i l e synaptosomal membrane assay m i x t u r e s  contained  an i n c u b a t i o n time o f 5 minutes was used. preparation, ouabain-sensitive Na -K +  a t i n g the r e a c t i o n mixture  +  60-80 y g membrane p r o t e i n and  I n the case o f the c a r d i a c membrane  ATPase was a l s o assayed by i n c u b -  w i t h o u a b a i n (30 mM).  The d i f f e r e n c e between t h e  ATPase a c t i v i t y i n the presence and absence o f o u a b a i n i s taken as the ouabain-sensitive Na -K +  +  ATPase.  S p e c i f i c a c t i v i t i e s o f the enzyme were e x p r e s s e d as  moles o f i n o r g a n i c  phosphate l i b e r a t e d p e r mg p r o t e i n per hour. (ii)  Mg -dependent C a - s t i m u l a t e d ++  The r e a c t i o n m i x t u r e  + +  f o r determining  Mg -dependent ATPase i n c l u d e d :  (0.09 and  2  the C a - s t i m u l a t 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 M g C l  ATPase.  (192 mM), 0.24 ml ATP (25 mM), 0.1 ml EGTA (3mM), 0.1 ml C a C l  mM and 0.2 mM) ( c o r r e s p o n d i n g  to f i n a l free C a  + +  2  c o n c e n t r a t i o n o f 1.0  90 yM r e s p e c t i v e l y ) * , 0.6-0.8 mg e r y t h r o c y t e membrane p r o t e i n , and 1 ml  each o f v a r i o u s c o n c e n t r a t i o n s  o f t e s t drugs, a l l i n a f i n a l volume o f 3 m l .  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 m i x t u r e + +  without  calcium.  * Free C a  + +  concentrations  i n the EGTA-Ca  by a computer programme p r o v i d e d c e u t i c a l S c i e n c e , U.B.C.  ++  b u f f e r system were determined  by Dr. R. R o u f o g a l i s , F a c u l t y o f Pharma-  Page 28  Reactions  were t e r m i n a t e d  a f t e r 1 hour w i t h 1 ml i c e c o l d 8%  silcotungstic  a c i d i n 8% p e r c h l o r i c a c i d , a m i x t u r e which a l l o w e d 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  inter-  f e r e n c e 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 d e s c r i b e d Ca -stimulated  ATPase a c t i v i t y was  + +  (100).  o b t a i n e d 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 o f c a l c i u m and t h a t measured i n i t s absence.  c)  Acetylcholinesterase (i)  E f f e c t of A n t i a r r h y t h m i c s on the A c t i v i t y o f A c e t y l c h o l i n e s t e r a s e  F r e s h human b l o o d o b t a i n e d by venous puncture was fuged.  The  plasma was  removed and  h e p a r i n i z e d and  centri-  the e r y t h r o c y t e s were washed t w i c e w i t h  i s o t o n i c N a C l , f o l l o w e d by one wash w i t h 0.1 M phosphate b u f f e r , pH 8.0 centrifugation.  and  S i x t y m i c r o l i t e r s of the packed e r y t h r o c y t e s were resuspended  i n 12 ml o f 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 o f 1:200  for  the enzyme assays w i t h v a r i o u s 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  room temperature. M), pH 8.0, ( i n 0.1  The  M phosphate b u f f e r , pH 8.0),  r e a c t i o n was  immediately  i n c l u d e d 1.0 ml phosphate b u f f e r  0.6-0.8 ml phosphate b u f f e r (0.1 M), pH 8.0,  drugs to be t e s t e d and The  r e a c t i o n mixture  assayed s p e c t r o p h o t o m e t r i c a l l y at  0.2  ml DTNB (10  1.0 ml each o f v a r i o u s c o n c e n t r a t i o n s  ml e r y t h r o c y t e  s t a r t e d w i t h 0.1  0.1  (0.2  of  suspension.  ml 30 mM  acetylthiocholine chloride  a f t e r an i n i t i a l absorbance r e a d i n g was  a d d i t i o n o f the e r y t h r o c y t e s u s p e n s i o n .  mM)  The  recorded  r e a c t i o n was  f o l l o w i n g the  a l l o w e d t o 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 a c t i v i t i e s of the enzyme i n 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 i n order to follow as closely as possible the conditions used i n the assay of intact erythrocyte acetylcholinesterase. Dilutions of 1:200 and 1:8 were used for erythrocyte ghost membranes and brain synaptosomal membranes respectively.  (ii)  Kinetic Analysis of the Effects of Antiarrhythmics on the Enzyme  The effect of various drugs on the substrate kinetics of acetylcholinesterase was further studied with intact human erythrocytes or erythrocyte ghosts using each drug at a concentration producing a 35% inhibition of enzyme a c t i v i t y 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 i n each case.  Page 30  The  k i n e t i c parameters K  and V m  max  f o r each drug r e a c t i o n were d e t e r °  mined from the s l o p e and y - i n t e r c e p t o f the b e s t f i t l i n e computed f o r the experimental  (iii)  data expressed  i n the form o f an E a d i e  E f f e c t o f Transmembrane C h l o r i d e G r a d i e n t  plot.  on Drug I n h i b i t i o n of  the  Enzyme I n t a c t human e r y t h r o c y t e s 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 g r a d i e n t 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  investigated.  page 28 e x c e p t t h a t the two  E r y t h r o c y t e s were prepared  i s o t o n i c s a l i n e washes were o m i t t e d  the c e l l s were washed t h r e e times w i t h 0.1 f i n a l r e a c t i o n mixture  as d e s c r i b e d  and i n s t e a d  M phosphate b u f f e r , pH 8.0.  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  The  as d e s c r i b e d  page 28 w i t h the e x c e p t i o n t h a t the 1.0 ml phosphate b u f f e r (0.2 M), pH c o n t a i n e d e i t h e r 0.45  M NaCl or 0.3  c o n c e n t r a t i o n s o f 150 mM Barr-Yaakov  d)  and 100 mM  M Na2S0^ t o g i v e f i n a l  on  on  8.0,  salt  r e s p e c t i v e l y as d e s c r i b e d by L i v n e  and  (104).  p-Nitrophenylphosphatase B a s a l (no Mg  ), Mg  (NPPase)  -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 s t u d i e d on e r y t h r o c y t e ghosts and  c a r d i a c plasma  membranes i n the absence and presence o f 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  f o r a s s a y i n g K - s t i m u l a t e d Mg  o f e r y t h r o c y t e ghosts c o n s i s t e d of the f o l l o w i n g : 1.0 b u f f e r , pH 7.4,  0.1  ml p - n i t r o p h e n y l phosphate (90 mM),  ++  -dependent NPPase ml 0.15 0.1  M  imidazole-HCl  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 w i t h water.  o f t e s t drugs, a l l made up t o a f i n a l volume o f 3 ml  The Mg -dependent NPPase was determined as above except t h a t ++  KC1 was o m i t t e d from the r e a c t i o n m i x t u r e . but i n the absence o f b o t h KC1 and  B a s a l NPPase was measured as above  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 , i n c u b a t e d f o r 1 hour at 37+0.5°C and t e r m i n a t e d w i t h 1 ml i c e c o l d 20% (w/v) TCA. were c e n t r i f u g e d (40,000 x g, 5 min.) t o remove membrane m a t e r i a l . (3.0 m l ) o f supernatant  was added t o 1 ml o f 1.5 M T r i s s o l u t i o n .  o f p - n i t r o p h e n o l i n the a l k a l i n i z e d s u p e r n a t a n t  Mixtures An a l i q u o t The amount  was q u a n t i f i e d s p e c t r o p h o t o -  m e t r i c a l l y by measuring the absorbance o f 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 o f membrane NPPase was e x p r e s s e d  as umoles p - n i t r o p h e n o l  l i b e r a t e d per hour per mg p r o t e i n . All  t h r e e NPPase a c t i v i t i e s o f c a r d i a c plasma membranes were determined by  the same procedure but 0.3-0.5 mg c a r d i a c membrane p r o t e i n and i n c u b a t i o n times o f 10 minutes were used.  I n 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 o f t h e s u b s t r a t e .  CHEMICAL PROBE STUDIES a)  G e l F i l t r a t i o n o f Membranes  labelled with Trinitrobenzenesulfonic acid  (TNBS) Two m i l l i l i t e r s r e a c t i o n mixture  o f e r y t h r o c y t e membranes were l a b e l l e d w i t h TNBS i n a  c o n t a i n i n g 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 o f each t e s t drug a t v a r i o u s c o n c e n t r a t i o n s , and water t o  Page 32  make a f i n a l volume o f 20 m l .  Samples were i n c u b a t e d at 37+0.5 C f o r 1 hour  and r e a c t i o n s stopped by a d d i t i o n o f 1 M HC1.  Samples were c e n t r i f u g e d at  40,000 x g f o r 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 fugation.  P e l l e t s were resuspended i n 1.5 ml w a t e r , 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 t o d i a l y s i s sacs and d i a l y z e d a g a i n s t pH 7.5, at 4 C f o r 70 ho u r s .  5 mM EDTA-5 mM 2 - m e r c a p t o e t h a n o l ,  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 t o a 16 x  100 mm column o f Sephadex G-200 and e l 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% NaN -0.05 M NH HC0 3  4  3  as d e s c r i b e d  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 o f 15-20 seconds p e r drop and the absorbance at 335 nm o f each f r a c t i o n was measured.  b)  Incorporation  of 5 , 5 - d i t h i o b i s - ( 2 - n i t r o b e n z o i c 1  The e f f e c t o f a n t i a r r h y t h m i c s  a c i d ) (DTNB)  on the membrane s u r f a c e  r e a c t i v i t y was a n a l y z e d by s t u d y i n g  s u l f h y d r y l group  the i n c o r p o r a t i o n o f DTNB.  Each  sample  contained  1.0 ml 0.15 M i m i d a z o l e 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 o f 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 c o n c e n t r a t i o n s  of  t e s t d r u g , 0.6-0.8 mg o f membrane p r o t e i n and water i n a f i n a l volume 3 m l . I n 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. at 37+0.5°C f o r 30 m i n u t e s , c e n t r i f u g e d  The samples were i n c u b a t e d  at 40,000 x g f o r 10 minutes and the  absorbance o f t h e s u p e r n a t a n t at 412 nm determined.  The number o f 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 t o the c o n t r o l was c a l c u l a t e d u s i n g 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 ( 9 2 ) .  T o t a l s u l f h y d r y l group t i t e r o f the  membranes was determined by d i s r u p t i n g t h e membranes w i t h SDS at a f i n a l concentration  o f 1% (w/v) i n the absence o f 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 o f t h a t d e s c r i b e d  by  Machleidt  e t a l (106). A l l g l a s s w a r e s 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  t o remove any t r a c e o f d e t e r g e n t which would i n f l u e n c e the  h e m o l y s i s of r e d c e l l s .  F r e s h human b l o o d was withdrawn i n t o a h e p a r i n i z e d  s y r i n g e and the b l o o d was c e n t r i f u g e d at t o p speed f o r 5 minutes u s i n g a c l i n i c a l centrifuge.  Erythrocytes  were washed t h r e e times i n 0.9% NaCl/15 mM  T r i s HCL, pH 7.0, u s i n g f o u r volumes o f b u f f e r per volume o f packed cytes.  A f t e r the f i n a l wash, 2 ml o f packed c e l l s  the i s o t o n i c b u f f e r .  erythro-  were suspended i n 32 ml o f  The h e m o l y s i s 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 a t  room temperature o f 0.2 ml e r y t h r o c y t e  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) c o n t a i n i n g v a r i o u s  concentrations  o f t e s t drugs.  T h i s was f o l l o w e d by a h y p o t o n i c c h a l l e n g e 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 t h e same c o n c e n t r a t i o n o f t e s t drug as was p r e s e n t i n the p r e v i o u s incubation.  F o l l o w i n g a 10 minute i n c u b a t i o n at room t e m p e r a t u r e , 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 o f s u p e r n a t a n t at 540 nm was determined and t h i s v a l u e e x p r e s s e d as a p e r c e n t o f t o t a l water.  h e m o l y s i s o f 0.2 ml e r y t h r o c y t e s  Each experiment was performed i n t r i p l i c a t e  as mean + S.E.M.  was  in distilled  and r e s u l t s were e x p r e s s e d  Page 34  RESULTS  The  o b j e c t i v e o f t h i s study was t o d e s c r i b e the membrane p e r t u r b a t i o n a l  e f f e c t s of several d i f f e r e n t pharmacological  agents u s i n g the human e r y t h r o -  c y t e membrane as a model system and u t i l i s i n g a v a r i e t y o f p h y s i o c h e m i c a l 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 o f c o n v e n t i o n a l 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 p r o p a n o l o l were compared t o those of p r a c t o l o l , a 3 " b l o c k e r w h i c h , u n l i k e p r o p r a n o l o l , appears t o l a c k d i r e c t cardiodepressant  p r o p e r t i e s (107); p r a n o l i u m , a q u a t e r n a r y  analogue of  p r o 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 o f 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 o f v e r a p a m i l which may e x e r t a n t i a r r h y t h m i c e f f e c t s , a t l e a s t i n p a r t , by v i r t u e o f i t s a b i l i t y t o b l o c k slow C a  + +  channels  a c t i v a t e d d u r i n g the p l a t e a u phase o f the c a r d i a c 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 q u a t e r n a r y f i n a l l y , QX 572, a q u a t e r n a r y  a d r e n e r g i c neurone b l o c k e r ( 4 2 ) , and  a n t i a r r h y t h m i c r e l a t e d t o 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 o f v a r i o u s a n t i a r r h y t h m i c drugs i s shown i n F i g u r e s 1 and 2.  A l l the  drugs examined produced some degree o f 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.  A n t i h e m o l y t i c e f f e c t s o f q u i n i d i n e , D-600, l i d o c a i n e and practolol. These experiments were performed u s i n g chromic acid-washed g l a s s w a r e e x a c t l y as d e s c r i b e d p r e v i o u s l y ( 5 2 ) . Each p o i n t r e p r e s e n t s the mean o f t r i p l i c a t e experiments performed on a minimum o f t h r e e d i f f e r e n t b l o o d samples o b t a i n e d by v e n i p u n c t u r e from h e a l t h y v o l u n t e e r s .  Page 36  Page 37  FIGURE 2.  Antihemolytic e f f e c t s of propranol, pranolium, b r e t y l i u m . See legend t o F i g u r e 1.  QX 572,  and  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 l y s i s i n 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 s t a b i l i z a t i o n 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 components and on the a c t i v i t y of membrane-associated enzymes. Before the studies with various chemical probes are presented, i t i s necessary to comment on the homogeneity of membrane preparations employed i n these experiments.  ANALYSIS OF MITOCHONDRIAL CONTAMINATION AND COMPOSITIONAL CHARACTERISTICS Succinic dehydrogenase was used as an indication of mitochondrial contamination i n the cardiac membrane preparation from guinea pig hearts. Enzyme  Page 40  a c t i v i t y i n v a r i o u s s u c r o s e g r a d i e n t f r a c t i o n s o b t a i n e d u s i n g two d i f f e r e n t methods o f membrane p r e p a r a t i o n i s r e p o r t e d i n T a b l e 1.  A progressive  decrease i n the s p e c i f i c a c t i v i t y o f s u c c i n i c dehydrogenase F^ was  observed i n f r a c t i o n s o b t a i n e d by e i t h e r method.  i n bands  to  However, the  s u c r o s e g r a d i e n t f r a c t i o n s o b t a i n e d by the procedure d e s c r i b e d i n the Methods ( t h a t i s , the combined methods o f H u i e t a l (96) and Sulakhe e t a l (97)) exhibited  a relatively  lower a c t i v i t y o f the enzyme as compared t o the c o r r e s -  ponding f r a c t i o n s o b t a i n e d by the method o f Sulakhe e t a l i n d i c a t i n g a l e s s e r degree o f m i t o c h o n d r i a l c o n t a m i n a t i o n when membranes were p r e p a r e d by the former combined method.  The a c t i v i t y o f M g - d e p e n d e n t N a - K ++  plasma membrane marker enzyme o f a l l f r a c t i o n s was The combined F^ and F^ f r a c t i o n was a c t i v i t y than F^ or  ++  +  +  a l s o assayed ( T a b l e I I ) . +  ( t o the e x t e n t o f a p p r o x i m a t e l y 2 0 % ) .  ATPase, a  +  found t o have h i g h e r N a - K  f r a c t i o n and i t was  s e n s i t i v e Mg -dependent N a - K  +  +  ATPase  found t o be o u a b a i n s e n s i t i v e  The s p e c i f i c a c t i v i t y of ouabain  ATPase found i n the p r e s e n t membrane  p r e p a r a t i o n (combined F^ and F^ f r a c t i o n s ) ( 9 . 2 ymoles Pi/mg/hr) was comparable  to that reported e a r l i e r  very  by Sulakhe e t a l (111) f o r the same  f r a c t i o n s which were termed "plasma membrane e n r i c h e d f r a c t i o n s " . Guinea p i g b r a i n synaptosomal membranes were a l s o assayed f o r s u c c i n i c dehydrogenase  activity.  I t was  found t h a t the synaptosomal membranes were  v i r t u a l l y d e v o i d of measurable m i t o c h o n d r i a l c o n t a m i n a t i o n and thus c o n s t i t u t e d a more homogeneous membrane p r e p a r a t i o n than t h a t o b t a i n e d from myocard 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 o f the a c t i v i t y o f s u c c i n i c dehydrogenase i n guinea p i g m y o c a r d i a l membrane f r a c t i o n s prepared by v a r i o u s procedures  A c t i v i t y (A absorbance/mg/15 min.) Membrane Fraction  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 e x p r e s s e d as mean + SEM was o b t a i n e d from f i v e d i f f e r e n t p r e p a r a t i o n s i n the case o f p r o c e d u r e 1, u s i n g the method d e s c r i b e d by Sulakhe e t a l ( 9 7 ) , and from seven d i f f e r e n t p r e p a r a t i o n s employing procedure 2, which r e p r e s e n t s our m o d i f i c a t i o n o f t h e methods o f H u i e t a l (96) and Sulakhe e t a l (97). Note t h a t the crude f r a c t i o n o b t a i n e d from procedure 2 had a lower a c t i v i t y than p r o c e d u r e 1.  Page 42  Table I I  O u a b a i n - s e n s i t i v e Mg  -dependent Na , K - s t i m u l a t e d  ATPase a c t i v i t y o f guinea p i g m y o c a r d i a l f r a c t i o n s prepared  membrane  by the combined procedures o f H u i  e t a l ( 9 6 ) and Sulakhe e t a l ( 9 7 )  Fraction  M g ( N a - K ) ATPase + +  +  +  activity  ( y moles Pi/mg/hr) Crude  2.2 + 0.2  F-L  4.6 + 0.6  F  2  F  3  5.7+0.9 + 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 d e s c r i b e d i n the Methods. R e s u l t s a r e t h e mean v a l u e s +_ S.E.M. f o r a minimum o f t h r e e different preparations.  Page  43  c o m p o s i t i o n a l c h a r a c t e r i s t i c s o f b r a i n s y n a p t i c membranes were t h e r e f o r e analyzed  and compared w i t h those of human e r y t h r o c y t e membrane.  shows the v a l u e s o f t o t a l p h o s p h o l i p i d , c h o l e s t e r o l and e r y t h r o c y t e and synaptosomal membrane. plasma membranes, p h o s p h o l i p i d and equimolar amounts. were p r e s e n t  I n e r y t h r o c y t e membranes, as i n most  c h o l e s t e r o l are p r e s e n t  i n g r e a t e r a b s o l u t e q u a n t i t i e s on a per mg  g r e a t e r as w e l l .  a c i d i n both  in  approximately  I n s y n a p t i c membranes, b o t h p h o s p h o l i p i d and c h o l e s t e r o l  the e r y t h r o c y t e membrane and  same.  sialic  Table I I I  The  p r o t e i n b a s i s than i n  the molar r a t i o o f 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  amount of s i a l i c  a c i d i n b o t h p r e p a r a t i o n s was  These p r e l i m i n a r y c o m p o s i t i o n a l a n a l y s e s  suggested t h a t membranes  e x 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 erythrocytes.  Phospholipids  l i p i d components were apparent on the TLC ents were seen w i t h synaptosomes.  and  Four d i f f e r e n t phospho-  o f e r y t h r o c y t e s w h i l e f i v e compon-  I n d i v i d u a l p h o s p h l i p i d s were i d e n t i f i e d  v a l u e s i n the s o l v e n t system employed.  v a l u e s f o r a l l the components are p r e s e n t e d (phosphatidylethanolamine,  of  from b o t h membranes were f u r t h e r s e p a r a t e d  examined by means of t h i n l a y e r chromatography (TLC).  the b a s i s of t h e i r known  the  i n T a b l e IV.  Four  p h o s p h a t i d y l c h o l i n e , sphingomyelin  The  on  R^  phospholipids and  phospha-  t i d y l s e r i n e ) were common to b o t h e r y t h r o c y t e and synaptosomal membranes.  In  synaptosomal membrane, the f i f t h spot which appeared on top o f the phosphat i d y l e t h a n o l a m i n e on the TLC was  probably  cerebroside  (a g a l a c t o l i p i d found  o n l y i n b r a i n t i s s u e s ) on the b a s i s of i t s r e l a t i v e l y h i g h R  value.  Page 44  Table I I I  C o m p o s i t i o n a l d a t a o f human e r y t h r o c y t e and guinea p i g b r a i n synaptosomal membranes.  Membrane  Total Phospholipids (u moles/mg)  Cholesterol (u moles/mg)  P Lipid Choi  r a  tj.  0  S i a l i c Acid (n moles/mg)  Erythrocyte  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  R e s u l t s were o b t a i n e d from a minimum o f s i x t e e n d i f f e r e n t p r e p a r a t i o n s f o r human e r y t h r o c y t e and s i x f o r b r a i n synaptosomal membranes. V a r i o u s c o m p o s i t i o n a l a n a l y s e s a r e d e s c r i b e d i n the s e c t i o n o f Methods. Data a r e 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  %  Phosphatidylserine  0.138+0.004  15.7+0.8  0.135+0.012  14.8+1.3  Sphingomyelin  0.205+0.006  9.5+1.5  0.201+0.018  2.6+1.1  Phosphatidylcholine  0.339 + 0.009  29.4 + 0.5  0.307 + 0.014  36.2 + 2.8  Phosphatidylethanolamine  0.468+0.008  43.8+1.8  0.447+0.018  46.5+2.0  0.578 +0.017  0.8+0.3  Cerebroside  *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 o f each i n d i v i d u a l p h o s p h o l i p i d a r e a l s o r e p o r t e d IV).  I n b o t h e r y t h r o c y t e s and synaptosomes, p h o s p h a t i d y l e t h a n o l a m i n e  p h o s p h a t i d y l c h o l i n e were the predominant p h o s p h o l i p i d s p r e s e n t .  (Table and  However,  b r a i n synaptosomes possessed r e l a t i v e l y g r e a t e r amounts o f these two phosphol 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 t h a t b r a i n membrane has h i g h e r  q u a n t i t i e s o f these two p h o s p h o l i p i d s o t h e r workers (112-113).  i s i n accordance w i t h the r e s u l t s o f  The minute q u a n t i t y o f c e r e b r o s i d e s p r e s e n t  i n the  membrane i s a l s o c o n s i s t e n t w i t h the f i n d i n g s o f the above workers (112-113).  MEMBRANE PERTURBATIONAL PROPERTIES OF THE ANTIARRHYTHMIC AGENTS a)  E f f e c t s o f A n t i a r r h y t h m i c s on L a b e l l i n g o f 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 o f t h e a n t i a r r h y t h m i c s were m o n i t o r e d  i n terms o f a l t e r a t i o n s produced i n the r e a c t i o n o f membrane s u l f h y d r y l groups w i t h 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 r e p o r t e d  the i n t e g r i t y o f membrane-protein s u l f h y d r y l groups i s a c r i t i c a l determining  the h e m o l y t i c b e h a v i o u r  o f erythrocytes (114).  have shown t h a t a n t i h e m o l y t i c c o n c e n t r a t i o n s  that  factor i n  Godin e t a l ( 5 2 )  o f p r o p r a n o l o l cause a p r o g r e s -  s i v e i n c r e a s e i n t h e r e a c t i v i t y o f e r y t h r o c y t e 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 t o a n a l y z e 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 o f other a n t i a r r h y t h m i c The  r e s u l t s o f these experiments a r e p r e s e n t e d  agents. i n F i g u r e s 3 and 4.  With  most d r u g s , a p r o g r e s s i v e i n c r e a s e 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 o f membrane s u l f h y d r y l groups towards DTNB i s observed i n the range o f drug  Page 47  FIGURE 3.  M o d i f i c a t i o n o f e r y t h r o c y t e 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 o f antiarrhythmics. Each p o i n t r e p r e s e n t s t h e mean o f e x p e r i m e n t s performed on a minimum o f f o u r d i f f e r e n t membrane p r e p a r a t i o n s . R e s u l t s a r e e x p r e s s e d as t h e i n c r e m e n t a l i n c r e a s e 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 o f drug. Mean c o n t r o l v a l u e +_ S.E.M. was 23.6 + 0.2 umoles SH/mg p r o t e i n . 1  Page 48  Page 49  FIGURE 4.  M o d i f i c a t i o n o f e r y t h r o c y t e 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 o f antiarrhythmics. See l e g e n d t o F i g u r e 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 t h e a b i l i t i e s o f drugs t o p e r t u r b t h e -3 membrane a r e apparent. pranolium D-600.  A t a c o n c e n t r a t i o n o f 10  M, QX 572, p r o p a n o l o l and  produce a h i g h e r degree o f 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  The two e x c e p t i o n s  to t h i s general  i n c r e a s e o f DTNB i n c o r p o r a t i o n a r e  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 ( F i g u r e 4 ) .  Their i n t e r a c t i o n with the  e r y t h r o c y t e membrane a t a n t i h e m o l y t i c drug c o n c e n t r a t i o n s a l t e r t h e m o d i f i c a t i o n o f membrane s u l f h y d r y l s by DTNB.  does n o t a p p r e c i a b l y To i n v e s t i g a t e t h e  i n f l u e n c e o f 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 o f i n t a c t e r y t h r o c y t e s , i n c r e a s e s i n DTNB l a b e l l i n g produced by a n t i a r r h y t h m i c s were e x p r e s s e d r e l a t i v e t o t h e c o r r e s p o n d i n g agents.  a n t i h e m o l y t i c e f f e c t s o f these  When such r e l a t i o n s h i p s were e x p l o r e d  suggesting  a common m o l e c u l a r  ( F i g u r e 5 ) , e v i d e n c e was found  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 t h e s t a b i l i z i n g range o f drug c o n c e n t r a t i o n s 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. and  i t s quaternary  d e r i v a t i v e , pranolium,  Propranolol  ( F i g u r e 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 t o the i n i t i a l s t a b i l i z i n g component o f o t h e r arrhythmics  examined, a l t h o u g h  anti-  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.  I n p a r t i c u l a r , i t appeared t h a t f o r a g i v e n  degree o f s u l f h y d r y l p e r t u r b a t i o n , the q u a t e r n a r y duced a l e s s e r degree o f a n t i h e m o l y s i s  p r o p r a n o l o l analogue p r o -  than p r o p r a n o l o l .  The above r e s u l t s  suggest t h a t t h e r e 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 r t u r b a t i o n s and the a n t i h e m o l y s i s  induced by v a r i o u s 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 i n 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 •  6  a ••„.  UJ I h Z <  L I D O C A I N E  4 0  S O  Q X - 5 7 E  • Q U I N I D I N E  cr-°  D - B O O 5  IIMCREIVIEIMTAL  4  o  •  • 6  8  SULFHYDRYL  (ju M O L E S / m g  MODIFICATION  PROTEIN)  Page 54  FIGURE 6.  R e l a t i o n s h i p between the c o n c e n t r a t i o n - d e p e n d e n c e o f a n t i h e m o l y s i s and o f the i n c r e m e n t a l i n c r e a s e s i n DTNB m o d i f i c a t i o n o f e r y t h r o c y t e membranes produced by p r o p r a n o l o l and p r a n o l i u m . O r i g i n a l d a t a i n F i g u r e s 2 and 4.  Page 55  1  a  INCREMENTAL  4  1  I  B  8  SULFHYDRYL  I  IO  i IS  MODIFICATION  ( A J M O L E S / m g PROTEIN)  Page 56  and may f u r t h e r suggest t h a t the decrease i n a n t i h e m o l y s i s concentrations  involves progressive  at h i g h  drug  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 o f s u l f h y d r y l s i t e s . E f f e c t s o f the a n t i a 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 s t u d i e d i n a s y n a p t i c membrane obtained  preparation  from guinea p i g b r a i n and the r e s u l t s a r e shown i n F i g u r e  the e x c e p t i o n  7.  With  o f p r a n o l i u m , 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 p a t t e r n s observed i n e r y t h r o c y t e  ghost membranes.  as were  P r a n o l i u m d i f f e r e d from o t h e r s i n  t h a t 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 o f  p r a n o l i u m may be due t o i t s l e s s e r a b i l i t y t o permeate synaptosomal membranes or t o i t s l e s s e r a b i l i t y t o p e r t u r b  the environment s u r r o u n d i n g  membrane  s u l f h y d r y l s i t e s , or some c o m b i n a t i o n o f b o t h . b)  Effects of Antiarrhythmics The  on L a b e l l i n g o f the E r y t h r o c y t e Membrane by TNBS  f o r e g o i n g experiments u t i l i z i n g DTNB f o c u s s e d  b a t i o n a l e f f e c t s of antiarrhythmics  at the l e v e l o f membrane p r o t e i n s .  known, however, t h a t membrane p h o s p h o l i p i d s modifying  e n t i r e l y on the p e r t u r It is  a r e even more s u s c e p t i b l e t o the  influence of l i p i d - s o l u b l e anaesthetic  agents (50-51).  Previous  work o f G o d i n and co-workers had shown t h a t a n t i h e m o l y t i c p r o p e r t i e s o f prop 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 t o i n c r e a s e the i n c o r p o r a t i o n o f 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 o f 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 o f a n t i a r r h y t h m i c s . Each p o i n t r e p r e s e n t s t h e mean o f experiments performed on a minimum o f three d i f f e r e n t membrane preparations. R e s u l t s a r e e x p r e s s e d as t h e i n c r e m e n t a l i n c r e a s e 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 o f drug. Mean c o n t r o l v a l u e + S.E.M. was 26.8 + 2.3 umoles SH/mg Protein.  Page 58  Page 59  c y t e membrane s t r u c t u r a l components ( 5 2 ) . the m o l e c u l a r  T h e r e f o r e , we undertook t o a n a l y z e  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 o f o t h e r a n t i a r r h y t h m i c s i n terms  o f t h e i r i n f l u e n c e on t h e i n c o r p o r a t i o n o f TNBS i n t o e r y t h r o c y t e membrane p r o t e i n and p h o s p h o l i p i d amino groups. Membranes were l a b e l l e d w i t h TNBS i n the presence or absence o f a n t i arrhythmic  agents at v a r i o u s c o n c e n t r a t i o n s , 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 p h o s p h o l i p i d components r e s o l v e d by g e l f i l t r a t i o n u s i n g Sephadex G-200.  T h i s r e s u l t e d i n complete s e p a r a t i o n o f 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 b e g i n n i n g  at the v o i d  volume, from the p h o s p h o l i p i d components, which e l u t e d much l a t e r , thus e n a b l i n g the e f f e c t s o f a n t i a r r h y t h m i c agents on p r o t e i n and p h o s p h o l i p i d components t o be viewed The  independently.  d a t a from these e x p e r i m e n t s i n d i c a t e d t h a t a n t i a r r h y t h m i c agents  i n c r e a s e TNBS i n c o r p o r a t i o n i n t o b o t h p r o t e i n s and p h o s p h o l i p i d s and t h a t 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 a c t i o n s on the membrane p h o s p h o l i p i d and p r o t e i n components ( F i g u r e 8 ) . were most c o n v e n i e n t l y e x p r e s s e d  i n terms o f t h e s l o p e o f t h e l i n e a r  ship c h a r a c t e r i z i n g drug-stimulated as a f u n c t i o n o f c o r r e s p o n d i n g  i n c o r p o r a t i o n o f TNBS i n t o  relation-  phospholipids  i n c o r p o r a t i o n o f 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 (Table V).  These e f f e c t s  erythrocytes  The f a c t t h a t a l l s l o p e s were g r e a t e r than one r e f l e c t s t h e  g r e a t e r p e r t u r b a t i o n a l a c t i o n s o f these p h a r m a c o l o g i c a l phospholipids  as compared w i t h membrane p r o t e i n s .  agents on membrane  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 p h o s p h o l i p i d and p r o t e i n components o f e r y t h r o c y t e membranes i n t h e presence o f i n c r e a s i n g c o n c e n t r a t i o n s o f a n t 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 t h e presence o f a n t i a r r h y t h m i c s and t h e e x t e n t o f t r i n i t r o p h e n y l a t i o n o f p h o s p h o l i p i d and p r o t e i n components r e s o l v e d by g e l f i l t r a t i o n u s i n g 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 ( 5 2 ) . Each p o i n t r e p r e s e n t s the mean o f experiments performed on two d i f f e r e n t membrane p r e p a r a t i o n s .  Page 61  RELATIVE  PROTEIN  LABELLING  Page 62  Table V  E f f e c t s o f a n t i a r r h y t h m i c s on t h e s l o p e o f t h e 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 p h o s p h o l i p i d l a b e l l i n g by TNBS as a f u n c t i o n o f membrane p r o t e i n l a b e l l i n g  Drug  C o n c e n t r a t i o n Range  Correlation slope* (mean + S.E.M.)  Lidocaine  2.0-10  mM  2.6 + 0.6  Quinidine  0.1-0.8 mM  3.6 + 0.3  Propranolol  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 o f two d i f f e r e n t membrane p r e p a r a t i o n s . Slopes o f 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 i n c r e a s e s i n p h o s p h o l i p i d l a b e l l i n g as a f u n c t i o n o f p r o t e i n l a b e l l i n g were e v a l u a t e d u s i n g 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  a n t i h e m o l y t i c p r o p e r t i e s o f t h e drugs examined and t h e i r membrane p e r t u r b a t i o n a l a c t i o n s were assessed  by e x p r e s s i n g  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 p h o s p h o l i p i d  components (as determined by absorbance at 335  nm) a t each drug c o n c e n t r a t i o n as a f u n c t i o n o f e r y t h r o c y t e h e m o l y t i c i z a t i o n produced a t t h e same c o n c e n t r a t i o n ( F i g u r e 9 ) .  These  r e v e a l e d t h a t q u i n i d i n e , QX 572, p r o p r a n o l o l and p r a n o l i u m  stabil-  analyses  a l l produce  i d e n t i c a l e f f e c t s on t h e i n c o r p o r a t i o n o f TNBS i n t o p r o t e i n s ( l e f t  panel,  F i g u r e 9 ) , w h i l e l i d o c a i n e and D-600 d e v i a t e from t h i s p a t t e r n , w i t h t h e former e x h i b i t i n g t h e g r e a t e s t 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 o f a l l the agents and  t h e l a t t e r a c t u a l l y c a u s i n g an i n i t i a l d e c r e a s e i n TNBS i n c o r p o r a t i o n  r e l a t i v e t o t h a t i n t h e absence o f added drug.  When t h e drug-induced  i n c r e a s e s i n TNBS l a b e l l i n g o f e r y t h r o c y t e membrane p h o s p h o l i p i d s analyzed,  a completely  were  d i f f e r e n t spectrum o f l a b e l l i n g p r o f i l e s was o b t a i n e d  (right panel, Figure 9).  The two q u a t e r n a r y  agents, pranolium  and QX 572,  produced e q u i v a l e n t e f f e c t s w h i c h , on t h e b a s i s o f t h e i r s h a r p i n c r e a s e drug c o n c e n t r a t i o n , appeared c o o p e r a t i v e  i n nature.  with  Q u i n i d i n e 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 o f l i d o c a i n e , w i t h p r o p r a n o l o l o c c u p y i n g an i n t e r m e d i a t e two groups.  p o s i t i o n between these  The s m a l l e f f e c t s o f b r e t y l i u m and p r a c t o l o l on TNBS i n c o r p o r -  a t i o n were o n l y observed a t r a t h e r h i g h drug c o n c e n t r a t i o n s  although  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 t o possess hemolytic  p r o p e r t i e s ( F i g u r e s 1 and 2 ) .  both anti-  This apparent l a c k o f p e r t u r b a t i o n a l  a c t i o n s 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 o f  Page 64  FIGURE 9.  E f f e c t s o f a n t i a r r h y t h m i c s on t h e l a b e l l i n g o f p r o t e i n and p h o s p h o l i p i d components o f e r y t h r o c y t e membranes. The e x t e n t o f t r i n i t r o p h e n y l a t i o n was a s s e s s e d as d e s c r i b e d i n F i g u r e 8. Each p o i n t i s the mean o f experiments performed on two d i f f e r e n t membrane p r e p a r a t i o n s .  Page 65  7 /o A N T I H E M O L Y S I S  Page 66  a n t i a r r h y t h m i c agents might suggest t h a t 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 a r e produced by a m o l e c u l a r  mechanism which d i f f e r s from t h a t o f the o t h e r  s u b s t a n c e s examined. The  s t r u c t u r a l b a s i s o f the d e s t a b i l i z i n g o r l y t i c phase o f a n t i -  arrhythmics-erythrocyte  i n t e r a c t i o n was examined u s i n g two drugs w i t h  pronounced l y t i c a c t i o n s a t h i g h 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 ) .  I n b o t h c a s e s , a sharp i n c r e a s e i n p h o s p h o l i p i d l a b e l l i n g was  observed a t l y t i c drug c o n c e n t r a t i o n s . l a b e l l i n g was a l s o seen a t l y t i c  A corresponding  concentrations  increase i n protein  o f q u i n i d i n e but n o t D-600.  I t would appear, t h e r e f o r e , t h a t , as the experiments u s i n g DTNB had suggested (Figure 5), the molecular  c h a r a c t e r i s t i c s o f drug-induced l y s i s o f e r y t h r o -  c y t e s by q u i n i d i n e and D-600 a r e n o t i d e n t i c a l .  EFFECTS OF ANTIARRHYTHMICS ON ACTIVITY OF MEMBRANE-ASSOCIATED ENZYMES R e s u l t s from the f o r e g o i n g experiments have p r o v i d e d arrhythmics  evidence that  anti-  d i f f e r considerably i n t h e i r perturbational c h a r a c t e r i s t i c s i n  e r y t h r o c y t e membranes and t h a t these d i f f e r i n g p a t t e r n s o f s t r u c t u r a l  alter-  a t i o n s may be r e l e v a n t t o the a n t i h e m o l y t i c p r o p e r t i e s o f these agents i n intact  erythrocytes.  Another approach t o a n a l y z e t h e f u n c t i o n a l consequences o f these  molecular  i n t e r a c t i o n s i n v o l v e d a study o f t h e e f f e c t s o f i n c r e a s i n g c o n c e n t r a t i o n s o f a n t i a r r h y t h m i c s on t h e a c t i v i t y o f s e v e r a l membrane-associated e n z y m a t i c processes.  When t h e e f f e c t s o f a n t i a r r h y t h m i c agents on t h e a c t i v i t i e s o f  Page 67  Mg  -independent ( o r b a s a l ) and Mg  - s t i m u l a t e d NPPases o f e r y t h r o c y t e  ghosts were s t u d i e d , most drugs showed a s m a l l 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 c o n c e n t r a t i o n o f drug, so t h a t , f o r c o n v e n i e n c e , o n l y an average value f o r i n h i b i t i o n over the whole range o f drug c o n c e n t r a t i o n s (Table V I ) .  i s reported  I t i s c l e a r from these d a t a t h a t the i n h i b i t o r y p r o p e r t i e s of  these drugs a r e v i r t u a l l y e q u i v a l e n t i n the s t a b i l i z i n g range o f concent 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 a p p r o x i m a t e l y 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  two-fold  greater  producing  l y s i s o f 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 b o t h 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 a c t i o n s o f p r a c t o l o l  ( F i g u r e 10) were  v e r y much g r e a t e r and showed a c o n c e n t r a t i o n dependency compared t o other agents examined ( T a b l e V I ) b u t were q u a l i t a t i v e l y s i m i l a r , i n t h a t the b a s a l NPPase a c t i v i t y was a p p r o x i m a t e l y  t w i c e as s u s c e p t i b l e t o i n a c t i v a t i o n  Mg -stimulated  I n the s m a l l i n h i b i t o r y e f f e c t s  ++  NPPase a c t i v i t y .  as the  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 t o be r e v e r s e d  so t h a t  b a s a l a c t i v i t y was l e s s s u s c e p t i l e t o i n h i b i t i o n ( F i g u r e 10). The a c t i v i t y o f the o u a b a i n - s e n s i t i v e K - s t i m u l a t e d +  component o f  Mg -dependent NPPase (K -NPPase) was v e r y much more s u s c e p t i b l e t o ++  +  p r o g r e s s i v e i n h i b i t i o n by i n c r e a s i n g drug c o n c e n t r a t i o n s .  The agents shown i n  F i g u r e 11 appeared t o share an i n i t i a l common i n h i b i t o r y component which was f o l l o w e d at h i g h e r c o n c e n t r a t i o n s by i n h i b i t o r y p a t t e r n s 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  o f p r o p r a n o l o l on the a c t i v i t y o f K -NPPase +  (right  Page 68  Table V I  E f f e c t s o f a n t i a r r h y t h m i c s on a c t i v i t y o f b a s a l (Mg and M g - s t i m u l a t e d + +  e r y t h r o c y t e membrane  Average Drug*  basal  -independent)  p-nitrophenylphosphatase  Inhibition** Mg -stimulated + +  Bretylium  8.0 + 1..3  3.5 + 1.1  Lidocaine  11.1 + 1 .7  6.7 + 1.0  13.7 + 3..4 37.1 + 3 .0  6.6 + 2.1 14.9 + 1.2  13.2 + 1..3 15.7 + 3 .2  6.5 + 0.8 11.5 + 1.5  10.2 + 1..0  5.6 + 2.1  4.1 + 0..9  9.2 + 0.8  Quinidine s t a b i l i z i n g range d e s t a b i l i z i n g range D-600 s t a b i l i z i n g range d e s t a b i l i z i n g range Pranolium Propranolol  *A11 drugs were t e s t e d i n the range o f c o n c e n t r a t i o n s p r o d u c i n g s t a b i l i z a t i o n o f i n t a c t e r y t h r o c y t e s (see F i g s . 1 and 2) u n l e s s o t h e r w i s e i n d i c a t e d . * * R e s u l t s a r e e x p r e s s e d as mean + S.E.M. o f e x p e r i m e n t s u s i n g t h r e e membrane p r e p a r a t i o n s .  different  Page 69  FIGURE 10.  I n h i b i t i o n o f b a s a l ( M g - i n d e p e n d e n t ) and M g - d e p e n d e n t p - n i t r o p h e n y l p h o s p h a t a s e (NPPase) by p r a c t o l o l and p r o p r a n o l o l i n r e l a t i o n t o the a n t i h e m o l y t i c e f f e c t s o f these a g e n t s . Each p o i n t r e p r e s e n t s t h e mean o f experiments performed u s i n g t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . ++  ++  Page 70  %  ANTIHEMOLYSIS  Page 71  FIGURE 11.  I n h i b i t i o n o f the K - s t i m u l a t e d component o f Mg -dependent p - n i t r o p h e n y p h o s p h a t a s e (K -NPPase) by a n t i a r r h y t h m i c s i n r e l a t i o n t o the a n t i h e m o l y t i c e f f e c t s o f these agents. Each p o i n t r e p r e s e n t s the mean o f experiments performed u s i n g t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . +  ++  +  Page 72  %  ANTIHEMOLYSIS  Page 73  p a n e l , F i g u r e 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 p a n e l o f the same f i g u r e ( F i g u r e 1 1 ) .  Both b r e t y l i u m  and p r a c t o l o l a g a i n 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 o f the other p h a r m a c o l o g i c a l  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 o f c o n c e n t r a t i o n i n the range s t u d 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 widely  distributed  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 u n d e r t a k e n to compare the i n h i b i t o r y e f f e c t s o f a n t i a r r h y t h m i c s on the enzyme from human e r y t h r o c y t e 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 d e r i v e d from guinea p i g h e a r t  (96-97).  The m y o c a r d i a l  f o u r o f the f i v e drugs t e s t e d , a l t h o u g h c y t e enzyme.  enzyme was shown t o be i n h i b i t e d by  t o a l e s s e r degree than the e r y t h r o -  F u r t h e r , i n most c a s e s , a s t i m u l a t o r y component was noted at  lowest drug c o n c e n t r a t i o n s w i t h the m y o c a r d i a l  K -NPPase ( F i g u r e 1 2 ) . +  A g a i n , d i s t i n c t groupings o f drugs emerged w i t h q u i n i d i n e and D-600 a p p a r e n t l y s h a r i n g a common r e l a t i o n s h i p which d i f f e r e d from t h a t f o r e i t h e r p r o p r a n o l o l or p r a n o l i u m ,  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 m y o c a r d i a l  enzyme over the e n t i r e range o f c o n c e n t r a t i o n s s t u d i e s on m y o c a r d i a l  tested.  These p r e l i m i n a r y  sarcolemmal membranes were a g a i n i n d i c a t i v e o f p e r t u r b -  a t i o n a l a c t i o n s of a n t i a r r h y t h m i c s which d i f f e r e d w i t h the  pharmacological  agent i n q u e s t i o n and the i n h i b i t o r y p a t t e r n s i n the two systems s t u d i e d  Page 74  FIGURE 12.  R e l a t i o n s h i p between e r y t h r o c y t e and m y o c a r d i a l membrane K -NPPase i n h i b i t i o n by v a r i o u s a n t i a r r h y t h m i c s . The e r y t h r o c y t e data are those p r e s e n t e d i n F i g . 11 and t h e m y o c a r d i a l data were o b t a i n e d u s i n g f o u r d i f f e r e n t sarcolemmal p r e p a r a t i o n s (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 f o r b o t h p r e p a r a t i o n s (52) except t h a t r e a c t i o n times o f 60 minutes and 10 minutes were used f o r the e r y t h r o c y t e and cardiac preparations r e s p e c t i v e l y . +  Page 75  % INHIBITION  ERYTHROCYTE  K  +  -NPPase  Page 76  suggested t h a t the s t r u c t u r a l consequences not e q u i v a l e n t  of drug-membrane i n t e r a c t i o n are  i n the e r y t h r o c y t e membrane and the plasma membrane o f the  myocardium. The f i n a l membrane enzyme system s t u d i e d was Mg  ++  -dependent Ca  ++  s t i m u l a t e d ATPase w h i c h , i n the e r y t h r o c y t e membrane, c o n s i s t s o f the two d i f f e r e n t components, one s t i m u l a t e d by Ca  ++  at concentrations  (low a f f i n i t y C a - A T P a s e ) and a second component a c t i v a t e d by ++  t r a t i o n s of C a (52,115).  + +  -3  M  concen-  i n the range of 10 ^ M ( h i g h a f f i n i t y C a - A T P a s e ) ++  The e x t e n t t o which each o f 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  energization of active C a adenosine t r i p h o s p h a t e (115-116).  of 10  + +  e x t r u s i o n from e r y t h r o c y t e s as the r e s u l t o f  (ATP) h y d r o l y s i s has not as y e t been f i r m l y e s t a b l i s h e d  The i n h i b i t o r y p r o p e r t i e s o f a n t 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 ( F i g u r e 13).  With p r o p r a n o l o l and p r a n o l i u m  (both o f 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 s t i m u l a t e d by low c o n c e n t r a t i o n s  of C a  + +  (termed the h i g h 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 .  I n 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 b o t h a c t i v i t e s to about the same e x t e n t ( F i g u r e 13). R e s u l t s w i t h b r e t y l i u m and p r a c t o l o l are not shown as b o t h of them d i d not a p p r e c i a b l y m o d i f y the a c t i v i t y o f e i t h e r component.  EFFECTS OF ANTIARRHYTHMICS ON ACTIVITY OF ACETYLCHOLINESTERASE As an e x t e n s i o n 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 t i o n i n terms o f the f u n c t i o n a l consequences  alter-  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 o f h i g h 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 ( C a - A T P a s e ) from e r y t h r o c y t e membranes by v a r i o u s a n t i a r r h y t h m i c s . The assays f o r h i g h 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 t h a t a Ca -EGTA b u f f e r system was used t o a c h i e v e c o n c e n t r a t i o n s o f f r e e C a i n the media o f 1 and 90 M r e s p e c t i v e l y . Each p o i n t r e p r e s e n t s t h e mean of experiments performed u s i n g t h r e e d i f f e r e n t membrane preparations. + +  ++  ++  ++  + +  Page 78  Page 79  the influence of antiarrhythmics on the a c t i v i t y of membrane-associated acetylcholinesterase was studied.  This enzyme has been well characterized i n  erythrocytes and is known to be located on the external surface of the erythrocyte membrane (93).  Thus, the a c t i v i t y of this enzyme can be measured i n  both intact erythrocytes and i n hemoglobin-free erythrocyte membranes using the method described by Ellman (117) wherein acetylthiocholine i s employed as substrate and the rate of formation of thiocholine i s measured colorimetrically using 5,5 -dithio-bis-(2-nitrobenzoic acid). 1  These experiments  enabled us to determine the extent to which the membrane actions of antiarrhythmics i n isolated erythrocyte membranes are relevant to those i n the membrane of the intact c e l l s .  Furthermore, the presence of acetylcholin-  esterase i n plasma membrane fractions derived from guinea pig brain tissue has enabled a direct comparison of the membrane perturbations produced by the antiarrhythmic agents i n excitable membranes with those i n 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 e l e c t r i c a l s t a b i l i z a t i o n i n excitable tissues (54). Antiarrhythmics were shown to cause progressive inhibition of erythrocyte acetylcholinesterase at concentrations producing s t a b i l i z a t i o n 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  (Figure 14).  A g a i n , 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 s e p a r a t e group. exception  was l i d o c a i n e which a p p a r e n t l y  above c l a s s e s .  The  d i d not b e l o n g to e i t h e r one o f the  This f i n d i n g suggested the p o s s i b i l i t y t h a t d i f f e r e n c e s may  e x i s t i n t h e m o l e c u l a r b a s i s o f enzyme i n h i b i t i o n induced by these agents.  various  P r a n o l i u m c o u l d not be s t u d i e d as i t was found t o cause h e m o l y s i s o f  intact erythrocytes  under the e x p e r i m e n t a l  c o n d i t i o n s o f the enzyme assay.  The i n h i b i t o r y e f f e c t s o f a n t i a r r h y t h m i c s intact erythrocytes,  erythrocyte  on the a c e t y l c h o l i n e s t e r a s e o f  ghost membranes and guinea p i g b r a i n  synaptic  membranes were compared u s i n g 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 bretylium, propranolol r e l a t i o n s h i p s provide  and l i d o c a i n e i n F i g u r e 15.  The s l o p e s o f these l i n e a r  a measure o f the c o o p e r a t i v i t y and/or the s t 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 t h e i n h i b i t o r y p r o c e s s (118) and the a n t i l o g o f the x - i n t e r c e p t represents  the K ^ v a l u e o f the i n h i b i t o r ; t h a t i s , the  concentration  r e q u i r e d t o produce 50% i n h i b i t i o n o f 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 . r e s u l t s o f these s t u d i e s a r e summarized i n T a b l e V I I .  The  No marked d i f f e r e n c e s  between the a c t i o n s o f these agents i n the i n t a c t e r y t h r o c y t e  and i t s i s o l a t e d  membrane were a p p a r e n t , a l t h o u g h f o r a few drugs, namely, l i d o c a i n e and practolol, K  values  f o r enzyme i n h i b i t i o n were somewhat g r e a t e r  membranes than f o r the i n t a c t c e l l s . K ^ values  Considerably  greater differences i n  were noted when e r y t h r o c y t e membranes and b r a i n  membranes were compared ( T a b l e V I I ) .  f o r the  synaptic  T h i s was p a r t i c u l a r l y so i n the case o f  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, s u g g e s t i n g  t h a t 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 o f t h e i n h i b i t i o n o f e r y t h r o c y t e membrane and s y n a p t i c 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 p o i n t r e p r e s e n t s t h e mean + S.E.M. o f experiments u s i n g t h r e e d i f f e r e n t e r y t h r o c y t e membrane p r e p a r a t i o n s and two d i f f e r e n t s y n a p t i c membrane p r e p a r a t i o n s .  Page 84  SYNAPTIC MEMBRANE  ERYTHROCYTE MEMBRANE  BRETYLIUM  BRETYLIUM  PROPRANOLOL  PROPRANOLOL  ,6 L I D O C A I N E ° LIDOCAINI •  1  -S  LOG  DRUG  COIMC.  Page 85  T a b l e V I I I n h i b i t o r y e f f e c t s o f a n t i a r r h y t h m i c s 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 o f 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  E r y t h r o c y t e membranes  Erythrocyte  5 (mM)  K  Hill Slope  .5 (mM)  K  Hill  S y n a p t i c Membranes  Slope  .5 (mM)  K  Hill Slope  Bretylium  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  Practolol  0. 70+.03  0. 84+.03  0.99+.05  1.03+.03  1.58+.03  1.01+.19  Propranolol  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 d a t a f o r e r y t h r o c y t e and s y n a p t i c membranes were o b t a i n e d u s i n g a minimum o f two d i f f e r e n t membrane p r e p a r a t i o n s and the experiments w i t h i n t a c t e r y t h r o c y t e s were performed i n t r i p l i c a t e on a minimum o f t h r e e d i f f e r e n t b l o o d samples. I n each c a s e , i n h i b i t i o n d a t a 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 c o n s t a n t s e v a l u a t e d from r e g r e s s i o n l i n e s determined u s i n g 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 a r e e x p r e s s e d as mean + S.E.M.  Page 86  environments where these studied.  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  E x a m i n a t i o n o f H i l l p l o t s l o p e s f o r the i n h i b i t i o n r e v e a l e d an  apparent l a c k o f c o o p e r a t i v i t y or o f s t o i c h i o m e t r y g r e a t e r than one i n t h e i n h i b i t o r y e f f e c t s o f most a n t i a r r h y t h m i c s so t h a t H i l l were o b s e r v e d i n most cases (118).  c o e f f i c i e n t s of u n i t y  However, two e x c e p t i o n s were observed f o r  p r o p r 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 ( b u t not e r y t h r o c y t e membranes) was c h a r a c t e r i z e d by a s l o p e s i g n i f i c a n t l y g r e a t e r than one. In order t o determine the n a t u r e arrhythmics  o f the i n h i b i t i o n e x e r t e d by the a n t i -  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 s t u d i e s were performed  u s i n g drug c o n c e n t r a t i o n s p r o d u c i n g  the same degree o f a c e t y l c h o l i n e s t e r a s e  inhibition.  A v a l u e o f 35% was a r b i t r a r i l y chosen as a c o n v e n i e n t  l e v e l of  inhibition.  Since the i n f l u e n c e o f drugs on the a c t i v i t y o f a c e t y l c h o l i n -  e s t e r a s e i n i n t a c t c e l l s was not found to d i f f e r from t h a t of the ghost membranes, o n l y i n t a c t e r y t h r o c y t e s were used f o r the k i n e t i c The v a l u e s  of K  m  (Michaelis-Menten  study.  c o n s t a n t , an i n d i c a t i o n o f s u b s t r a t e  c o n c e n t r a t i o n which g i v e s h a l f the n u m e r i c a l v a l u e o f maximal v e l o c i t y ) and V  ( t h e maximal r a t e o f enzymatic r e a c t i o n at h i g h s u b s t r a t e 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 o f drug were o b t a i n e d by E a d i e 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 F i g u r e 16 where the s l o p e and the y - i n t e r c e p t o f t h e l i n e a r s h i p g i v e the v a l u e o f K r  &  ized i n Table V I I I .  m  and V max  respectively. r  relation-  The r e s u l t s a r e summar-  3  A l l drugs except p r a c t o l o l showed an i n c r e a s e d  for  Page 87  FIGURE 16.  E a d i e p l o t a n a l y s i s of the n a t u r e of the i n h i b i t i o n of i n t a c t e r y t h r o c y t e 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 v a l u e s were o b t a i n e d from e x p e r i m e n t s performed i n the absence o f any added d r u g . Each p o i n t r e p r e s e n t s the means _+ S.E.M. o f experiments u s i n g two d i f f e r e n t samples o f b l o o d .  Page 88  Page 89  Table VIII  Drug  Influence of antiarrhythmics on the enzyme-substrate kinetics of intact erythrocytes* 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  +  QX-571  .411  +  .130  .065  +  0 .004  *The data for intact erythrocytes were obtained using a minimum of two different blood samples. Experimental details were described i n the Methods section. In each case, data were plotted as shown i n Fig. 16 and kinetic constants were derived from Eadie plots and evaluated from regression lines determined using a Compucorp (140) s t a 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 a c t i v i t y of acetylcholinesterase 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 p a r a l l e l s h i f t from the control i n the Eadie plot (Figure 16) . which gave a decreased V t i c a l K m for the substrate,  of the acetylcholinesterase but an idenmax  J  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 a c t i v i t y of intact erythrocytes i n the presence of antiarrhythmics was assayed i n a high chloride medium or i n 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 i n 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 o f transmembrane c h l o r i d e g r a d i e n t on t h e i n h i b i t i o n o f i n t a c t e r y t h r o c y t e 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 . Each p o i n t r e p r e s e n t s t h e means + S.E.M. o f experiments u s i n g a minimum o f two d i f f e r e n t samples o f b l o o d .  Page 92  111 (fl <  a  90  R  LU  • BRETYLIUM / o LIDOCAINE j\ a D-600  h  tf)•  111 111 7  Z 1-i  2  Q  • I 111 U • 50 J a  PROPRANOLOL  > •  111 i a < u ° 3  Z •  h  E i z  i  (D I  ,  a  eo  a a e a  7 INHIBITION 10  (LOW  BO  IOO  SO  40  EO  BO  IOO  ACETYLCHOLINESTERASE CHLORIDE MEDIUM)  Page 93  The fact that the membrane actions of antiarrhythmics i n the present model system vary i n their dependence on transmembrane potential may point to possible differences i n the molecular basis by which these compounds modify the functional properties of e l e c t r i c a l l y excitable tissues.  Page 94  DISCUSSION  A l a r g e number of s t u d i e s e x i s t which have been concerned w i t h the e f f e c t s o f drugs on the p r o p e r t i e s o f membranes or membrane-associated p r o c e s s e s . and  l a r g e , these s t u d i e s have been of a d e s c r i p t i v e n a t u r e w i t h  d e t a i l e d i n f o r m a t i o n of the m o l e c u l a r  mechanism g o v e r n i n g  By  little  the i n t e r a c t i o n .  T h i s s i t u a t i o n i s p a r t i c u l a r l y t r u e i n the case of a n t i a r r h y t h m i c s , where s t u d i e s 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 s t u d i e s molecular neglected.  aspects The  governing  the a c t i o n s of these drugs have been v e r y much  experiments r e p o r t e d  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 of p h a r m a c o l o g i c a l l y  i n t h i s study p r o v i d e  molecular I t has  ample e v i d e n c e f o r  f u n c t i o n a l consequences of the i n t e r a c t i o n  d i v e r s e a n t i a r r h y t h m i c agents w i t h a s i m p l e model  membrane system, namely t h a t of the human e r y t h r o c y t e . these o b s e r v a t i o n s  of  may  I t i s suggested t h a t  u l t i m a t e l y allow a b e t t e r understanding  b a s i s by which these substances modify m y o c a r d i a l  of  the  function.  g e n e r a l l y been assumed t h a t 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  a m p h i p a t h i c m o l e c u l e s to produce d i r e c t m y o c a r d i a l  depression,  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 by these compounds of e r y t h r o c y t e s a g a i n s t h y p o t o n i c  sometimes stabilization  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 h e r e , 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,  c o n s i d e r a b l e degree of a n t i h e m o l y s i s  ( F i g u r e s 1 and  d i d produce a  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 s t a b i l i z a t i o n (Figure 1) but unlike propranolol, a related g-adrenergic antagonist, practolol i s v i r t u a l l y devoid of direct cardiodepressant a c t i v i t y (119-120).  For several  other agents (lidocaine, quinidine, QX 572 and D-600) a typical pattern of hypotonic s t a b i l i z a t i o n at low concentrations followed by c e l l l y s i s at higher concentrations was observed.  Such biphasic actions of l i p i d soluble anaes-  thetics on red c e l l s t a b i l i t y have been reviewed i n 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 concentrations (54,56).  Thus, i t seems safe to assume that protection of red cells  from l y s i s i s 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 s o l u b i l i t i e s of the anaesthetic molecules and an excellent correlation exists between hydrophobicity and anaesthetic potency as well as with the a b i l i t y of general and local anaesthetics to f l u i d i z e membrane lipids (54).  However, i t i s not known i f both types of anaesthetics  interact with a common hydrophobic site i n neuronal membranes. The i n v e s t i 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 i n biological membranes.  Seeman suggested that  proteins figure prominently i n 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 o f o c c u p a t i o n  and the e x t e n t o f  membrane e x p a n s i o n produced by membrane s t a b i l i z e r s i n e r y t h r o c y t e and synaptosome membranes but n o t 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 t h a t p r o t e i n s a r e important  i n the p r o c e s s o f membrane e x p a n s i o n (54,56).  There i s much e v i d e n c e showing t h a t drug-membrane  i n t e r a c t i o n causes p e r t u r -  b a t i o n s i n membrane s t r u c t u r a l components w i t h b o t h p r o t e i n and p h o s p h o l i p i d components b e i n g i n v o l v e d (54). protein perturbations  Experiments u s i n g DTNB t o d e t e c t  drug-induced  i n e r y t h r o c y t e membranes showed t h a t most o f the drugs  i n v e s t i g a t e d here produced an i n c r e a s e 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 ( F i g u r e s 3 and 4).  The o n l y drugs t h a t d i d not  e x h i b i t any enhancement o f DTNB l a b e l l i n g o f e r y t h r o c y t e 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 .  I n c o n t r a s t t o the marked s t i m u l -  a t o r y e f f e c t s o f p r o p r a n o l o l ( F i g u r e 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 o f DTNB i n t o e r y t h r o c y t e membranes ( F i g u r e 3). absence o f d i r e c t c a r d i o d e p r e s s a n t  The  e f f e c t s o f p r a c t o l o l might have some  b e a r i n g on i t s i n a b i l i t y t o enchance DTNB l a b e l l i n g o f p r o t e i n components i n e r y t h r o c y t e membranes.  However, the observed membrane s t a b i l i z i n g a c t i o n s o f  p r a c t o l o l ( F i g u r e 1) may be r e l e v a n t t o the a b i l i t y o f t h i s m o l e c u l e t o a n t a g o n i z e o u a b a i n - i n d u c e d arrhythmias  (120,123) p o s s i b l y as the r e s u l t o f a  depressant a c t i o n at the l e v e l o f a d r e n e r g i c nerves (124).  I t i s significant  t h a t b r e t y l i u m , which i s a l s o b e l i e v e d t o a c t a t l e a s t i n p a r t by  depressing  c a r d i a c a d r e n e r g i c nerve f u n c t i o n (42), a l s o e x e r t e d 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 e r y t h r o c y t e s which were not a s s o c i a t e d w i t h an i n c r e a s e d  incorporation  Page 97  o f DTNB ( F i g u r e 4) i n t o e r y t h r o c y t e membranes.  Our f i n d i n g s suggest t h a t t h e  molecular  mechanisms by which these two a n t i a r r h y t h m i c agents e x e r t  hemolytic  e f f e c t s i n i n t a c t e r y t h r o c y t e s may d i f f e r from those by which t h e  other antiarrhythmics  studied s t a b i l i z e red c e l l s .  anti-  As f o r the group o f agents  t h a t e x e r t e d a b i p h a s i c a c t i o n on t h e s t a b i l i z a t i o n o f e r y t h r o c y t e from hypot 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 o f DTNB i n c o r p o r a t i o n i n t o e r y t h r o c y t e membrane s u l f h y d r y l s was common to a l l o f them a t c o n c e n t r a t i o n s  i n the s t a b i l i z a t i o n range ( F i g u r e 5) b u t , a t  concentrations  l y s i s , the above mentioned drugs d i f f e r e d  o f drug p r o d u c i n g  from each o t h e r i n t h e 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 h e m o l y s i s ( F i g u r e 5 ) .  The f a c t t h a t l i d o c a i n e and  QX 572 d i d not appear t o 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 t h e l i g h t o f t h e c l o s e s t r u c t u r a l resemblance between these two m o l e c u l e s and  t h e i r comparable a n t i a r r h y t h m i c a c t i o n s (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 w i t h 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  antihemolysis, the quaternary sulfhydryl perturbation.  ( F i g u r e 6 ) . However, a t a g i v e n degree o f  d e r i v a t i v e produced a g r e a t e r degree o f membrane  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 , t h e s i m i l a r i t i e s between p r a n o l i u m  and p r o p r a n o l o l a r e  c o n s i s t e n t w i t h the s i m i l a r s p e c t r a o f a n t i a r r h y t h m i c a c t i v i t y observed when these agents a r e used t o a n t a g o n i z e o u a b a i n - i n d u c e d a r r h y t h m i a s , b e l i e v e d t o be governed s o l e l y by the m e m b r a n e - s t a b i l i z i n g m o l e c u l e s (126)  an a c t i o n  p r o p e r t i e s o f these  Page 98  When p r o t e i n p e r t u r b a t i o n was s t u d i e d i n guinea p i g b r a i n membranes u s i n g the same approach, q u i n i d i n e , D-600 and QX 572 a l l produced an e q u i v a l e n t enhancement o f s u l f h y d r y l l a b e l l i n g when these agents were s t u d i e d i n t h e same c o n c e n t r a t i o n range as had been used i n the e r y t h r o c y t e s t u d i e s .  U n l i k e the  p a t t e r n o b s e r v e d i n t h e e r 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 p r a n o l i u m i n t h a t t h e q u a t e r n a r y d e r i v a t i v e produced a l e s s e r i n c r e a s e i n DTNB i n c o r p o r a t i o n than p r o p r a n o l o l .  L i d o c a i n e produced p r a c t i c a l l y no s t i m u l a t i o n o f DTNB  i n t o the s u l f h y d r y l groups o f b r a i n synaptosomal membranes i n t h e concent r a t i o n range t h a t 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 .  T h i s d i f f e r e n c e cannot be  a t t r i b u t e d t o d i f f e r i n g a b s o l u t e amounts o f s u l f h y d r y l groups i n synaptosomal membranes as compared w i t h e r y t h r o c y t e s because t h e t o t a l number o f s u l f h y d r y l groups i n b o t h p r e p a r a t i o n s ( r e s u l t s n o t shown).  ( e x p r e s s e d on a per mg p r o t e i n b a s i s ) was the same  R a t h e r , i t would seem t h a t 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 o f lidocaine-membrane i n t e r a c t i o n i n t h e two systems. T h i s i n t u r n would suggest t h e p o s s i b i l i t y t h a t fundamental d i f f e r e n c e s may e x i s t i n t h e m o l e c u l a r b a s i s o f membrane s t a b i l i z a t i o n by a n t i a r r h y t h m i c s i n e x c i t a b l e and n o n - e x c i t a b l e t i s s u e s . Experiments r e l a t i n g the a n t i h e m o l y t i c p r o p e r t i e s o f a n t i a r r h y t h m i c s t o t h e i r e f f e c t s on t h e m o d i f i c a t i o n o f membrane s u l f h y d r y l groups by DTNB ( F i g u r e 3-6) and on t h e a c t i v i t y o f membrane enzymes ( F i g u r e 11, T a b l e V I ) suggested t h a t a l l drugs, w i t h t h e p r e v i o u s l y noted e x c e p t i o n o f b r e t y l i u m and p r a c t o l o l , produce a comparable degree o f membrane p r o t e i n p e r t u r b a t i o n f o r a given l e v e l of antihemolysis  at concentrations  i n t h e 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 o f b o t h p h 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 a t i o n a l e f f e c t s u s i n g the amino group t r i n i t r o p h e n y l a t i n g r e a g e n t TNBS ( 5 2 ) , 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 o f b o t h membrane p r o t e i n s and p h o s p h o l i p i d s became apparent ( F i g u r e 8 and 9, T a b l e V ) . The l a c k o f o b v i o u s 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 p h o s p h o l i p i d l a b e l l i n g i n d i c a t e d t h a t a c o n s i d e r a b l e degree o f 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 p h o s p h o l i p i d s t r u c t u r a l components.  The f a c t t h a t DTNB ( F i g u r e 5) and TNBS  ( F i g u r e 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 t h e 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 o f d r u g - i n d u c e d p r o t e i n m o d i f i c a t i o n suggested t h a t the e f f e c t s o f 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 t h a t these two r e a g e n t s 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 t h a t a n t i a r r h y t h m i c s a r e  a b l e t o e x e r t w i d e s p r e a d 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 o f membrane p r o t e i n and l i p i d components.  I n order  to obtain  more d e t a i l e d i n f o r m a t i o n r e g a r d i n g t h e s t r u c t u r a l consequences o f membranedrug i n t e r a c t i o n , the i n f l u e n c e o f s e v e r a l a n t i a r r h y t h m i c s stimulated, ouabain-sensitive  +  component o f p - n i t r o p h e n y l p h o s p h a t a s e  ( K - N P P a s e ) ( 1 2 7 ) and on the C a - s t i m u l a t e d +  on the K -  + +  component o f Mg -dependent ++  ATPase ( C a - A T P a s e ) ( 1 1 6 ) - two enzyme systems whose r o l e i n a c t i v e i o n ++  t r a n s p o r t 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 p h o s p h o l i p i d was examined.  Page 100  W i t h the e x c e p t i o n o f p r a c t o l o l and b r e t y l i u m ( F i g u r e 11, T a b l e V I ) , whose membrane a c 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 c o n s i d e r a b l y more i n h i b i t o r y towards the K - s t i m u l a t e d +  p-nitrophenylphosphatase Mg  + +  component of Mg -dependent ++  as compared w i t h the a c t i v i t i e s i n the presence of  alone or the b a s a l a c t i v i t y i n the absence o f added M g  Table V I ) .  ( F i g u r e 10,  T h i s presumably r e f l e c t s the g r e a t e r dependence o f the o u a b a i n -  sensitive K -stimulated +  membrane.  + +  component on the s t r u c t u r a l i n t e g r i t y o f the  The more marked i n h i b i t i o n o f K -NPPase a l s o suggests t h a t +  these  agents may e x e r t 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 o u t e r s u r f a c e o f the e r y t h r o c y t e membrane where the K - s t i m u l a t e d +  i s l o c a t e d (128).  component o f the enzyme complex  I n h i b i t o r y a c t i o n s o f a n t i a r r h y t h m i c s have p r e v i o u s l y been  demonstrated - f o r example by Lowry e t a l (129) who found t h a t 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 , h e m o g l o b i n - f r e e human e r y t h r o +  c y t e and beef h e a r t membrane. The experiments w i t h K -NPPase a l l o w e d a d i r e c t comparison o f 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 o f v a r i o u s a n t i a r r h y t h m i c s on e r y t h r o c y t e membranes and c a r d i a c sarcolemmal membranes ( F i g u r e 12). W i t h the e x c e p t i o n o f l i d o c a i n e , h i g h c o n c e n t r a t i o n s o f a n t i a r r h y t h m i c s were i n h i b i t o r y , to v a r y i n g d e g r e e s , i n b o t h systems.  I n m y o c a r d i a l membranes, however, the  e f f e c t s o f l i d o c a i n e were s t i m u l a t o r y , a p r o p e r t y shared arrhythmics  at low c o n c e n t r a t i o n s .  albeit  by other  anti-  The common p r o p e r t i e s o f 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 o f p r o p r a n o l o l and p r a n o l i u m when compared i n these two systems tended t o p a r a l l e l the p h 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 e r y t h r o c y t e membrane ( F i g u r e 9) w h i l e the p r o p e r t i e s o f 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 b a l a n c e between p h 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 ( F i g u r e 9, T a b l e V ) . were important  The f o r e g o i n g  observations  i n e m p h a s i z i n g t h a t the consequences o f antiarrhythmic-membrane  i n t e r a c t i o n s a r e governed not o n l y by t h e s t r u c t u r e o f the drug i n q u e s t i o n but a l s o by the n a t u r e Antiarrhythmic  o f the membrane.  substances have been shown t o m o d i f y C a - d e p e n d e n t ++  p r o c e s s e s i n a v a r i e t y o f 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 o f t h e i r c a r d i o v a s c u l a r a c t i o n s ( 8 7 ) .  T h i s i s p a r t i c u l a r l y so i n the case o f D-600, a v e r a p a m i l  d e r i v a t i v e whose  a n t i a r r h y t h m i c p r o p e r t i e s a r e b e l i e v e d t o i n v o l v e the b l o c k a d e o f slow C a  + +  channels a c t i v a t e d d u r i n g the p l a t e a u phase o f the c a r d i a c a c t i o n p o t e n t i a l (109,133). provided  The C a - s t i m u l a t e d + +  ATPase system o f e r y t h r o c y t e membranes  a c o n v e n i e n t model w i t h which t o i n v e s t i g a t e t h e i n f l u e n c e o f v a r i o u s  a n t i a r r h y t h m i c s 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 t h a t a n t i a r r h y t h m i c s 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 t o i n h i b i t the h i g h and low a f f i n i t y components ( a c t i v a t e d by 10 ^ and 10 concentrations  of free C a  + +  an e x p e r i m e n t a l  determining  active C a  + +  M  r e s p e c t i v e l y ) suggested t h a t 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 m o l e c u l a r may p r o v i d e  4  s p e c i e s w i t h i n t h e membrane and these drugs  means o f a s s e s s i n g the r o l e o f each component i n  e x t r u s i o n from the e r y t h r o c y t e  (115-116).  g e n e r a l l y , however, the groupings o f drugs o b s e r v e d l i k e l y r e f l e c t p a t t e r n s o f membrane p r o t e i n and p h o s p h o l i p i d  More differing  p e r t u r b a t i o n s which v a r y i n  Page  terms of t h e i r i n f l u e n c e on membrane-Ca  102  interaction.  No  definitive  evidence c o u l d be found i n any of our experiments to i n d i c a t e t h a t the membrane p e r t u r b a t i o n a l a c t i o n s of D-600 s t u d i e d here have any d i r e c t to i t s a c t i o n i n b l o c k i n g C a  + +  c h a n n e l s i n the h e a r t .  relevance  However, the  capacity  of t h i s m o l e c u l e t o i n t e r a c t w i t h and p e r t u r b membrane s t r u c t u r a l components may  w e l l account f o r 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  k i n e t i c s of the C a - c a r r y i n g  system and on the s t e a d y s t a t e outward  i n cat p a p i l l a r y muscle (133)  and  + +  P u r k i n j e f i b e r s (134). anaesthetics  i t s a b i l i t y to m o d i f y K  Moreover, i t has  +  + +  local  concentrations  comparable t o those c a u s i n g b l o c k a d e of f a s t N a - c u r r e n t s (135).  The  +  suggestion  of the a u t h o r s t h a t these r e s u l t s may  current  fluxes i n canine  r e c e n t l y been shown t h a t  are a b l e t o i n f l u e n c e slow C a - c u r r e n t s at  the  r e f l e c t p e r t u r b a t i o n by  agents of a sarcolemmal l i p o p r o t e i n m a t r i x common t o b o t h channels (135) 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 d e s c r i b e d  probably represent  a u t o m a t i c i t y i s as y e t i n c o m p l e t e l y  (136)  and  t i s s u e s produced by  A l t h o u g h the i o n i c b a s i s  understood and  causing  such as l i d o c a i n e  (87,137) t o m o d i f y transmembrane f l u x e s of K  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 c a r d i a c a u t o m a t i c i t y . erythrocytes  (52) and  +  has  been  Model s t u d i e s i n  (87) have demonstrated t h a t p r o p r a n o l o l , at  s t a b i l i z a t i o n of e r y t h r o c y t e s  of  l i k e l y i s not i d e n t i c a l i n  of the h e a r t , the a b i l i t y o f a n t i a r r h y t h m i c s  propranolol  anti-  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 .  a l l regions  is  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 m y o c a r d i a l arrhythmics  these  concentrations  an enhancement of K  +  efflux  Page 103  from canine P u r k i n j e f i b e r s (137), produces an i n c r e a s e 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 o f 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 l e a d i n g t o the displacement  o f membrane-bound C a  + +  (87).  L i d o c a i n e does not share t h i s p r o p e r t y and q u i n i d i n e has been shown t o i n h i b i t the enhancement o f K  +  c e l l u l a r l e v e l s of C a  e f f l u x from e r y t h r o c y t e s caused by i n c r e a s e d + +  (132).  intra-  These f i n d i n g s a r e c e r t a i n l y i n accord  with  the d i f f e r i n g p r o p e r t i e s o f these t h r e e agents as r e v e a l e d i n the p r e s e n t study o f e r y t h r o c y t e membranes and suggest, by a n a l o g y , t h a t the m o l e c u l a r mechanisms by which these t h r e e a n t i a r r h y t h m i c s 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 .  T h i s i n t u r n may have a d i r e c t  bearing  on the d i v e r s e p a t t e r n s o f a n t i a r r h y t h m i c 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 c o n s i d e r a t i o n s serve t o  emphasize t h e i n a p p r o p r i a t e n e s s o f the g e n e r a l term q u i n i d i n e - l i k e a c t i o n when used t o d e s c r i b e the d i r e c t membrane e f f e c t s o f a n t i a r r h y t h m i c s other q u i n i d i n e on the h e a r t .  than  F u r t h e r m o r e , i f a p a r a l l e l e x i s t s between t h e  b i p h a s i c a c t i o n o f a n t i a r r h y t h m i c s i n e r y t h r o c y t e s ( F i g u r e s 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  f o l l o w e d by c a r d i o d e p r e s s i o n at  h i g h doses when these agents a r e 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 t h e m o l e c u l a r b a s i s o f t h e s t a b i l i z a t i o n and l y t i c phases i n e r y t h r o c y t e s (see R e s u l t s ) suggests the p o s s i b i l i t y t h a t these two phases o f a n t i 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 r e p r e s e n t a continuum but might be t o some e x t e n t 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 i n the structural and functional characteristics of erythrocyte membranes (52) at concentrations producing hypotonic stabilization of intact erythrocytes (52,54) and e l e c t r i c a l stabilization of nerves (56,140) suggests that an analysis of drug-induced perturbations i n isolated erythrocyte membranes might provide meaningful information regarding the mechanism by which pharmacological agents alter the functional properties of excitable tissues. I t therefore becomes important to ascertain whether or not molecular aspects of drug action studies i n isolated membranes are relevant to the situation i n 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 i n isolated erythrocyte membranes or i n solubilized preparations of the enzyme. I t 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 glyceraldehyde 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 i n terms of the  Page 105  l o s s o f membrane l i p i d s and p r o t e i n s i n the presence o f h y p e r t o n i c s a l i n e solution.  Burger e t a l (144) a l s o demonstrated t h a t b o v i n e e r y t h r o c y t e ghosts  l o s t most o f 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 o f t h e 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 d u r i n g h y p o t o n i c h e m o l y s i s . o b t a i n e d from c h e m i c a l  Information  l a b e l l i n g experiments have f u r t h e r p r o v i d e d e v i d e n c e t o  show t h a t membranes of i n t a c t e r y t h r o c y t e s and ghosts d i f f e r from each o t h e r i n terms o f 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 p h o s p h a t i d y l s e r i n e r e a c t e d 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 o n l y the p h o s p h a t i d y l e t h a n o l a m i n e  of the i n t a c t  membranes was l a b e l l e d by the agent (145), s u g g e s t i n g  t h a t the ghost membranes  possessed h i g h e r a c c e s s i b i l i t y t o d i n i t r o f l u o r o b e n z e n e . study w i t h a c e t i c a n h y d r i d e ,  erythrocyte  I n a double l a b e l l i n g  the h i g h e r l i p i d r e a c t i v i t y observed i n the ghost  membranes but n o t i n i n t a c t c e l l s was b e l i e v e d t o 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 p r e p a r a t i o n of the ghost membranes (146). serum a l b u m i n - s p i n  l a b e l complex was t i g h t l y bound t o the ghost membranes but  not t o i n t a c t c e l l s . due  Landsberger e t a l (147) found t h a t the b o v i n e  They suggested t h a t such d i f f e r e n c e i n b i n d i n g may be  t o the a c c e s s i b i l i t y o f the i n n e r s u r f a c e o f the ghost membrane t o l a r g e  m o l e c u l e s and/or an a l t e r a t i o n o f the ghost membrane s u r f a c e . i s o l a t e d e r y t h r o c y t e membranes may be c o n s i d e r e d  Therefore,  t o be c l o s e d e r i v a t i v e s o f  the membrane o f i n t a c t c e l l s , r e t a i n i n g many o f 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 m e d i a t i n g  ion transport, etc.  However, i n a d d i t i o n t o r e t e n t i o n o f i t s osmotic p r o p e r t i e s and l i p i d  content,  Page  106  human h e m o g l o b i n - f r e e e r y t h r o c y t e membranes prepared by h e m o l y s i s and washings w i t h h y p o t o n i c  salt solutions, s t i l l  repeated  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 e r y t h r o c y t e (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 t o 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 o u t e r s u r f a c e of the membrane  (93)  a l l o w s 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 p r o p e r t y has p r o v i d e d  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 a c t i o n s of a n t i a r r h y t h m i c s on b o t h the i n t a c t and membrane of the The  isolated  cell  erythrocyte.  experiments i n the p r e s e n t  s t u d i e s have shown t h a t 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 d e s c r i b e d  f o r l i n o l e n o y l s o r b i t o l (141)  phenomenon ( F i g u r e 15, T a b l e V I I ) . q u i n i d i n e , and QX  572  i s not a  general  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 ,  on e r y t h r o c y t e 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 , s m a l l 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 c o n c e n t r a t i o n of drug p r o d u c i n g versus  i n t a c t c e l l s were noted.  50% i n h i b i t i o n (K  These d i f f e r e n c e s may  i n membrane a r c h i t e c t u r e produced d u r i n g h y p o t o n i c Expression corresponding  v a l u e ) i n membranes a r i s e from  lysis  (148-149).  of the enzyme i n h i b i t i o n by a n t i a r r h y t h m i c s antihemolytic effects  degree of membrane o c c u p a t i o n  alterations  r e l a t i v e to t h e i r  ( F i g u r e 14) i l l u s t r a t e d t h a t f o r a g i v e n  by drug ( 5 4 ) , 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 v a r y w i t h the s t r u c t u r e of the agent i n q u e s t i o n .  The  acetyl-  Page  107  c h o l i n e s t e r a s e of e r y t h r o c y t e membrane has been shown to be a l i p o p r o t e i n (143) whose p r 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  s t u d i e s w i t h TNBS p r e s e n t e d here as w e l l as work  by G o d i n e t a l (52) have shown t h a t a n t i a r r h y t h m i c s a b i l i t y to p e r t u r b  d i f f e r markedly i n t h e i r  e r y t h r o c y t e 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 groups by the amino-group probe, TNBS. t h i s regard  and  B r e t y l i u m was  phospholipid  minimally  t h i s f i n d i n g , when t a k e n w i t h the pronounced and  amino  disruptive in virtually  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 ( T a b l e V I I ) may  indicate  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 q u a t e r n a r y m o l e c u l e on the enzyme, poss i b l e at the a n i o n i c s i t e n o r m a l l y  i n v o l v e d i n the b i n d i n g o f 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 m o l e c u l e . e f f e c t s of the other  antiarrhythmics  I t would seem l i k e l y t h a t the  on membrane a c e t y l c h o l i n e s t e r a s e  diverse activity  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 t o i n t e r a c t w i t h m o d i f y p r o t e i n and p h o s p h o l i p i d ther experimentation  s t r u c t u r a l components of the membrane.  w i l l be r e q u i r e d b e f o r e a d e t a i l e d a n a l y s i s of  m o l e c u l a r b a s i s 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  and Fur-  the  experiments  are  i m p o r t a n t i n e m p h a s i z i n g t h a t l i p i d - s o l u b l e a m p h i p h a t i c s u b s t a n c e s , which have g e n e r a l l y been assumed t o p r o t e c t e r y t h r o c y t e s  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 ( 5 4 ) , not e x e r t i d e n t i c a l p e r t u r b a t i o n a l a c t i o n s on the e r y t h r o c y t e membrane. these f i n d i n g s i n e r y t h r o c y t e s  do  While  suggest a c o r r e s p o n d i n g degree of c o m p l e x i t y  in  Page 108  the d i r e c t membrane a c t i o n s o f a n t i a r r h y t h m i c s o l a t i o n of r e s u l t s obtained w i t h erythrocytes  on m y o c a r d i a l  tissues, extrap-  t o 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 u n d e r t a k e n w i t h c o n s i d e r a b l e  caution.  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 o f a n t i a r r h y t h m i c s  This  on the  K -NPPase a c t i v i t y o f e r y t h r o c y t e and h e a r t membranes ( F i g u r e 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  coefficients for cholinesterase  i n h i b i t i o n noted f o r some drugs when e r y t h r o c y t e and s y n a p t i c membrane a t i o n s were compared d i r e c t l y ( T a b l e V I I ) .  S i n c e membrane p a r t i t i o n  prepar-  coeffic-  i e n t s f o r drugs i n e r y t h r o c y t e s and synaptosomal membranes are g e n e r a l l y equivalent  (74) and the m o l e c u l a r  properties of cholinesterase i n erythrocytes  comparable t o those o f the enzyme d e r i v e d from e x c i t a b l e t i s s u e s ( 1 5 2 ) ,  these  d i f f e r e n c e s p o i n t t o the importance o f 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  t h e f u n c t i o n a l consequences o f 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 o f i n h i b i t i o n o f a c e t y l c h o l i n e s t e r a s e e x e r t e d by the a n t i a r r h y t h m i c s was e x p l o r e d , a type o f mixed i n h i b i t i o n , w h e r e i n b o t h enzymes u b s t r a t e 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 o b s e r v e d .  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 o f 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 as were apparent i n our e x p e r i m e n t s i n v o l v i n g TNBS and DTNB. P r a c t o l o l was the o n l y drug which showed a n o n - c o m p e t i t i v e type o f inhibition.  I t i s p o s s i b l e t h a t 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 f e a t u r e s which determine i t s unique 8 - b l o c k i n g a c t i o n s which a r e a s s o c i a t e d w i t h m i n i m a l d i r e c t c a r d i o d e p r e s s a n t  properties  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 e r y t h r o c y t e membranes d e s c r i b e d p r e v i o u s l y . E r y t h r o c y t e s have been shown t o 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 u s i n g f l u o r e s c e n t c y a n i n e dyes whose d i s t r i b u t i o n a c r o s s t h e membrane i s p o t e n t i a l - s e n s i t i v e (153) o r by means o f d i r e c t m i c r o e l e c t r o d e the case o f salamander g i a n t r e d c e l l s (154). magnitude  measurements i n  I n human e r y t h r o c y t e s t h e  o f t h i s p o t e n t i a l , which i s l a r g e l y d e t e r m i n e d 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 a p p r o x i m a t e l y -9 mV ( 1 5 3 ) , has been shown t o govern t h e i n h i b i t i o n o f e r y t h r o c y t e 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 p r e s e n t s t u d i e s have shown t h a t 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 o f the c h l o r i d e g r a d i e n t a c r o s s the e r y t h r o c y t e membrane ( F i g u r e 17).  On the other hand, t h e i n h i b i t o r y e f f e c t s o f q u i n i d i n e  and p r o p r a n o l o l were enhanced chloride gradient.  i n the presence o f an i n c r e a s e d  transmembrane  A l t h o u g h i t i s known t h a t t h e i n t e r a c t i o n o f p r o p r a n o l o l  w i t h i n t a c t e r y t h r o c y t e s causes an i n c r e a s e i n the membrane p e r m e a b i l i t y t o K  +  ( 8 7 ) , an a c t i o n which might f u r t h e r m a g n i f y the p o t e n t i a l d i f f e r e n c e s  a c r o s s the membrane o f e r y t h r o c y t e s 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 t o be shared by q u i n i d i n e w h i c h , r a t h e r , tends t o decrease membrane p e r m e a b i l i t y t o K  +  (132,155).  The g r e a t e r  inhibitory  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 o f 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 o f 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). 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