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Isolation and phosphorylation of guinea pig cardiac sarcolemma Hui, Chi Wai 1976

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ISOLATION AND PHOSPHORYLATION OF GUINEA PIG CARDIAC SARCOLEMMA by Hui Chi Wai A.B., University of C a l i f o r n i a , L.A., 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of PHARMACOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1976 © Hui Chi Wai, 1976 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t ten pe rm i ss i on . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 Date i ABSTRACT Plasma membranes were prepared from guinea pig heart by homogenizing the tissue i n a Polytron homogenizer, followed by four cycles of washing and d i f f e r e n t i a l c e n t r i -fugation using low g forces (380 x g/10 minutes to 120 x g/10 minutes). The crude p a r t i c l e s thus obtained were extracted with KC1 (1.25 M) , followed by isopycnic centrifugation i n a discontinuous sucrose gradient. Adenylate cyclase was p u r i f i e d 10-15 f o l d over the whole homogenate with a spec-i f i c a c t i v i t y of 3.6±0.72 nmoles/mg/minute. Ouabain-sensitive Mg + +-dependent Na++K+-ATPase, another plasma membrane specif-i c enzyme, was enriched by 4 fo l d , with a s p e c i f i c a c t i v i t y of 107+8.2 nmoles/mg/minute. Cytochrome C oxidase, an enzyme predominantly of mitochondrial o r i g i n , was recovered i n low y i e l d . These membrane "marker" enzyme studies indicated that the i s o l a t e d membrane preparation consisted of highly p u r i f i e d plasma membranes. Functional studies with the cardiac sarcolemma indicated that an ATP-dependent C a + + binding system as well as Ca + +-dependent ATPases were present. I n t r i n s i c protein kinase a c t i v i t i e s and membrane-bound substrates for phosphor-y l a t i o n were also found to be associated with these membranes, which were phosphorylated by endogenous or added protein kinase. Membrane p h o s p h o r y l a t i o n was s t i m u l a t e d by c y c l i c AMP (1 jjM) and was r e v e r s e d by a membrane-bound phosphoprotein phosphatase, i n d i c a t i n g the presence o f a p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n system i n c a r d i a c sarcolemma. Plasma membranes, when phosphor-y l a t e d , were capable of accumulating twice as much C a + + as: c o n t r o l p r e p a r a t i o n s . For each nanomole of phosphate i n c o r p -o r a t e d i n t o the membrane, there were 15 nanomoles of net i n c -rease i n C a + + uptake. These data are c o n s i s t e n t with the p o s s i b i l i t y t h a t c y c l i c AMP may f a c i l i t a t e C a + + movement i n t o the c a r d i a c c e l l v i a a p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n mechanism. i i i TABLE OF CONTENTS Page PART I: CARDIAC SARCOLEMMA - ISOLATION AND CHARACTERIZATION A. Introduction 1 B. Experimental Procedures 6 a. Materials 6 b. Methods 6 1. Adenylate cyclase assay 6 2. Enzyme assays - ATPase assay 8 3. Other enzyme assays 8 C. Results 9 1. Membrane Preparations from guinea pig. heart 9 2. Characterization of cardiac sarcolemma by "marker" enzymes 2 2 D. Discussion 29 PART I I : CARDIAC SARCOLEMMA - PHOSPHORYLATION AND CALCIUM UPTAKE A. Introduction 34 B. Experimental Procedures 4 9 a. Materials 49 b. Methods 1. Isolation of cardiac sarcolemma 49 2. Calcium binding and uptake 50 3. Phosphorylation of sarcolemma 50 C. Results 53 1. Calcium binding and uptake i n 53 c a r d i a c sarcolemma 2. Ca -stimulated ATPase of cardiac sarcolemma 53 3. Phosphorylation of sarcolemma by exogenous protein kinase 56 4. Endogenous protein kinase and s e l f -phosphorylation of cardiac sarcolemma 59 5. C a + + accumulation by phosphorylated cardiac sarcolemma 64 6. R e v e r s i b i l i t y of membrane phosphoryl-ation and d i s t r i b u t i o n of phospho-protein phosphatase 72 D. Discussion 79 REFERENCES 86 i v LIST OF TABLES Page I Effectiveness of various s a l t s i n i s o l a t i o n of cardiac sarcolemma 16 II E f f e c t s of d i t h i o t h r e i t o l (DTT) on the a c t i v i t y and y i e l d of adenylate cyclase during i s o l a -t i o n of cardiac sarcolemma 18 III E f f e c t s of d i f f e r e n t buffers and the number of washes on removal of protein from heart homogenate 20 IV D i s t r i b u t i o n and a c t i v i t i e s of marker enzymes i n various fractions obtained during i s o l a t i o n of cardiac sarcolemma 24 V D i s t r i b u t i o n and a c t i v i t i e s of 5' Nucleotidase i n fractions obtained during i s o l a t i o n of cardiac sarcolemma 28 VI Calcium binding and uptake a c t i v i t i e s of cardiac sarcolemma 54 VII Ca— +-stimulated ATPase a c t i v i t i e s of cardiac sarcolemma 55 VIII E f f e c t of d i f f e r e n t preincubation conditions on C a + + uptake by cardiac sarcolemma 65 IX D i s t r i b u t i o n of phosphoprotein phosphatase i n fractions obtained during i s o l a t i o n of cardiac sarcolemma 78 V LIST OF FIGURES Page 1. Scheme o f i s o l a t i o n o f plasma membranes from g u i n e a p i g h e a r t s 13 2. D i s t r i b u t i o n o f enzyme a c t i v i t i e s i n s u c r o s e g r a d i e n t f r a c t i o n s 27 3. P h o s p h o r y l a t i o n o f c a r d i a c sarcolemma by p u r i f i e d p r o t e i n k i n a s e 58 4. H i s t o n e p h o s p h o r y l a t i o n by c a r d i a c sarcolemma 61 5. S e l f - p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma by endogenous p r o t e i n k i n a s e 63 6. E f f e c t o f c y c l i c AMP and p r o t e i n k i n a s e on c a l c i u m uptake by c a r d i a c sarcolemma 68 7. E f f e c t o f C a C ^ . o n p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma by exogenous p r o t e i n k i n a s e 71 8. R e l a t i o n s h i p between p h o s p h o r y l a t i o n and s t i m u l a t i o n o f c a l c i u m uptake by c a r d i a c sarcolemma 74 9. P h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma 76 v i A B B R E V I A T I O N S A T P a d e n o s i n e 5 1 - t r i p h o s p h a t e c y c l i c A M P a d e n o s i n e 3', 5 ' - c y c l i c m o n o p h o s p h a t e 5' A M P a d e n o s i n e 5 ' - m o n o p h o s p h a t e 3 ' - A M P a d e n o s i n e 3 * - m o n o p h o s p h a t e E D T A e t h y l e n e d i a m i n e t e t r a a c e t a t e E G T A e t h y l e n e g l y c o l b i s ( B - a m i n o e t h y l e t h e r ) N , N ' - t e t r a a c e t a t e T r i s t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e T C A t r i c h l o r o a c e t i c a c i d D T T d i t h i o t h r e i t o l v i i ACKNOWLEDGEMENTS I wish to thank P r o f e s s o r George I. Drummond f o r h i s guidance d u r i n g the course of t h i s work. I am p a r t i c u l a r l y g r a t e f u l to him f o r encouraging me to engage i n a combined med-i c a l and graduate s t u d i e s program. I wish t o thank my wife f o r her p a t i e n c e and support d u r i n g my long years of U n i v e r s i t y e d u c a t i o n , and f o r t y p i n g the o r i g i n a l manus-c r i p t o f t h i s t h e s i s . The f i n a n c i a l a s s i s t a n c e by the Canadian Heart Foundation i n the form of a Me d i c a l S c i e n t i s t F e l l o w s h i p i s hereby acknowledged. PART I CARDIAC SARCOLEMMA - ISOLATION AND CHARACTERIZATION INTRODUCTION Plasma membranes of mammalian c e l l s can be viewed as an assembly o f d i v e r s e , but s p e c i f i c a l l y and g e n e t i c a l l y r e g u l -ated f u n c t i o n a l systems arr a y e d a t the c e l l p e r i p h e r y perform-in g i n t r i c a t e and c r u c i a l b i o l o g i c a l r o l e s . F u nctions now r e -cognized t o be mediated by the plasma membrane i n c l u d e : (1 ) r e g u l a t i o n o f c e l l u l a r p e r m e a b i l i t y and t r a n s p o r t o f smal l ions and numerous b i o - o r g a n i c substances i n v o l v i n g s p e c i f i c en-zymes and " c a r r i e r mechanisms" ( 1 , 2 ) ; ( 2 ) communication between the c e l l and i t s environment by c e l l s u r f a c e r e c e p t o r s f o r hor-mones, a n t i b o d i e s and other r e g u l a t o r y agents; and ( 3 ) v i a membrane-bound enzymes, s y n t h e s i z e r e g u l a t o r y molecules a c t i n g both e x t r a c e l l u l a r l y as w e l l as i n t r a c e l l u l a r l y . In order to understand how the plasma membrane per-forms these complex f u n c t i o n s , they must f i r s t be i s o l a t e d . While the aim of most procedures f o r the i s o l a t i o n of a p a r t i c u -l a r c e l l component i s to o b t a i n t h a t component in. i t s most n a t i v e form, t h i s i s c l e a r l y not p o s s i b l e i n the case of plasma memb-ranes s i n c e these must f i r s t be d i s r u p t e d i n order t o remove c e l l c o ntents. A p r e p a r a t i o n r e p r e s e n t i n g i n t a c t membranes i s the c e l l "ghosts" o b t a i n e d from e r y t h r o c y t e s ( 3 ) . T h i s c e l l has no nucleus and so one does not have to d i s r u p t the membrane ex-t e n s i v e l y to remove i n t r a c e l l u l a r o r g a n e l l e s . Furthermore, i t - 2 -i s d e v o i d o f e n d o p l a s m i c r e t i c u l u m , w h i c h f r e q u e n t l y c o m p l i ^ c a t e s t h e p r e p a r a t i o n o f s u r f a c e membranes from more complex c e l l s . A l t h o u g h t h e r e a r e undoubted advantages i n u s i n g r e d c e l l " g h o s t s " f o r s t u d y i n g membrane f u n c t i o n s , i t s u f f e r s t h e d i s t i n c t d i s a d v a n t a g e o f b e i n g a membrane p r e p a r a t i o n d e r i v e d from a c e l l s i m p l i f i e d f o r s p e c i a l i z e d f u n c t i o n s . The f i r s t u s e f u l method f o r t h e i s o l a t i o n o f a mam-m a l i a n plasma membrane f r a c t i o n from a s o l i d t i s s u e was t h a t d e s c r i b e d by N e v i l l e f o r l i v e r c e l l s (4, 5 ) . The p r o c e d u r e employed d i f f e r e n t i a l c e n t r i f u g a t i o n s o f t h e l i v e r homogenate i n a h y p o t o n i c b u f f e r f o l l o w e d by i s o p y c n i c c e n t r i f u g a t i o n i n s u c r o s e g r a d i e n t s . The N e v i l l e method, l a t e r m o d i f i e d by Emmelot e t a l (6) has been adapted f o r t h e i s o l a t i o n o f plasma membranes from hepatomas ( 7 ) , k i d n e y ( 8 ) , i n t e s t i n a l e p i t h e -l i u m ( 9 ) , t h y r o i d ( 1 0 ) , He La c e l l s (11, 12) , b o v i n e mammary g l a n d ( 1 3 ) , and a n t e r i o r p i t u i t a r y (14). W h i l e N e v i l l e ' s method may be adapted t o a number o f s o f t t i s s u e s , i t s a p p l i c a t i o n t o s t r i a t e d muscles has n o t been s u c c e s s f u l . M u s c l e s a r e h i g h l y s p e c i a l i z e d t i s s u e s w i t h a major p r o p o r t i o n o f the c e l l mass made up o f t h e c o n t r a c t i l e a p p a r a t u s . M y o f i b r i l s a r e bound t o g e t h e r by what was d e f i n e d by P e t e r s as a " c y t o s k e l e t o n " l o c a t e d w i t h i n elements o f t h e s a r c o p l a s m i c r e t i c u l u m and o f t h e Z membrane system w h i c h form complex i n t e r l o c k i n g networks (15). E l i m i n a t i o n o f the myo-f i b r i l s can be a c c o m p l i s h e d by b r e a k i n g t h i s c y t o s k e l e t o n w i t h v a r i o u s c h e m i c a l and p h y s i c a l t e c h n i q u e s . Kono and C o l o w i c k (16) - 3 -were f i r s t to describe a procedure for the i s o l a t i o n of sarco-lemma from rat s k e l e t a l muscle. The method involved extraction of c e l l fragments with 1.0 M KC1 solution to remove the contrac-t i l e proteins, and the tubular membranes were separated from other c e l l p a r t i c l e s by d i f f e r e n t i a l centrifugation i n a 25% KBr solution. This method has been modified by Severson et a l (17) who used 0.4 M LiBr instead of 1.0 M KC1 to extract cont-r a c t i l e proteins. Membranes thus i s o l a t e d contained no s t r i a -tions and appeared as empty, transparent s a c - l i k e structures. Adenylate cyclase, generally accepted as the best plasma mem-brane "marker" enzyme, was enriched approximately 15-fold over the whole homogenate with a y i e l d of a c t i v i t y of about 30%. Other procedures reported i n the l i t e r a t u r e for p u r i f i c a t i o n of s k e l e t a l sarcolemma employed one or several of the following: "ageing" of the muscle c e l l segments, washing i n various s a l t solutions i n concentrations ranging from millimolar to molar l e v e l s , or incubation at 37°C within a limited pH range, followed by d i f f e r e n t i a l centrifugation to y i e l d sarcolemmal tubules or sheets (18-21). More recently, techniques were des-cribed to obtain, instead of sac-like sarcolemma, plasma mem-branes i n the form of v e s i c l e s . Plasma membranes were separ-ated from myofibrils by strong s a l t solutions or by f i l t r a t i o n . The f i n a l p u r i f i c a t i o n was obtained by centrifugation on sucrose density gradients (22-24). Attempts to i s o l a t e plasma membranes from cardiac muscle are less numerous. Portius and Repke (25) were the f i r s t - 4 -to apply the methods developed for the i s o l a t i o n of sarcolemma from s k e l e t a l muscle (16, 18) to prepare plasma membranes from the myocardium. The sarcolemmal preparation obtained had an 2+ + .+. active Mg -dependent Na +K -ATPase, but electron microscopy revealed s i g n i f i c a n t amounts of extraneous material. Stam et a l (26) described a cardiac plasma membrane preparation i n which the crude homogenate was extracted up to 16 hours in,1 M potassium ..iodide. Although s t r i a t i o n s were apparently re-moved from these cardiac f i b e r s a f t e r such treatment, i t was clear that the use of such powerful structure-disrupting s a l t s had destroyed many important membrane functions (27). Katz et a l (2 7) and Kidwai et a l (28) t r i e d to avoid using high con-centrations of s a l t , and employed density gradient ultracent-r i f u g a t i o n to subfractionate a microsomal preparation of the heart into sarcoplasmic reticulum and plasma membrane frac t i o n s , but the y i e l d of plasma membrane was extremely low and cross contamination by other i n t r a c e l l u l a r membranes was s i g n i f i c a n t . More recently, Tada et a l (2 9) attempted to i s o l a t e plasma membranes from guinea pig heart by exposing i s o l a t e d c e l l seg-ments to osmotic shock, followed by extraction of actomyosin i n 1 M KC1. These preparations contained as much as 15% of the protein i n the o r i g i n a l homogenate, and when examined by phase contrast microscopy, many s t r i a t i o n s were seen. Furthermore, 2+ + + both adenylate cyclase and Mg -dependent Na' + K -ATPase . were enriched only 2-3-fold over the orig n a l homogenate, in d i c a t i n g that the p u r i f i c a t i o n was far from complete. - 5 -I t i s c l e a r t h a t i n order to i s o l a t e c a r d i a c plasma o membranes, the f o l l o w i n g must be achieved: (1) e x t r a c t i o n of c o n t r a c t i l e p r o t e i n s , which or g a n i z e themselves f i r m l y i n the cytoplasm as a complex i n t e r l o c k i n g network; (2) removal of m i t o c h o n d r i a , which are abundant and deeply embedded i n the myofibrils;, ; ; (3) s e p a r a t i o n o f plasma membranes from other mem-branes of i n t r a c e l l u l a r o r i g i n ; and (4) p r e s e r v a t i o n of the f u n c t i o n a l i n t e g r i t y of the plasma membrane. Most attempts c i t e d above e i t h e r used harsh procedures t o achieve e x t r a c t i o n o f c o n t r a c t i l e p r o t e i n s a t the expense of the i n t e g r i t y of the plasma membrane, or too g e n t l e methods y i e l d i n g sarcolemmal p r e p a r a t i o n s contaminated by l a r g e amounts of i n t r a c e l l u l a r m a t e r i a l s . As a r e s u l t , f u n c t i o n a l s t u d i e s w i t h these memb-rane p r e p a r a t i o n s were e i t h e r d i f f i c u l t t o c a r r y out, or l i m i t e d t o i s o l a t e d a s p e c t s o f membrane a c t i v i t y which s u r v i v e d the i s o l a t i o n procedure. The aim of t h i s p r o j e c t was t o d e v e l o p a b e t t e r method f o r the i s o l a t i o n of c a r d i a c sarcolemma i n order to study i t s f u n c t i o n i n a manner t h a t i s p h y s i o l o g i -c a l l y meaningful. For example, an i d e a l c a r d i a c sarcolemmal p r e p a r a t i o n would a l l o w one not o n l y t o i n v e s t i g a t e how hormones a c t i v a t e the plasma membrane, but a l s o t o t r a c e the m e t a b o l i c events t h a t may subsequently take p l a c e which u l t i m a t e l y l e a d t o changes of p e r m e a b i l i t y and i o n i c movements. In the f i r s t p a r t o f the t h e s i s , a procedure i s presented which employs d i f -f e r e n t i a l c e n t r i f u g a t i o n and i s o p y c n i c c e n t r i f u g a t i o n t o y i e l d a h i g h l y p u r i f i e d plasma membrane f r a c t i o n from guinea p i g - 6 -v e n t r i c l e s . Some res u l t s i n d i c a t i n g the l i m i t a t i o n s of various s a l t s and agents used i n i s o l a t i o n of plasma membranes w i l l also be presented, and the concept of plasma membrane "marker" en-zyme w i l l be b r i e f l y discussed. EXPERIMENTAL PROCEDURES A. M a t e r i a l s C y c l i c 3[H]AMP (22 C i per mmole) , 4 5 C a C l 2 (20 mCi per 14 r T mg) and uniformly l a b e l l e d [Cj ATP (400mCi per mmole) were ob-tained from New England Nuclear. Ethanol was removed from the solution under vacuum and the radioactive materials were d i l u t e d with water to the desired concentration and stored at -20°C. Unlabelled nucleotides were obtained from Calbiochem and Sigma. Pyruvate Kinase (from rabbit muscle) and 2-phosphoenol pyruvate (trisodium s a l t ) , cytochrome C, d i t h i o t h r e i t o l and protein kinase i n h i b i t o r were obtained from Sigma. Ouabain (strophanthin G) was purchased from Calbiochem, and sucrose (density gradient grade - ribonuclease free) was obtained from Schwarz/Mann. ATP ( t r i s salt) was purchased from Sigma. B. Methods Adenylate cyclase - Assay A; Membrane fractions (150-400 ug protein) were incubated in a medium (t o t a l volume 150 ul) containing 40 mM Tris-HCl, pH 7.5, 8 mM theophylline, 8 mM NaF, 6 mM KC1, 15 mM MgSO., 20 mM phosphoenol pyruvate, 21.7 ug - 7 -pyruvate kinase, and 0.3 mM [c]ATP (20 uCi per pmole). Assay tubes were equilibrated at 37°C and the reaction started by the addition of membrane protein. Incubations were carried out for 10 minutes and were terminated by placing the tubes i n a b o i l i n g water bath for 3 minutes. Control tubes contained water instead of membranes. The tubes were centrifuged at 8,000 x g for 10 minutes to remove denatured protein, and 100 u l of the super-natant was streaked over 2 cm on Whatman 3 MM chromatography paper. Descending paper chromatograms were developed for 18-22 hours at room temperature using 1 M ammonium acetate: 95% ethanol (15:35) as solvent. After drying, the area corresponding to c y c l i c AMP was v i s u a l i z e d under u l t r a v i o l e t l i g h t , cut out and placed i n 18 ml of s c i n t i l l a t i o n f l u i d (4 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis-2-(5-phenyloxazole)-benzene dissolved i n 1 l i t e r of toluene) and the r a d i o a c t i v i t y was measured i n a Nuclear Chicago S c i n t i l l a t i o n spectrometer. The amount of radioactive c y c l i c AMP formed was calculated from the s p e c i f i c a c t i v i t y of 14 r T the LCJATP substrate aft e r correction for r a d i o a c t i v i t y present i n the c y c l i c AMP area of the control. Assay B: for adenylate cyclase was i d e n t i c a l to assay A except that instead of 0.3 mM 14 r T |_CjATP, 1 mM unlabelled ATP ( t r i s - s a l t ) was used and the cyc-l i c AMP formed was determined according to the s p e c i f i c binding protein method of Gilman (30). Except for r e s u l t s i n Table I, a l l c y c l i c AMP determinations i n t h i s thesis were done according to assay B. - 8 -ATPases - Mg -dependent ATPase a c t i v i t y 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 (150-200 ug) i n a medium ( f i n a l volume 0.2 ml) c o n t a i n i n g 50 mM T r i s - H C l pH 7.5, 5 mM M g C l 2 , 5 mM NaN 3, 0.5 mM EGTA and 3 mM T r i s ATP. Mg 2 +-depend-ent Na ++K +-ATPase was measured by the i n c l u s i o n o f 100 mM NaCl and 10 mM KC1. I n t h e p r e s e n c e o f 0.1 mM o u a b a i n , p a r t o f t h e 2+ + + Mg -dependent Na +K -ATPase a c t i v i t y was i n h i b i t e d . The d i f -f e r e n c e i n the ATPase a c t i v i t y i n t h e pr e s e n c e and absence o f + + 2+ ou a b a i n i s t a k e n as t h e o u a b a i n - s e n s i t i v e Na +K -ATPase. Ca s t i m u l a t e d MgATPase was d e t e r m i n e d i n t h e same medium as f o r MgATPase, e x c e p t t h a t EGTA was o m i t t e d and 50 uM C a C l 2 was i n -c l u d e d . To measure CaATPase, t h e o n l y i o n i c s p e c i e s p r e s e n t i n th e medium was 5 mM C a C l 2 and 0.5 mM EDTA was s u b s t i t u t e d f o r EGTA. The r e a c t i o n was s t a r t e d by a d d i t i o n o f s u b s t r a t e and a f t e r i n c u b a t i o n f o r 5 minutes a t 37°C, t e r m i n a t e d by t h e a d d i -t i o n o f 0.2 ml o f c o l d 12% t r i c h l o r o a c e t i c a c i d . The p r e c i p i -t a t e d p r o t e i n was removed by c e n t r i f u g a t i o n and t h e i n o r g a n i c phosphate c o n t e n t o f t h e s u p e r n a t a n t was d e t e r m i n e d by t h e method o f Ames (31). Under a l l c o n d i t i o n s used, not more than 30% o f the s u b s t r a t e was h y d r o l y z e d . Other a s s a y s : 5 1 - n u c l e o t i d a s e was assayed by i n c u b a t -i n g membrane p r o t e i n (100-150 jug) i n a medium ( f i n a l volume 0.2 ml) c o n t a i n i n g 50 mM 2 - a m i n o - 2 - m e t h y l - l , 3 - p r o p a n e d i o l , pH 9.0, 2.5 mM M g C l 2 , 100 mM KC1 and 8 mM 5'-AMP. A p p r o p r i a t e c o n t r o l s were performed i n w h i c h 5'-AMP was r e p l a c e d w i t h 3'-AMP o r w a t e r . The r e a c t i o n was s t a r t e d by a d d i t i o n o f s u b s t r a t e and a f t e r i n -c u b a t i o n a t 37°C f o r 10 m i n u t e s , t e r m i n a t e d by a d d i t i o n o f 0.2 - 9 -ml cold 12% t r i c h l o r o a c e t i c acid. The pr e c i p i t a t e d protein was removed by centrifugation and the inorganic phosphate content of the supernatant was determined by the method of Ames (31). Cyto-chrome C oxidase a c t i v i t y was determined as described by Cooper-s t e i n and Lazarow (33). Protein determinations were made by the method of Lowry et a l (34), using bovine serum albumin as stand-ard. RESULTS 1. Membrane preparations from guinea pig heart Numerous attempts were made to obtain a preparation of cardiac sarcolemma f u l f i l l i n g a l l the c r i t e r i a l i s t e d above. The f i n a l method s e t t l e d upon w i l l be described f i r s t , followed by more detailed explanation o f each step and o f attempts which proved unsatisfactory. Preparation of washed p a r t i c l e s - Guinea pigs were { k i l l e d by a sharp blow to the head, bled from the neck for 30 seconds to 1 minute, and the hearts were promptly excised and perfused with 10 ml of warm (30°-35°C) Krebs-Ringer bicarbonate solution delivered by syringe. A l l the following procedures were performed at 4°C. A t r i a l tissue, fat and large vessels were removed and discarded; v e n t r i c u l a r tissue was s l i c e d to 4-5 pieces with scissors and weighed. Usually 2.0-2.5 g of tissue from 2 to 3 guinea pigs was used. The tissue was suspended i n 5 volumes ( a l l volumes were based on the o r i g i n a l wet weight - 10 -of the tissue) of hyptonic buffer containing 2 mM d i t h i o t h r e i t o l , 10 mM Tris-HCl, pH 7.5, and homogenized i n a Polytron PT 10 Homo-genizer for 15 seconds at one t h i r d maximal v e l o c i t y , followed by 2 seconds at maximal v e l o c i t y . The homogenate was d i l u t e d by adding 5 volumes of the same buffer, and after passing through a 250 ym nylon mesh under mild suction, centrifuged at 620 x g for 10 minutes. The supernatant f l u i d was removed and discarded. A d i s t i n c t layer, predominantly of mitochondria and c a p i l l a r i e s as determined by phase contrast microscopy, sedimented on the upper surface of the p e l l e t . This layer could be loosened and separated from the p e l l e t by the addition of 1 ml of buffer f o l -lowed by rocking the centrifuge tube gently; the slur r y was then removed with a pasteur pipette. Great care was exercised to leave the p e l l e t undisturbed, which was washed by re-suspending i n 10 volumes of buffer and centrifuged at 380 x g for 10 minutes. This washing procedure was repeated 3 more times with c e n t r i f u -gation at decreasing g r a v i t a t i o n a l forces (successively: 196 x g, 120 x g, and 120 x g, each for 10 minutes), to y i e l d the washed p a r t i c l e s . KCl-extracted p a r t i c l e s - Washed p a r t i c l e s were sus-pended i n 10 volumes of i s o t o n i c buffer containing 2 50 mM suc-rose, 2 mM d i t h i o t h r e i t o l and 10 mM Tris-HCl, pH 7.5 and were homogenized i n a Polytron PT 10 Homogenizer for 30 seconds at maximal v e l o c i t y . These p a r t i c l e s were then subjected to s a l t extraction by adding dropwise an equal volume of a solution containing 2.5 M KC1, 250 mM sucrose, 2 mM d i t h i o t h r e i t o l in 10 mM Tris-HCl, pH 7.5, so that the f i n a l concentration of KC1 was 1.25 M. The solution was gently s t i r r e d for 5 minutes - 11 -and then centrifuged at 100,000 x g for 30 minutes. The pelleted p a r t i c l e s were thoroughly washed with 15 volumes of isotonic buf-fer and centrifuged at 37,000 x g for 15 minutes. Sucrose gradient centrifugation - Discontinuous sucrose gradients containing layers of 5 ml each of 50%, 55%, 60% and 65% sucrose i n 2 mM d i t h i o t h r e i t o l , 10 mM Tris-HCl, pH 8.2 were prep-ared 4 hours before use and stored at 4°C. KCl-extracted p a r t i c l e s obtained from the previous step were homogenized i n 10 ml of a s o l -ution containing 250 mM sucrose, 2 mM d i t h i o t h r e i t o l i n 10 mM T r i s -HCl, pH 8.2, using a Potter-Elvehjem (10 strokes with a l o o s e - f i t -ting pestle, and 10 strokes with a t i g h t - f i t t i n g p e s t l e ) . The sus-pended p a r t i c l e s were layered on the sucrose gradients and c e n t r i -fuged at 40,000 x g for 1 hour i n a SW 25.1 swinging bucket rotor. The material sedimenting at the d i f f e r e n t interfaces was separately c o l l e c t e d with Pasteur pipettes and d i l u t e d 3-fold with a buffer containing 2 mM d i t h i o t h r e i t o l i n 10 mM Tris-HCl, pH 7.5 before being centrifuged at 100,000 x g for 1 hour. The f i n a l p e l l e t s were suspended i n 250 mM sucrose, 2 mM d i t h i o t h r e i t o l i n 10 mM Tris-HCl, pH 7.5, and were used for various studies within 2 hours. A scheme of the procedure i s provided i n Figure 1. Before we arrived at the f i n a l procedure described above, our i n i t i a l approach was to apply the techniques developed by Severson and Drummond for the i s o l a t i o n of s k e l e t a l s a r c o l -emma (17) to cardiac tissues. Adenylate cyclase was used as plasma membrane "marker" enzyme, and i n each step of i s o l a t i o n , phase contrast microscopy was used to check the presence or FIGURE 1 Scheme o f i s o l a t i o n o f plasma membranes from guinea p i g h e a r t s . D e t a i l s of the procedure are d e s c r i b e d i n t e x t under R e s u l t s i n P a r t I o f t h i s t h e s i s . - 13 -Homogenizat ion Polytron PTIO Max. Sp., 2 sec. |GUINEA PIG VENTRICLES J CRUDE H O M O G E N A T E Hypotonic Washings (5x) Centrifuge with decreasing g forces 600g to I20g/I0min. SUPERNATANT containing mito. capillaries etc. DISCARDED Re-homogenization Polytron PTIO Max.Sp., 30 sec. W A S H E D PARTICLES R E - H O M O G E N I Z E D PARTICLES KCI (1.25M) Extraction (5min.) Centrifuge at I O O , O O O g / 3 0 min. H KCI SUPERNATANT containing solubilized protein.DISCARDED. EXTRACTED PARTICLES Sucrose Gradient SUCROSE GRADIENT FRACTIONS SAMPLE' 5 0 % 5 5 % 6 0 % 6 5 % 40 ,000 g I HOUR Fl F2 F3 F4 removal of s t r i a t i o n s . Some of these i n i t i a l attempts and the res u l t s obtained are b r i e f l y described below. Heart s l i c e s were homogenized for 30 seconds i n 15 mM Tris-HGl, pH 7.5 with a S o r v a l l Omnimixer (rheostat set at 120). The homogenate was strained through a nylon seive (pore size 2 1 mm ) to remove connective tissue and centrifuged at 1,000 x g for 10 minutes. The 1,000 x g p e l l e t was washed twice by sus-pending i n 15 volumes of 10 mM Tris-HCl, pH 7.5 and centrifug-ing at 1,000 x g> for 10 minutes to y i e l d the washed p a r t i c l e s . Preparation of lithium-bromide (LiBr) extracted part-i c l e s : Washed p a r t i c l e s obtained from above were suspended i n 15 volumes of 10 mM Tris-HCl, pH 7.5 and LiBr (4.0 M solution) was added dropwise to a f i n a l concentration of 0.4 M. The solu-t i o n , gently s t i r r e d for 45 minutes with a magnetic s t i r r e r , was d i l u t e d with an equal volume of 10 mM Tris-HCl, pH 7.5 and cent-rifuged at 2,000 x g for 10 minutes. The extracted p a r t i c l e s were washed twice by suspending i n 10 mM Tris-HCl, pH 7.5 followed by centrifugation at 2,000 x g for 10 minutes. I n i t i a l r e s u l t s with LiBr extraction were encouraging. As shown i n Table I, 75% of the protein i n the o r i g i n a l homo-genate was removed after LiBr extraction. Adenylate cyclase a c t i v i t y was enriched about 2.7 f o l d over the o r i g i n a l homoge-nate with a y i e l d of 70% of enzyme a c t i v i t y . Cross s t r i a t i o n s were largely removed, but much r e f r a c t i l e material arranged l o n g i t u d i n a l l y along the long axis of the muscle f i b e r s could s t i l l be seen. We therefore attempted to extract further the - 15 -LiBr-extracted p a r t i c l e s . The agents tested included various con-centrations of KCI (0.6 to 3.5 M), 0.5 M NaSCN, 1 M LiBr, 0.5 M NaClG 4, 25% KBr and 1 mM ATP. The LiBr-extracted p a r t i c l e s were thoroughly suspended for about 1 minute i n 15 volumes of 10 mM Tris-HCl pH 7.5 solution containing one of the above-mentioned agents. The suspension was immediately d i l u t e d 3-fold with buf-fer and centrifuged at 7,000 x g for 20 minutes. The membranes thus obtained were washed by suspending i n 10 mM Tris-HCl pH 7.5 followed by centrifugation at 2,000 x g for 10 minutes to y i e l d the f i n a l plasma membrane f r a c t i o n . Results of further treatment of LiBr-extracted part-i c l e s by the various agents mentioned above were generally poor. As shown i n Table I, although the protein content of the f i n a l membrane f r a c t i o n was reduced to 6-8%, there was no concomitant enrichment of adenylate cyclase a c t i v i t y and the f i n a l y i e l d of enzyme a c t i v i t y was 20% or less. Treatment of LiBr-extracted p a r t i c l e s with KCI seemed to be most promising. As the concent-rati o n of KCI was increased from 0.6 to 3.5 M, protein content of the f i n a l membrane f r a c t i o n was progressively reduced, and t h i s was accompanied by an increase i n enrichment of adenylate cyclase to a maximum of 5.3 f o l d with a y i e l d of 28%. These re s u l t s led us to believe that while LiBr was a good protein-s o l u b i l i z i n g agent, and was i t s e l f not d i r e c t l y damaging to ade-nylate cyclase a c t i v i t y , i t disrupted membrane structure i n such a manner that any further treatment of the membrane would lead to extensive damage. Attempts were made to substitute LiBr TABLE I Effectiveness of various s a l t s i n the i s o l a t i o n of cardiac sarcolemma as determined by adenylate cyclase a c t i v i t y i n the various stages of i s o l a -t i o n . Adenylate cyclase a c t i v i t y was measured according to Assay A (see Methods f o r d e t a i l ) and i n the presence of f l u o r i d e . S p e c i f i c a c t i v i t y and recovery of adenylate cyclase and recovery of p r o t e i n were expressed i n percentage r e l a t i v e to the homogenate (taken as 100) to f a c i l i t a t e comparison of d i f f e r e n t agents used to t r e a t LiBr-extracted p a r t i c l e s . Numbers were at l e a s t duplicate preparations i n each case. Adenylate Cyclase Protein Membrane Fractions S p e c i f i c A c t i -v i t y i n percent Recovery i n percent Recovery i n percent (a) Crude Homogenate 100 100 100 (b) Washed P a r t i c l e s 131 81 59 (c) LiBr-extracted P a r t i c l e s 268 70 26 F i n a l Membrane a f t e r ex-t r a c t i o n of LiBr-extracted p a r t i c l e s with (d) 0.6 M KC1 290 39 14 (e) 2.0 M KC1 330 22 6 (f) 3.5 M KC1 534 28 5 .(g) 0.5 M NaSCN 335 20 6 (h) 1.0 M Li B r 255 20 8 (i) 0.5 M NaCIO,, 4 270 19 8 (j) 25% KBr 240 16 7 (k) 1.0 mM ATP 112 11 11 with s a l t s such as Nal or Kl which had been employed by e a r l -i e r workers (26, 35). Unfortunately, re s u l t s with these s a l t s were equally, i f not more disappointing with respect to adeny-late cyclase a c t i v i t y , both i n terms of s p e c i f i c a c t i v i t y and t o t a l recovery. It was soon apparent that due to the unique morpho-logy and i n t e r n a l organization of the myocardial c e l l , extrac-t i o n of i n t r a c e l l u l a r contents, p a r t i c u l a r l y mitochondria and c o n t r a c t i l e proteins, was dependent not only on the choice of chemical agents, but also on the method of homogenization. In t h i s respect, both the S o r v a l l Omnimixer and the Waring Blender were found to be unsuitable for the cardiac tissue because i n order to achieve the degree of disruption necessary for e f f i c i -ent extraction, r e l a t i v e l y long periods (1 minute or longer) of homogenization were required and t h i s was found to be ext-remely damaging to the i n t e g r i t y of the plasma membrane as indicated by the low recovery of "marker" enzymes. With a Poly tron PT 10 Homogenizer, i t was found that, provided the tissue was thoroughly dispersed by preliminary low-speed homogeniza-t i o n , a period of homogenization of exactly 2 seconds was s u f f i cient to e f f e c t i v e l y disrupt the f i b e r s . D i t h i o t h r e i t o l (2 mM) was found to have a "protective" e f f e c t on the membranes since i t improved both the s p e c i f i c a c t i v i t y and the recovery of ade-nylate cyclase a c t i v i t y . The e f f e c t of t h i s agent on adenylate cyclase i s shown i n Table I I . When 2 mM d i t h i o t h r e i t o l was omitted i n the i n i t i a l homogenization, the difference i n the TABLE II Eff e c t s of d i t h i o t h r e i t o l (DTT) on the a c t i v i t y and y i e l d of aden-ylate cyclase during i s o l a t i o n of cardiac sarcolemma. Results were the means with S.E.M. of 3 membrane preparations. Fractions • -DTT +DTT (2 mM) Protein (%) Adenylate Cyclase Protein (%) Adenylate Cyclase nmoles/mg/min %recovery nmoles/mg/min % recovery Crude Homogenate 100 .17 + .02 100 100 .23 + .03 100 Washed P a r t i c l e s 42 '+_ 2 .67 + .08 159 + 15 41 + 3 1.1 + .12 164 + 13 KCl-extracted Membranes 15 + 1 .54 + .12 31 + 16 13 + 1 2.1 + .38 89 + 10 - 19 -s p e c i f i c a c t i v i t y of the whole homogenate was "soticeable (compare 0.23 nmoles/mg/minute with DTT to 0.17 nmoles/mg/minute without DTT). In the washed p a r t i c l e s , the protective e f f e c t of d i t h i o t h r e i t o l was even more s i g n i f i c a n t , as evidenced by the a l -most 2-fold difference i n adenylate cyclase a c t i v i t y . It appeared that the presence of t h i s agent was p a r t i c u l a r l y c r i t i c a l during homogenization, and was less c r u c i a l in the subsequent washing procedures. Its presence was again of c r i t i c a l importance when the washed p a r t i c l e s were homogenized before extraction with KC1 as well as during extraction. As seen i n Table I I , adenylate cyclase a c t i v i t y i n the KCl-extracted p a r t i c l e s previously homo-genized i n the presence of d i t h i o t h r e i t o l was 4 times higher than those homogenized i n the absence of t h i s agent. As w i l l be seen 2 + l a t e r , d i t h i o t h r e i t o l did not appear to "protect" the Mg -dep-endent Na++K+-ATPase whose a c t i v i t y was d r a s t i c a l l y reduced during extraction with KC1. Protein recovery was not affected by d i t h -i o t h r e i t o l . At the concentrations used, th i s reagent did not i n t e r f e r e with the adenylate cyclase assay or the protein binding assay for c y c l i c AMP. The main purpose for subjecting the crude homogenate to several cycles of washings and centrifugations was to remove soluble proteins, endoplasmic reticulum, "exposed" c o n t r a c t i l e proteins and mitochondria etc., and to further break up the "cytoskeleton". Both hypotonic and isot o n i c solutions have been employed by e a r l i e r workers. Results shown i n Table III i n d i c -ate that the effectiveness of either 10 mM Tris-HCl, pH 7.5 or - '20 -TABLE III E f f e c t of d i f f e r e n t buffers and the number of washes on removal of proteins from heart homogenate. Results are means of duplicate preparations and the numbers are percentages of protein remaining i n the p e l l e t r e l a t i v e to the homogenate taken as 100. Number of Washes Buffers Homogenate I II III IV V VI 10 mM Tris-HCl pH 7.5 100 67 53 47 42 39 37 10 mM Tris-HCl 250 mM sucrose 100 67 51 45 43 35 33 pH 7.5 0.1 M NaCIO lOmM Tris-HCl 100 53 46 38 34 31 30 pH 7.5 - 21 -250 mM sucrose i n 10 mM Tris-HCl, pH 7.5 to remove protein from the whole homogenate were i d e n t i c a l . Hypotonic medium had the advantage of lys i n g residual red blood c e l l s that were trapped i n the myocardial c a p i l l a r y beds. NaC104 (0.1 M) was by far the most e f f i c i e n t agent i n removing protein. After the f i r s t cycle of washing and centrifugation with t h i s agent, close to 50% of the protein was removed. But t h i s agent was not used in our f i n a l procedure due to i t s deleterious e f f e c t s on cert a i n enzyme a c t i v i t i e s . Regardless of the buffers used, protein removal tended to l e v e l o f f after 4 washes and further washing tended to decrease the y i e l d of "marker" enzymes without further removing protein. We therefore routinely used 4 cycles of washings and centrifuga-tions. The c e n t r i f u g a l forces chosen for the f i n a l procedure decreased from 620 x g/10 minutes to 120, x g/10 minutes. While i t i s generally accepted that mitochondria w i l l not sediment at 5 a g r a v i t a t i o n a l force less than 10 g-minute (time integrated force), cardiac mitochondria are larger and denser than those from other c e l l types and the decreasing g force was necessary to pre-vent the sedimentation of these p a r t i c l e s . The f i n a l step i n the procedure involved the use of discontinuous sucrose density gradients. The aim here was to comp-lete the separation of plasma membranes from any contaminating subcellular components which escaped the previous extraction procedures. One of the major problems encountered was the tendency of the membrane bands to overlap i n the gradient. In - 2 2/ the i n i t i a l attempts, sucrose gradients were prepared i n 10 mM Tris-HCl, pH 7.5 and 3 bands of membranous material were re-covered from the gradient. The bands were close together and adenylate cyclase and cytochrome C oxidase a c t i v i t i e s were d i s -tributed almost evenly i n a l l 3 bands. With the addition of 1 mM EDTA there was a dramatic improvement i n the resolution of gradient bands both i n terms of t h e i r physical separation on the gradient as well as d i s t r i b u t i o n of "marker" enzymes. The best resolution of gradient bands was found when the sucrose was prepared i n 10 mM Tris-HCl at pH 8.2 instead of pH 7.5. By simply a l t e r i n g the pH of the gradient, 4 d i s t i n c t bands were resolved and as w i l l be seen i n the following section, one of the bands was highly enriched i n plasma membranes. 2. Characterization of cardiac sarcolemma by "marker" enzymes The washed p a r t i c l e s , a f t e r 4 cycles of washing and centrifugations, were found to have as l i t t l e as 40% of the pro-t e i n i n the o r i g i n a l homogenate. The remaining 60% of the pro-t e i n was released i n the supernatant which, i n the main, con-tained c a p i l l a r i e s , residual red blood c e l l s , endoplasmic r e t i -culum, soluble proteins and mitochondria. As much as 50% of the cytochrome C oxidase a c t i v i t y , a mitochondrial "marker" en-zyme, was released into the supernatant by t h i s procedure. On the other hand, adenylate cyclase and ouabain-sensitive Na++K+-ATPase a c t i v i t i e s , enzymes considered to be predominantly l o -c a l i z e d i n plasma membranes (36) , were enriched i n the washed 2 3 -p a r t i c l e s , as indicated by the 4-fold and 2-fold increase i n t h e i r respective s p e c i f i c a c t i v i t i e s (Table IV). When viewed under the phase contrast microscope, these washed p a r t i c l e s were seen to contain some s t r i a t i o n s , i n d i c a t i n g the incomplete re-moval of c o n t r a c t i l e proteins. After homogenization and extrac-t i o n with KCl, another 30% of the protein was removed. There was a further reduction of cytochrome C oxidase a c t i v i t y by 30%. Adenylate cyclase was enriched i n these extracted membranes up to 8-fold, having a s p e c i f i c a c t i v i t y of 2 nmoles/mg/minute, 2+ but a s i g n i f i c a n t portion, as much as 65% of the Mg -dependent + + Na +K -ATPase was l o s t . The s p e c i f i c a c t i v i t y . o f t h i s enzyme was reduced from 56 nmoles/mg/minute i n the washed p a r t i c l e s to 41 nmoles/mg/minute a f t e r extraction. This loss was probably due to i n a c t i v a t i o n of the enzyme by the high s a l t concentration since repeated attempts to recover the a c t i v i t y from the KCl supernatant were not successful. The KCl-extracted p a r t i c l e s , containing 10% of the protein of the o r i g i n a l homogenate, was re-solved by sucrose density gradient centrifugation into 4 d i s t -i n c t bands (Figure 1). The d i s t r i b u t i o n of enzyme a c t i v i t i e s i n these fract i o n s i s shown i n Figure 2. The material i n Band 1 on the top of the gradient was highly enriched i n cytochrome C oxidase (5-fold increase i n s p e c i f i c a c t i v i t y as shown i n Table IV). I t contained 60% of the t o t a l cytochrome C oxidase a c t i -2+ v i t y and only 10% each of the t o t a l adenylate cyclase and Mg -dependent Na++K+-ATPase a c t i v i t i e s recovered from the gradient, i n d i c a t i n g that the material was predominantly of mitochondrial TABLE IV D i s t r i b u t i o n and a c t i v i t i e s of "marker" enzymes i n various f r a c t i o n s obtained during i s o l a t i o n of heart sarcolemma. A l l values were the means with S.E.M. of 6 d i f f e r e n t cardiac membrane preparations Protein Adenylate Cyclase Ouabain-sensitive Cytochrome C Oxidase Fractions ++ Mg (Na++K )-ATPase nmoles cAMP percent nmoles ATP percent nmoles/mg/ percent . . . % formed/mg/ recovery hydrolysed recovery minute recovery minute mg/minute Homogenate 100 .26 + .03 100 26 + 1.6 100 166 ± 17 100 Washed P a r t i c l e s 40 ± 2.8 1.1 + .12 164 ± 13 56 + 3.4 87 ± 6.0 196 ± 5.7 48 ± 10 KCl-extracted P a r t i c l e s 12 ± 1.7 2.1 + .38 89 ± 10 41 + 3.7 19 ± 2.2 308 ± 27 21 ± 3.0 Gradient Band 1 2.3 ± .16 1.3 + .11 11 ± 88 48 + 7.3 3.1± .55 829 ± 124 11 ± 1.4 Gradient Band 2'. 2.1 ± .18 2.8 + .48 22 ± 2.3 98 + 13 5.6 ± 1.5 318 ± 32 4.3 ± .26 Gradient Band 3 2.6 ± .45 3.6 + .72 33 ± 4.0 107 + 8.2 7.5 ± 2.0 164 ± 21 2.6 ± .52 Gradient Band 4 5.1 ± .27 2.2 + .27 44 ± 4.6 60 + 8.9 7.8 ± 1.1 93 ± 14 2.5 ± .35 o r i g i n . The material i n Band 2 which sedimented at the interface between 50-60% sucrose represented a p a r t i a l enrichment of memb-ranes of sarcolemmal o r i g i n as indicated by the increase i n spe-2+ + + c i f i c a c t i v i t i e s of adenylate cyclase and Mg -dependent Na*+K -ATPase. But t h i s f r a c t i o n was also s i g n i f i c a n t l y contaminated by mitochondria since 20% of the cytochrome C oxidase a c t i v i t y recovered from the gradient was located here. Material sedi-mented i n Band 3 at the interface between 55-60% sucrose and i n Band 4 at the interface between 60-65% sucrose contained mem-branes that were highly enriched i n sarcolemma. Adenylate cyc-lase s p e c i f i c a c t i v i t y was increased 14-fold i n Band 3 with a s p e c i f i c a c t i v i t y of 3.6 nmoles/mg/minute, and even the p a r t i -2+ + + a l l y inactivated Mg -dependent Na +K -ATPase was enriched over 4-fold. Together, Bands 3 and 4 contained over 65% of the t o t a l 2+ + + adenylate cyclase and 60% of the t o t a l Mg -dependent Na +K -ATPase a c t i v i t i e s recovered from the gradient. These two fr a c -tions were minimally contaminated by mitochondria. S p e c i f i c a c t i v i t y for cytochrome C oxidase associated with these memb-ranes were low, 164 and 93 nmoles/mg/minute respectively for Bands 3 and 4. Only 2% of the t o t a l cytochrome C oxidase a c t i -v i t y i n the whole homogenate were recovered from each of these f r a c t i o n s . 5' Nucleotidase, commonly used as a plasma memb-rane marker, was found not to be s i g n i f i c a n t l y enriched (1.3-fold) i n sucrose gradient Bands 3 and 4. In fact, as shown i n Table V, the enzyme appeared to associate with a l l the fractions obtained during the i s o l a t i o n of the sarcolemma, in d i c a t i n g that FIGURE 2 D i s t r i b u t i o n o f enzyme a c t i v i t i e s i n sucrose g r a d i e n t f r a c t i o n s . For each enzyme, the t o t a l a c t i v i t y from the g r a d i e n t i s taken as 100%, and the recovery from each g r a d i e n t f r a c t i o n i s expressed as percent of the t o t a l . 60 < O o Sf 40 >-> t— U < mmJ • t— o 50 r -30 h-20 h-10 h" (A) M g + + ( N a + - K + ) ATPase (B) Adenylate Cyclase (C) 5' Nucleotidase (D) -Cytochrome C Oxidase I II III IV I II III IV I II III IV GRADIENT FRACTIONS I II III IV - 2 8 -TABLE V D i s t r i b u t i o n and a c t i v i t i e s of 5'Nucleotidase i n f r a c t i o n s obtained during i s o l a t i o n of cardiac sarcolemma. Protein y i e l d and recovery of enzyme a c t i v i t i e s were expressed as percent r e l a t i v e to the homogenate. A l l values were means with S.E.M. of 6 d i f f e r e n t membrane preparations. Protein 5'Nucleotidase Fractions . Recovery ^ nmoles 5'AMP Recovery '': i n hydrolyzed/ i n Percent mg/minute Percent Homogenate 100 18 + .50 100 Washed P a r t i c l e s 40 + 2.8 21 + .95 47 + 2.2 KCl-extracted P a r t i c l e s 12 + 1.7 18 + 1.3 11 + .80 Gradient Band 1 2.3 + .16 18 + 3.1 2.4 + .21 Gradient Band 2 2.1 + .18 19 + 1.6 2.3 + .19 Gradient Band 3 2.6 +. .45 23 + 2.9 3.1 + .33 Gradient Band 4 5.1 + .27 23 + 2.8 6.4 ± .44 - 29 -the plasma membrane i s not the exclusive source of the enzyme, at least i n the guinea pig myocardium. We have routinely com-bined Bands 3 and 4 as our f i n a l sarcolemmal preparation. Each gram of v e n t r i c u l a r tissue (wet weight) yielded approximately 7-8 mg of membrane protein, providing adequate material for various enzymatic studies. DISCUSSION Although we have used phase contrast microscopy to examine various membrane fractions during the i s o l a t i o n proced-ure, the technique was of limited use i n view of the fact that the membranes assumed vesicular forms aft e r extensive homogen-izat i o n s and thus l o s t t h e i r physical i d e n t i t y . We therefore based our c r i t e r i a of membrane purity on "marker" enzymes. In t h i s respect, we had to r e l y heavily on data published for other tissues to choose the appropriate "marker" enzymes since only very l i t t l e data were available related s p e c i f i c a l l y to cardiac sarcolemma. In rat l i v e r , adenylate cyclase has been shown to associate with the plasma membrane to the exclusion of a l l other subcellular components (37). Marinetti et a l (38) re-ported p u r i f i c a t i o n of the enzyme up to 100-fold i n rat l i v e r plasma membranes. Wolff and Jones prepared plasma membranes from bovine thyroid and found that the enzyme was enriched from 80 to 150-fold over the whole homogenate (39). Enrichment i n adenylate cyclase a c t i v i t y to various degrees has also been demonstrated i n plasma membranes i s o l a t e d from erythrocytes (40), adrenal c e l l s (41), anterior p i t u i t a r y (14) and f a t c e l l s (47). If no enzyme has yet been proven to be an exclusive and univer-s a l enzyme marker of plasma membranes, these data indicate that adenylate cyclase appears to be the closest possible candidate for t h i s q u a l i f i c a t i o n . Results i n Table IV show that t h i s enzyme a c t i v i t y was enriched more than 10-fold i n the present cardiac sarcolemmal preparation. The small amount of adenylate cyclase a c t i v i t y that was found to associate with gradient bands 1 and 2 might represent contaminating plasma membranes i n these fr a c t i o n s , or they could be due to i n t r i n s i c adenylate cyclase a c t i v i t y associated with membranes not of sarcolemmal o r i g i n . Sulakhe et a i (43) and Katz et al (44) have recently provided some i n d i r e c t evidence that a minor portion of the adenylate cyclase a c t i v i t y i n the myocardium may be associated with the microsomal f r a c t i o n . As mentioned above, 2 mM d i t h i o t h r e i t o l was necessary to preserve the adenylate cyclase a c t i v i t y i n the course of the i s o l a t i o n of sarcolemma. This enzyme has been reported to be associated.with cer t a i n e s s e n t i a l sulfhydryl group(s). In pur-i f i e d bovine thyroid plasma membranes, Wolff et a_l (39) found that both TSH- and fl u o r i d e -stimulated adenylate cyclase a c t i -v i t y were equally sens i t i v e to su l f h y d r y l reagents such as N-ethyl-maleimide and p-chloromercuribenzoate. Similar findings were reported by Schramm et a l (45) who studied the adenylate cyclase of rat parotid gland. Strong evidence was also provided - '31 -by s t u d i e s w i t h the s o l u b l e a d e n y l a t e c y c l a s e from S t r e p t o - c o c c u s s a l i v a r i u s . T h i s enzyme has been p u r i f i e d 3 2 0 0 - f o l d and has been shown t o be s t r o n g l y i n h i b i t e d by s u l f h y d r y l r e -ag e n t s . The i n h i b i t i o n can be r e v e r s e d by agents such as c y s t e i n e , m e r c a p t o e t h a n o l o r d i t h i o t h r e i t o l ( 46). I t i s t h e r e -f o r e q u i t e p r o b a b l e t h a t e s s e n t i a l s u l f h y d r y l s a r e a f e a t u r e o f a l l a d e n y l a t e c y c l a s e s . I n t h e i n t a c t t i s s u e , components o f the c e l l membrane may p r o t e c t t h e s e groups t o some e x t e n t , b u t i n t h e c o u r s e o f t i s s u e f r a c t i o n a t i o n , l a b i l i z a t i o n o f membrane s t r u c t u r e s can presumably l e a d t o exposure o f s u l f h y d r y l groups w h i c h , u n l e s s p r o t e c t e d by r e d u c i n g agents such as d i t h i o t h r e i t -o l , can e a s i l y be o x i d i z e d . The p u r i t y o f the p r e s e n t sarcolemmal p r e p a r a t i o n i s a l s o s u p p o r t e d by t h e 4 - f o l d e n r i c h m e n t o f t h e o u a b a i n - s e n s i t -i v e Na ++K +-ATPase a c t i v i t y . T h i s enzyme has been shown t o be s p e c i f i c a l l y a s s o c i a t e d w i t h plasma membranes from s k e l e t a l muscle (17, 22, 23, 47) as w e l l as c a r d i a c muscle (28, 4 8 ) . 2+ A l t h o u g h c o - p u r i f i c a t i o n o f a d e n y l a t e c y c l a s e and Mg -depend-ent Na ++K +-ATPase has been r e p o r t e d i n i s o l a t e d plasma memb-rane fragments from a number o f t i s s u e s , t h e degree o f p u r i f i -c a t i o n f o r the two enzymes was seldom the same. Such d i f f e r -ences have been o b s e r v e d i n membrane p r e p a r a t i o n s from b o v i n e t h y r o i d (39), HeLa c e l l s ( 4 9 ) , l i v e r (50) and a n t e r i o r p i t u i t -2+ a r y ( 1 4 ) . Tada e t a l (29) r e p o r t e d a 2 - f o l d i n c r e a s e in-Mg -dependent Na ++K +-ATPase and a 4 - f o l d i n c r e a s e i n a d e n y l a t e c y c l a s e a c t i v i t i e s i n t h e i r p a r t i a l l y p u r i f i e d c a r d i a c sarccv-- 32 -lemma. That th e s e two enzymes were s i m u l t a n e o u s l y e n r i c h e d t o a much g r e a t e r e x t e n t i n the p r e s e n t sarcolemmal p r e p a r a t i o n 2+ ( 1 0 - f o l d f o r a d e n y l a t e c y c l a s e and 4 - f o l d f o r Mg -dependent Na ++K +-ATPase) emphasized i t s h i g h degree o f p u r i t y . C o n t a m i n a t i o n by m i t o c h o n d r i a was m i n i m a l ; t h i s was i n d i c a t e d by th e low r e c o v e r y o f cytochrome C o x i d a s e a c t i v i t y i n t h e f i n a l sarcolemmal p r e p a r a t i o n , and as w i l l be seen i n a l a t e r s e c t i o n , t h e a z i d e - i n s e n s i t i v e Ca uptake by t h e memb-ra n e s . I n t h e absence o f any a c c e p t e d s p e c i f i c marker enzyme f o r s a r c o p l a s m i c r e t i c u l u m , i t was not p o s s i b l e t o det e r m i n e t h e d i s t r i b u t i o n o f t h i s s u b c e l l u l a r f r a c t i o n . But i n vi e w o f t h e s e r i e s o f washes and low speed c e n t r i f u g a t i o n s employed, contam-i n a t i o n by microsomes i s n ot l i k e l y t o be s i g n i f i c a n t . I n p r e v i o u s s t u d i e s w i t h s k e l e t a l sarcolemma, Sulak h e and Drummond (32) have demonstrated t h e e f f e c t i v e n e s s o f d i f f e r e n t i a l c e n t r i f u g a t i o n a t low g r a v i t a t i o n a l f o r c e s i n s e p a r a t i n g microsomes from sarcolemma. When an exces s o f microsomes was mixed w i t h i s o l a t e d sarcolemma, t h e s e two d i f f e r e n t membrane f r a c t i o n s were c o m p l e t e l y s e p a r a t e d and i n d i v i d u a l l y i d e n t i f i e d a f t e r a one- s t e p c e n t r i f u g a t i o n a t 20,000 g-minute. I n the p r e s e n t p r o c e d u r e , t h e sarcolemmal memb-ranes were d e r i v e d from a 1,200 g-minute p e l l e t . I n c o n c l u s i o n , by c a r e f u l l y s e l e c t i n g t h e c o n d i t i o n s f o r p h y s i c a l d i s r u p t i o n and c h e m i c a l e x t r a c t i o n o f c a r d i a c muscle f i b e r s , and by com b i n i n g d i f f e r e n t i a l and i s o p y c n i c c e n t -r i f u g a t i o n t e c h n i q u e s , a membrane f r a c t i o n h i g h l y e n r i c h e d i n sarcolemma was i s o l a t e d . The p r e p a r a t i o n was r e l a t i v e l y un-c o n t a m i n a t e d by o t h e r c e l l u l a r o r g a n e l l e s as i n d i c a t e d by marker enzyme s t u d i e s ; and most i m p o r t a n t o f a l l , t h e p r e p a r a t i o n con-t a i n e d a good y i e l d o f a d e n y l a t e c y c l a s e and, as w i l l be seen l a t e r , i m p o r t a n t b i o c h e m i c a l p r o c e s s were p r e s e n t a l s o , i n d i c a t -i n g t h a t t h e f u n c t i o n a l i n t e g r i t y o f the sarcolemma was l a r g e l y p r e s e r v e d . PART I I : CARDIAC SARCOLEMMA: PHOSPHORYLATION AND CALCIUM UPTAKE INTRODUCTION Adenosine 3', 5' monophosphate ( c y c l i c AMP) was o r i g -i n a l l y discovered as the i n t r a c e l l u l a r mediator of the glyco-genolytic e f f e c t of epinephrine and glucagon i n the l i v e r ; i t has since been recognized as a 'second messenger"' mediating a variety of hormonal ef f e c t s (51). One of the most notable e f f e c t s of catecholamines on the heart i s t h e i r a b i l i t y to i n -crease c o n t r a c t i l i t y , and much research has been devoted i n the past decade to determine the precise r e l a t i o n s h i p between c y c l i c AMP and the hormone-induced inotropic response. Sutherland and his associates proposed four c r i t e r i a which, i f s a t i s f i e d , would constitute s u f f i c i e n t evidence to implicate c y c l i c AMP as an intermediary i n a given hormone-end organ system: (1) adenylate cyclase, the enzyme that catalyzes the formation of c y c l i c AMP from ATP, should be stimulated by the hormone i n in t a c t tissue; (2) a simi l a r e f f e c t should be ob-served i n broken c e l l preparations of the same tissue; (3) en-hanced end organ a c t i v i t y should be observed i n the presence of in h i b i t o r s of 3',5' c y c l i c nucleotide phosphodiesterase, the enzyme responsible for the breakdown of c y c l i c AMP, and (4) c y c l i c AMP should have the a b i l i t y to produce the end organ response d i r e c t l y . In the case of hormone-induced inotropism, i t may be - 35 -added t h a t there should be a proper temporal r e l a t i o n s h i p bet-ween i n t r a c e l l u l a r l e v e l s o f c y c l i c AMP and the mechanical response of the hea r t . The f i r s t and second c r i t e r i a to i m p l i c a t e c y c l i c AMP as a mediator i n the i n o t r o p i c response of c a r d i a c t i s s u e to catecholamines were s a t i s f i e d when i t was shown t h a t the administ-r a t i o n o f e p i n e p h r i n e i n c r e a s e d c y c l i c AMP l e v e l s i n i s o l a t e d , p e r f u s e d h e a r t s (52-54) as w e l l as i n h e a r t s i n v i v o (55). In-creases i n t i s s u e l e v e l s o f c y c l i c AMP i n response to epine-p h r i n e preceded the i n c r e a s e i n c o n t r a c t i l e f o r c e (52, 56, 57). Be t a - a d r e n e r g i c b l o c k i n g agents such as p r o n e t h a l o l or d i c h l o r o -i s o p r o t e r e n o l , which prevented the i n c r e a s e i n c y c l i c AMP, a l s o a b o l i s h e d the i n o t r o p i c response (52, 55). C y c l i c AMP l e v e l s i n h e a r t s l i c e s (58, 59) were a l s o increased.by i n c u b a t i o n w i t h r catecholamines, and the i n c r e a s e was prevented by p r o p a n o l o l (59). The observations i n i s o l a t e d i n t a c t t i s s u e s were confirmed by s i m i l a r s t u d i e s u s i n g p a r t i c u l a t e p r e p a r a t i o n s from r a t (60), c a t (61) and dog (62, 63) h e a r t s . B e t a - a d r e n e r g i c b l o c k i n g agents prevented the a c t i v a t i o n of adenylate c y c l a s e by c a t e -cholamines i n a l l these t i s s u e s . Furthermore, catecholamines s t i m u l a t e d adenylate c y c l a s e i n these p a r t i c u l a t e c e l l f r a c t i o n s q u a n t i t a t i v e l y i n the order of t h e i r i n o t r o p i c potency i n v i v o (60-62). P o s i t i v e support f o r the t h i r d c r i t e r i a i m p l i c a t i n g c y c l i c AMP as a mediator i n the i n o t r o p i c response was pr o v i d e d by R a i l and West who noted a g r e a t l y enhanced i n o t r o p i c e f f e c t - 36 -of n o r e p i n e p h r i n e i n the presence o f t h e o p h y l l i n e , a phospho-d i e s t e r a s e i n h i b i t o r (64). In was subsequently demonstrated t h a t c y c l i c AMP accumulation was w e l l c o r r e l a t e d w i t h t h i s augmented p o s i t i v e response a f t e r t h e o p h y l l i n e treatment (65, 66). Antagonism o f the methylxanthines by imidazole' , a potent stimulant o f phosphodiesterase, p r o v i d e d f u r t h e r evidence f o r c y c l i c AMP mediation (67) . The f o u r t h and l a s t c r i t e r i a to .be s a t i s f i e d i n order to e s t a b l i s h a c a u s e - a n d - e f f e c t r e l a t i o n s h i p between c y c l i c AMP and the p o s i t i v e i n o t r o p i c response was found to be more d i f -f i c u l t to e s t a b l i s h because of the low myocardial p e r m e a b i l i t y to c y c l i c AMP. U n t i l the d i b u t y r y l d e r i v a t i v e of c y c l i c AMP became a v a i l a b l e , success i n the use of exogenous c y c l i c AMP to reproduce the a c t i o n of hormone was l i m i t e d to one r e p o r t by Levine e t a l (68) who observed an i n c r e a s e d c a r d i a c output and h e a r t r a t e a f t e r i n t r a c a r d i a c a d m i n i s t r a t i o n of high doses of c y c l i c AMP i n u n a n e s t h e t i z e d dogs. The i n t r o d u c t i o n of the d i b u t y r y l d e r i v a t i v e of c y c l i c AMP, an analog o f c y c l i c AMP t h a t i s more r e f r a c t o r y to the degrading a c t i o n of phosphodiesterase and more l i p i d s o l u b l e than c y c l i c AMP i t s e l f allowed the i n v e s t -i g a t i o n of the e f f e c t o f t h i s agent on c a r d i a c c o n t r a c t i l i t y . S k e l t o n e t a l (69) demonstrated t h a t d i b u t y r y l c y c l i c AMP ex-h i b i t e d a p o s i t i v e i n o t r o p i c e f f e c t on i s o l a t e d c a t p a p i l l a r y muscle d r i v e n e l e c t r i c a l l y , and these responses were not a l t e r e d by p r o p a n o l o l . Kukovetz and Poch (70) showed t h a t d i b u t y r y l c y c l i c AMP, as w e l l as i t s d i h e x a n o y l d e r i v a t i v e , produced - 37 -increases i n rate and amplitude of contractions measured i n hearts of rats, guinea pigs and rabbits. Drummond and Hemmings (71) also provided evidence that t h i s c y c l i c AMP analog has actions on the beating rat heart q u a n t i t a t i v e l y s i m i l a r to those of adrenergic amines. Their data also indicated that cyc-l i c AMP, formed by a deacylation reaction, was i n fact the act-ive component i n the action of di b u t y r y l c y c l i c AMP. These studies, which have s a t i s f i e d a l l the four c r i t -e r i a , provide very strong evidence for a cause-and-effect re-lationship between c y c l i c AMP and the inotropic action of cate-cholamines. However, several experiments reported i n the l i t -erature have indicated that such an association i s not indisput-able. For example, Benfey and his associates (72, 73) observed that while dopamine, phenylephrine and norepinephrine increased c o n t r a c t i l i t y , only norepinephrine stimulated adenylate cyclase and increased c y c l i c AMP l e v e l s . More recently, the same authors demonstrated that phenoxybenzamine prevented the inotropic ef-fect of 5-hydroxytryptamine, but did not i n h i b i t the r i s e i n c y c l i c AMP accumulation. A single i n j e c t i o n of reserpine 24 hours before the experiment did not i n h i b i t the inotropic e f f e c t of 5-hydroxytryptamine but prevented the r i s e i n c y c l i c AMP formation (74). These authors concluded that cardiac cont-r a c t i l i t y may be increased without a r i s e i n c y c l i c AMP form-ation and, conversely, c y c l i c AMP accumulation need not be accompanied by a r i s e i n c o n t r a c t i l i t y . Shanfeld e_t a l (75) , using i s o l a t e d perfused rat hearts, reported that at low doses of catecholamines, s i g n i f i c a n t increases i n c o n t r a c t i l e tension - 38 -could be demonstrated without measurable increase i n c y c l i c AMP lev e l s . Furthermore, they demonstrated that the blocking agent isopropylmethoxamine abolished the r i s e i n c y c l i c AMP without a f f e c t i n g the increased c o n t r a c t i l i t y produced by norepinephrine. I t was concluded that under proper conditions, increased cardiac c o n t r a c t i l i t y produced by catecholamines could take place with-out changes i n c y c l i c AMP. In contrast, Wastila et a l (76) found that a sim i l a r blocking agent, N-tertiary-butyl-methoxamine, when infused into open chest dogs, produced a dose dependent decrease i n norepinephrine-induced c o n t r a c t i l i t y , c y c l i c AMP levels and phosphorylase a l e v e l s , i n d i c a t i n g that increase i n c y c l i c AMP level s could not be dissociated from the c o n t r a c t i l e response. S i m i l a r l y , the support gained from the use of methyl-xanthines has been seriously challenged because these agents have t h e i r own i n t r i n s i c actions on cardiac tissue which may account for effects attributed to c y c l i c AMP. For example, theophylline i s known to a l t e r i n t r a c e l l u l a r calcium binding by the sarcoplasmic reticulum (77) , mitochondrial accumulation of calcium (78) as well as membrane transport of calcium (79). Under these circumstances, the increased c o n t r a c t i l i t y observed may be due to elevation of free i n t r a c e l l u l a r calcium lev e l s rather than a l t e r a t i o n s i n cardiac c y c l i c AMP l e v e l . The issue i s further complicated by the fact that methylxanthines have been shown to release catecholamines from both the brain (80) and, heart (81). Theophylline has also been observed to exert a pos i t i v e inotropic e f f e c t at concentrations i n s u f f i c i e n t to i n h i b i t phosphodiesterase (82). Thus, ef f e c t s of these agents which appear to mimic actions of catecholamines might be due to catecholamine l i b e r a t i o n or a l t e r a t i o n of calcium l e v e l s or some other yet unspecified mechanisms rather than f a c i l i t a t i o n of i n t r a c e l l u l a r accumulation of c y c l i c AMP v i a i n h i b i t i o n of phosphodiesterase. In view of these considerations, several investigators have adopted d i f f e r e n t approaches whereby i n t r a c e l l u l a r c y c l i c AMP may be elevated by mechanisms that bypass the adrenergic re-ceptor. Dimethylsulfoxide (DMSO) i s an agent that f a c i l i t a t e s the d i f f u s i o n of. a wide variety of compounds into c e l l s . Kjekshus et a l (83) studied changes i n cardiac c o n t r a c t i l i t y during per-fusion of isola t e d hearts with c y c l i c AMP i n the presence of DMSO. Transformation of phosphorylase b to phosphorylase a was used as an index of the entry of c y c l i c AMP. Under these condit-ions, both c y c l i c AMP and i t s d i b u t y r y l analog were e f f e c t i v e i n transforming phosphorylase b to a. However, there was no change in myocardial c o n t r a c t i l i t y despite the fact that the hearts r e t -ained normal inotropic response to epinephrine. Thus th i s study indicated that although c y c l i c AMP enters the c e l l , i t does not influence cardiac mechanics under those circumstances. Another study car r i e d by Langlet and 0ye (84) showed that nucleotide entrance into myocardial c e l l s i s f a c i l i t a t e d by perfusing r a t hearts at low temperatures. At 16°C, both epinephrine and c y c l i c AMP enter the c e l l . However, only epinephrine exhibits positive inotropic and chronotropic e f f e c t s , suggesting that when c y c l i c AMP enters the c e l l and achieves a concentration s u f f i c i e n t to - 40 -transform phosphorylase b to phosphoylase a, i t does not augment c o n t r a c t i l i t y i n preparations capable of responding mechanically as well as metabolically to epinephrine. While an all-encompassing explanation for these contro-v e r s i a l data i s not yet available, i t has been suggested by some authors (85) that there might e x i s t i n the c e l l a l o c a l i z e d and c r i t i c a l i n t r a c e l l u l a r pool of c y c l i c AMP which influences cont-r a c t i l i t y when i t i s augmented under physiological circumstances i n response to catecholamines. Recently, some very ex c i t i n g studies have been reported by Tsien and his colleagues (86) who examined the e l e c t r i c a l e f f e c t s of iontophoretic i n j e c t i o n of c y c l i c AMP (concentration used estimated to be 0.6 mM) i n spontaneously active Purkinje f i b e r s . Compared to control experiments, f i b e r s injected with c y c l i c AMP showed increased frequency of spontaneous a c t i v i t y as a r e s u l t of a steeper pacemaker depolarization and a shortened plateau phase i n the action p o t e n t i a l . These effects of c y c l i c AMP were reproducible and f u l l y r e v e r s i b l e . On the other hand, iontophoretic i n j e c t i o n of 5'-AMP produced no change in the action potential or pacemaker depolarization. These re-sul t s provide d i r e c t support for the involvement of c y c l i c AMP i n the e l e c t r i c a l e f f e c t s of epinephrine i n the heart. Recently Brooker (87) studied the concentration of c y c l i c AMP within each myocardial contraction cycle and showed that myocardial c y c l i c AMP - 41 -c o n c e n t r a t i o n s o s c i l l a t e d u r i n g each c o n t r a c t i o n with peak con-c e n t r a t i o n s of c y c l i c AMP p r e c e d i n g peak development of s y s t o l i c t e n s i o n . Furthermore, t h i s o s c i l l a t i o n i s a l t e r e d i n the p r e s -ence o f e p i n e p h r i n e which i n c r e a s e d both d i a s t o l i c and s y s t o l i c c o n c e n t r a t i o n s of the c y c l i c n u c l e o t i d e as w e l l as f o r c e of cont-r a c t i o n . Wollenberger e t a l (150) conducted s i m i l a r s t u d i e s i n f r o g h e a r t v e n t r i c l e s and observed t h a t c y c l i c AMP. l e v e l s rose by 70% and c y c l i c GMP l e v e l s f e l l by 90% d u r i n g a p e r i o d of the c a r d i a c c y c l e corresponding to the l a t e n c y phase of c o n t r a c t i o n and e a r l y phase of mechanical s y s t o l e . A t the time j u s t a f t e r the v e n t r i c l e s had begun to r e l a x , both n u c l e o t i d e s r e t u r n e d to the p r e c e d i n g d i a s t o l i c l e v e l s . These t r a n s i e n t changes i n myo-c a r d i a l c y c l i c AMP and c y c l i c GMP suggest a p o t e n t i a l r o l e f o r the n u c l e o t i d e s as beat-to-beat r e g u l a t o r s o f myocardial c o n t r a c t -i l i t y . Although, c y c l i c AMP "has been proved or presumed to mediate a l a r g e number of p h y s i o l o g i c a l processes, r e l a t i v e l y l i t t l e i s known about the mechanism whereby t h i s n u c l e o t i d e a c t s . Evidence i s accumulating t h a t c y c l i c AMP e x e r t s i t s a c t i o n v i a a c t i v a t i o n of yet another enzyme: - t h e p r o t e i n k i n a s e ( s ) (88-91). The concept o f c y c l i c AMP-dependent p r o t e i n k i n a s e as a c l a s s o f r e g u l a t o r y enzymes grew out of the f i n d i n g s l a r g e l y of Krebs and h i s a s s o c i a t e s i n t h e i r e l u c i d a t i o n of the cascade of events i n v o l v e d i n the r e g u l a t i o n of g l y c o g e n o l y s i s by.epinephrine i n muscle. These authors showed t h a t c y c l i c AMP, formed as a r e s u l t of s t i m u l a t i o n of adenylate c y c l a s e by e p i n e p h r i n e , a c c e l e r a t e d - 42 -the rate of phosphorylation and ac t i v a t i o n of phosphorylase kinase. The l a t t e r enzyme then catalyzed the conversion of phosphorylase b to phosphorylase a, and i n the presence of calcium, led to glyco-gen breakdown. The nature of the phosphorylase kinase ac t i v a t i o n reaction was not clear u n t i l these authors found that c y c l i c AMP was i n t e r a c t i n g with another enzyme, a phosphorylase kinase kinase (92, 93). This kinase was i s o l a t e d from s k e l e t a l muscle and found to catalyze the ATP-dependent phosphorylation and a c t i v a t i o n of phosphorylase b kinase i n the presence of extremely low concentra-tions of c y c l i c AMP. Since the kinase was also capable of phosphor-y l a t i n g other proteins such as casein and protamine, i t was referred to as protein kinase (92). Based on studies ca r r i e d out using beef heart protein kinase (94), i t was found that the enzyme i s composed of a regulatory (R) and a c a t a l y t i c (C) subunit. The binding of the regulatory subunit to the c a t a l y t i c component res u l t s i n an e s s e n t i a l l y inactive holoenzyme (RC). C y c l i c AMP promotes dissoc-i a t i o n to y i e l d a regulatory subunit-cyclic AMP complex and a free enzymatically active, c a t a l y t i c subunit: RC + c y c l i c AMP . i R-cAMP + C (inactive) (active) The role of c y c l i c AMP-dependent protein kinase i n the mediation of hormonal action was expanded by observations made by Kuo and Greengard (95) of the widespread d i s t r i b u t i o n of t h i s enzyme i n various mammalian tissues and among d i f f e r e n t species including the bacterium Escherichia C o l l (96). This led to the - 43 -hypothesis (96, 97) that a l l physiological e f f e c t s of c y c l i c AMP are due to tne action of this enzyme. This concept embodied Sutherland's o r i g i n a l second messenger hypothesis, and extended the chain of events to include a protein phosphorylation step i n a sequence as follows: Hormonal or s i m i l a r signal » c y c l i c AMP production > protein kinase a c t i v a t i o n -» phosphorylation of functional protein(s) > a c t i v a t i o n or i n a c t i v a t i o n of functional protein(s) > physiological events The protein(s) that i s phosphorylated could be an enzyme, a nuc-lear protein, a membrane component or any other.protein involved at a c r i t i c a l control s i t e . For example, Soderling et aJL (98) and Schlender et a l (99) observed that c y c l i c AMP-dependent prot-ein kinase catalyzed the conversion of glycogen synthetase I to the D form. S i m i l a r l y , Corbin et a l (100) and Huttenen et a l (101) established that hormonal regulation of l i p o l y s i s . i n adipocytes could be ascribed to a c y c l i c AMP-dependent, protein kinase-mediated a c t i v a t i o n of t r i g l y c e r i d e l i p a s e . Phosphorylation of basic nuclear proteins by a c y c l i c AMP-dependent protein kinase was demonstrated by Langan (102) who subsequently showed, i n a highly s i g n i f i c a n t study, that histone phosphorylation occurs i n vivo i n response to c y c l i c AMP stimulation. Other physiological events involving c y c l i c AMP-dependent protein phosphorylation include the f a c i l i t o r y e f f e c t of protamine phosphorylation upon - 44 -i t s t r a n s p o r t from the c y t o p l a s m i c ribosomes t o . c h r o m a t i n -• (103); t h e i n s u l i n - a n d p r o l a c t i n - d e p e n d e n t p h o s p h o r y l a t i o n o f n u c l e a r plasma membrane and r i b o s o m a l p r o t e i n s d u r i n g mammary g l a n d d i f f e r e n t i a t i o n i n v i t r o (104, 105); t h e p h o s p h o r y l a t i o n o f r e t i c u l o c y t e ribosomes and i t s r o l e i n t r a n s l a t i o n a l r e g u l a t -o r y mechanisms (106); t h e p h o s p h o r y l a t i o n o f b o v i n e a d r e n a l p r o t -e i n s and r e g u l a t i o n o f s t e r o i d o g e n e s i s (107); r e g u l a t i o n o f n e u r o n a l f u n c t i o n s t h r o u g h p h o s p h o r y l a t i o n o f membrane fragments i s o l a t e d from ox (89) and r a t (90) b r a i n s ; t h e p o s s i b l e r o l e o f c a t i o n t r a n s p o r t i n t h e p h o s p h o r y l a t i o n o f e r y t h r o c y t e membrane (91, 109); p h o s p h o r y l a t i o n o f b r a i n t u b u l i n and i t s r o l e i n t h e r e g u l a t i o n o f n e u r o s e c r e t i o n (110); and t h e p h o s p h o r y l a t i o n o f t r o p o n i n and i t s p o s s i b l e e f f e c t on muscle c o n t r a c t i o n (111). The c y c l i c AMP-dependent p r o t e i n k i n a s e o r i g i n a l l y found i n r a b b i t muscle i s a s o l u b l e enzyme and i s l o c a t e d p r i m a r i l y i n t h e c y t o s o l (92). A s i m i l a r d i s t r i b u t i o n was found i n beef h e a r t muscle (94) and i n t h e l a c t a t i n g mammary g l a n d (112). I n r a t l i v e r , Chen and Walsh (113) found t h a t 89% o f t h e enzyme i s l o c a t e d i n t h e c y t o s o l , 6% i n t h e n u c l e i , 4% i n the microsomes and l e s s t h a n .1% i n t h e m i t o c h o n d r i a . I n c o n t r a s t t o what has been found i n s k e l e t a l muscle and l i v e r , Maeno e t a l found t h a t the c y c l i c AMP-dependent p r o t e i n k i n a s e from b r a i n was b o t h s o l -u b l e and p a r t i c u l a t e (88). P a r t i c u l a t e f r a c t i o n s from w h i c h p r o t e i n k i n a s e o f h i g h s p e c i f i c a c t i v i t y c o u l d be s o l u b i l i z e d i n c l u d e d t h e microsomes, synaptosomes, s y n a p t i c v e s i c l e s and s y n a p t i c membranes. F u r t h e r m o r e , . t h e s u b c e l l u l a r d i s t r i b u t i o n o f - 45 -p r o t e i n k i n a s e a c t i v i t y was c l o s e l y s i m i l a r t o t h a t o f a d e n y l a t e c y c l a s e and p h o s p h o d i e s t e r a s e . Both l a t t e r enzymes have been found t o be r i c h i n f r a c t i o n s c o n t a i n i n g microsomes and n erve e n d i n g s . These s t u d i e s i n d i c a t e t h a t c y c l i c AMP-dependent p r o t -e i n k i n a s e a c t i v i t y may be an i n t e g r a l p a r t o f membranes and r a i s e the p o s s i b i l i t y t h a t p h y s i o l o g i c a l phenomenon such as c e l l u l a r p e r m e a b i l i t y , s e c r e t o r y p r o c e s s e s and s y n a p t i c t r a n s m i s s i o n may be c o n t r o l l e d by a h i g h l y c o m p a r t m e n t a l i z e d system i n w h i c h en-zymes f o r t h e s y n t h e s i s and d e g r a d a t i o n o f t h e c y c l i c n u c l e o t i d e a r e found i n c l o s e p r o x i m i t y t o t h e enzyme r e s p o n s i b l e f o r i t s b i o l o g i c a l a c t i v i t y . Membrane-associated p r o t e i n k i n a s e s r e g u l a t -i n g endogenous p h o s p h o r y l a t i o n o f membrane s u b s t r a t e s have s i n c e been r e p o r t e d t o o c c u r i n s y n a p t i c v e s i c l e s (88-90), . e r y t h r o c y t e ghosts (91, 109), adenohypophyseal g r a n u l e s (114) and membranes (115), r e n a l m e d u l l a r y plasma membranes (116), c a r d i a c s a r c o -p l a s m i c r e t i c u l u m (117, 118) as w e l l as sarcolemma from s k e l e t a l muscle (119). I t i s now g e n e r a l l y a c c e p t e d t h a t c a t e c h o l a m i n e s , i n a d d i t i o n . t o i n c r e a s i n g c y c l i c AMP l e v e l s i n c a r d i a c - t i s s u e , a l s o i n c r e a s e s t h e transmembrane C a + + i n f l u x ; . t h e l a t t e r phenomenon has been demonstrated by a number o f e l e c t r o p h y s i o l o g i c a l and t r a c e r s t u d i e s (120-122). Thus, i n c r e a s e i n . t h e . i n t r a c e l l u l a r l e v e l s o f c y c l i c AMP and the transmembrane C a + + i n f l u x appear t o be c o n c u r r e n t e f f e c t s o f c a t e c h o l a m i n e s on m y o c a r d i a l c e l l s . However, the n a t u r e o f t h e r e l a t i o n s h i p between c y c l i c AMP ac-c u m u l a t i o n and C a + + movement remains c o n t r o v e r s i a l . Two d i f f e r e n t - 46 -models have been proposed: i n the f i r s t model, stimulation of beta-adrenergic.receptors by catecholamines could simultaneously but independently cause an a c t i v a t i o n of adenylate cyclase and an a l t e r a t i o n . o f sarcolemmal permeability to C a + + , so that c y c l i c AMP and C a + + both acted as second messengers. In the second model, stimulation of the beta-adrenergic receptor led to forma-t i o n of c y c l i c AMP, which i n turn caused a change i n C a + + perme-a b i l i t y of the membrane. In t h i s second type of i n t e r a c t i o n , c y c l i c AMP would function as the second messenger and C a + + would serve as the t h i r d messenger. Entman et aJL (123) studied the effects of epinephrine and glucagon on C a + + accumulation by a microsomal f r a c t i o n of canine myocardium.. They reported that each of these agents produced a concentration-dependent increase i n C a + + accumulation, and t h e i r e f f e c t s could be mimicked by using exogenous c y c l i c AMP. Furthermore, since, adenylate cyclase was present i n the microsomal preparation used, they observed that propanolol not only abolished the production.of c y c l i c AMP but also the accumulation of C a + + . Bloom and Sweat (151) studied the covariance of myocardial c y c l i c AMP and C a + + during beta-adrenergic stimulation i n vivo and noted a p a r a l l e l increase i n myocardial C a + + and c y c l i c AMP aft e r intraperitoneal injection, of isoprotere-nol i n rats. The maximum response for both C a + + and c y c l i c AMP was observed aft e r two minutes and the c o r r e l a t i o n c o e f f i c i e n t for the two responses was highly s i g n i f i c a n t (0.974). These f i n d -ings are consistent with the concept that the inotropic e f f e c t s of catecholamines occur as a r e s u l t of an accumulation of C a + + - 47 -mediated by c y c l i c AMP. R e c e n t l y Watanabe and Besch (124) s t u d i e d t h e r e l a t i o n s h i p between c e l l u l a r l e v e l s o f c y c l i c AMP and the slow i n w a r d C a + + c u r r e n t i n i s o l a t e d p e r f u s e d g u i n e a p i g h e a r t s i n w h i c h the f a s t N a + c h a n n e l s had been i n a c t i v a t e d e i t h e r by d e p o l a r i z a t i o n w i t h K + or b l o c k a d e w i t h t e t r o . d o t o x i n . They found t h a t e x c i t a b i l i t y and c o n t r a c t i l i t y c o u l d be r e s t o r e d t o t h e s e h e a r t s w i t h a v a r i e t y o f i n o t r o p i c agents w h i c h a l s o r a i s e d the l e v e l s o f c y c l i c AMP. A r i s e i n c y c l i c AMP l e v e l s was o b s e r v e d t o p r ecede r e s t o r a t i o n o f e x c i t a b i l i t y i n d e p o l a r -i z e d h e a r t s , and t h e magnitude o f t e n s i o n d e v e l o p e d by r e s t o r e d h e a r t s was d i r e c t l y r e l a t e d t o t h e e x t e r n a l C a + + c o n c e n t r a t i o n as w e l l as the c o n c e n t r a t i o n o f the i n o t r o p i c agent used. F u r t h e r -more, a h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was found between t h e magnitude o f C a + + - d e p e n d e n t t e n s i o n development and the l e v e l o f c y c l i c AMP i n t h e s e h e a r t s . U s i n g s p e c i f i c b l o c k a d e of s a r -colemmal C a + + c h a n n e l s w i t h Dggg o r verapamil., i t was shown t h a t the t e n s i o n . d e v e l o p m e n t by r e s t o r e d h e a r t s was a b o l i s h e d , a l -though the r i s e i n c y c l i c AMP l e v e l s i n r e s p o n s e t o hormone s t i m -u l a t i o n was n o t a t t e n u a t e d . These f i n d i n g s s u g g e s t e d t h a t c y c l i c AMP c o u l d a c t i v a t e slow C a + + c h a n n e l s i n t h e h e a r t and agreed w i t h t h o s e o f T s i e n who showed t h a t t h e i o n f l u x e s i n t h e p l a t e a u phase o f t h e a c t i o n p o t e n t i a l i n c a r d i a c p u r k i n j e f i b e r s were s e n s i t i v e t o c y c l i c AMP (125) and t h o s e o f M e i n e r t z . e t a l (126) who demonstrated t h a t d i b u t y r y l c y c l i c AMP, l i k e e p i n e p h r i n e , i n c r e a s e d c o n t r a c t i l e f o r c e and C a + + uptake by t h e b e a t i n g r a t a t r i a . These o b s e r v a t i o n s s uggest s t r o n g l y a c a u s a t i v e i n t e r -a c t i o n between augmentation o f c y c l i c AMP l e v e l s and i n c r e a s e - 48 -i n t r a n s a r c o l e m m a l C a + + i n f l u x , r e s u l t i n g i n a n e t i n c r e a s e i n c e l l u l a r C a + + l e v e l s w h i c h i n t u r n l e a d s t o a p o s i t i v e . i n o t r o p i c r esponse. I n v i e w o f the e v i d e n c e t h a t c y c l i c AMP c o n t r o l s many p h y s i o l o g i c a l p r o c e s s e s v i a t h e a c t i v a t i o n o f p r o t e i n k i n a s e , i t i s c o n c e i v a b l e t h a t t h e s t i m u l a t i o n o f C a + + movements by c y c l i c AMP may a l s o be mediated by membrane p h o s p h o r y l a t i o n . R e c e n t l y , K a t z e t a l (127, 140, 147-149) showed t h a t c a r d i a c microsomes, c o n s i s t i n g p r i m a r i l y o f s a r c o p l a s m i c r e t i c u l u m , c o u l d be phos-p h o r y l a t e d by p r o t e i n k i n a s e i n t h e p r e s e n c e o f c y c l i c AMP o r e p i n e p h r i n e . Membranes thus p h o s p h o r y l a t e d showed a marked en-hancement o f C a + + uptake and C a + + - a c t i v a t e d ATPase a c t i v i t y , and t h e n e t s t i m u l a t i o n o f C a + + uptake p a r a l l e l e d t h e amount o f p h o s p h o p r o t e i n f o r m a t i o n . I t was f u r t h e r shown t h a t t h e phos-p h o r y l a t i o n o c c u r r e d a t a membrane component o f low m o l e c u l a r w e i g h t (22,000 d a l t o n ) which was s e p a r a b l e from t h e C a + + t r a n s -p o r t p r o t e i n (100,000), but when p h o s p h o r y l a t e d c o u l d r e g u l a t e C a + + t r a n s p o r t by the c a r d i a c s a r c o p l a s m i c r e t i c u l u m . L a R a i a and M o r k i n (118) a l s o succeeded i n d e m o n s t r a t i n g i n c r e a s e d C a + + uptake by a c a r d i a c m i c r o s o m a l p r e p a r a t i o n a f t e r p h o s p h o r y l a t i o n by an endogenous p r o t e i n k i n a s e . These d a t a suggest v e r y s t r o n g l y t h a t c a t e c h o l a m i n e s can i n f l u e n c e i n t r a c e l l u l a r C a + + movements v i a membrane p h o s p h o r y l a t i o n a t the l e v e l o f the s a r c o p l a s m i c r e t i c u l u m , and may a c c o u n t f o r some o f the e f f e c t s o f t h e s e i n o t r o p i c agents on the c a r d i a c t i s s u e . But s i n c e t h e p o s i t i v e i n o t r o p i c r e s ponse has been c l e a r l y shown t o be r e l a t e d t o an increase of Ga i n f l u x across the plasma membrane (the slow i n -ward current), i t i s our purpose to investigate whether membrane phosphorylation catalyzed by c y c l i c AMP-dependent protein kinase also take place i n the plasma membrane. In t h i s connection, Sulakhe and Drummond (128), using a highly p u r i f i e d s k e l e t a l sar-colemmal preparation, showed that the membranes could be phos-phorylated by protein kinase and that the phosphorylated memb-ranes could accumulate more C a + + than control preparations. In the following section, data w i l l be presented to demonstrate that there i s an in t e r a c t i o n between c y c l i c AMP and C a + + v i a protein kinase-dependent phosphorylation at the l e v e l of the cardiac sarcolemma, and these interactions may provide a mechan-ism for the p o s i t i v e inotropic e f f e c t s of catecholamines, gluc-agon and other s i m i l a r agents. EXPERIMENTAL PROCEDURES A. Materials £ Y ~ 3 2Pj ATP (10 to 20 Ci per mmole) was obtained from New England Nuclear. Protein kinase (beef heart) and h i s -tone (calf thymus IIA) were purchased from Sigma. Other chemi-cals were obtained from si m i l a r sources as indicated i n the previous section. B. Methods Isol a t i o n of cardiac sarcolemma - was carried as des-cribed i n Part I of t h i s thesis. Unless indicated other-- 50 -wise, membranes were used for various.studies within 2 hours of i s o l a t i o n . Calcium binding and uptake - membrane protein (200-300 ug) was incubated i n a medium ( f i n a l volume 1.0 ml) containing 50 mM Tris-maleate, pH 6.0, 5 mM MgCl 2, 2 mM Tris-ATP, and 100 uM CaC l 2 (8,000-12,000 cpm/nmole). The reaction was started by addition of ATP, and afte r incubation for various times at 30°, terminated by f i l t e r i n g a 0.5 ml aliquot through a M i l l i p o r e f i l t e r (0.45 u, 25 mm) under l i g h t suction. The f i l t e r was washed with 5 ml of 100 mM Tris-maleate, pH 6.0, dried at 70° i n a s c i n t i l l a t i o n v i a l , and r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n spectrometry. Appropriate controls containing no ATP, no MgCl 2 or no protein were included. Ca binding was 45 calculated from the s p e c i f i c a c t i v i t y of the added CaCl 2, and the r a d i o a c t i v i t y retained by the membrane protein. In the ab-sence of membrane protein, retention of r a d i o a c t i v i t y by the f i l t e r was n e g l i g i b l e (32). Measurement of Ca + +.uptake was sim-i l a r to the above, except that 5 mM potassium oxalate was i n -cluded i n the medium. Phosphorylation of sarcolemma - membrane protein (100-200 ug) was incubated i n a medium (final.volume 0.2 ml) containing 50 mM sodium acetate, pH 6.0, 10 mM MgCl 2, 10 mM NaF, 2 mM theo-p h y l l i n e , 0.5 mM EGTA, 25 uM [ Y - 3 2 P ] A T P (500-2,000 cpm per picomole), 1 uM c y c l i c AMP, with or without bovine heart protein - 51 -k i n a s e (10-100 ug). P h o s p h o r y l a t i o n o f sarcolemma i n t h e absence o f added p r o t e i n k i n a s e i s due.to a u t o p h o s p h o r y l a t i o n by endogen-ous membrane-bound p r o t e i n k i n a s e . These v a l u e s were always sub-t r a c t e d from.those o b t a i n e d . i n t h e p r e s e n c e o f added enzyme. The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f ATP and a f t e r i n c u b a t i o n f o r v a r i o u s t i m e s a t 30°, was t e r m i n a t e d by a d d i t i o n o f 4.0 ml o f c o l d 10% t r i c h l o r o a c e t i c acid.. B o v i n e serum a l b u m i n . (0.2 ml o f a 1% s o l u t i o n ) was added as a p r o t e i n c a r r i e r . The t u b e s were k e p t i n i c e f o r a t l e a s t 10 m i n u t es b e f o r e c e n t r i f u g a t i o n . The s u p e r -n a t a n t was c a r e f u l l y a s p i r a t e d and the p e l l e t was washed 3 t i m e s w i t h 10% c o l d t r i c h l o r o a c e t i c a c i d . The f i n a l p e l l e t was d i s -s o l v e d i n 0.1 ml o f 1 N NaOH, d i l u t e d w i t h 0.2 ml w a t e r , an a l i q u o t o f t h e sample was d i s s o l v e d i n 20 ml o f Aquasbr^, and r a d i o a c t i v i t y was d e t e r m i n e d by s c i n t i l l a t i o n s p e c t r o m e t r y . 32 32 P r e p a r a t i o n o f P - h i s t o n e and B-sarcolemma. as s u b s t r a t e s f o r p h o s p h o p r o t e i n phosphatase - 3.34 mg o f h i s t o n e ( c a l f thymus II A ) o r 0.5 mg o f h e a t - t r e a t e d (100° f o r 15 m inutes) sarcolemma was i n c u b a t e d a t 30° f o r 60 minutes i n a medium ( f i n a l volume 1 ml) c o n t a i n i n g 50 mM sodium a c e t a t e , pH 6.0, 10 mM MgC^, 0.5 mM EGTA, 5 mM NaF, 2 mM t h e o p h y l l i n e , 1 uM c y c l i c AMP, 240 ug p u r i f i e d p r o t e i n k i n a s e , 2 5 uM ATP (500 mCi per mmole). The r e a c t i o n was s t a r t e d by a d d i t i o n o f r a d i o a c t i v e ATP and s t o pped by a d d i t i o n o f 6 ml o f 20% t r i c h l o r o a c e t i c a c i d . The t u b e s were c e n t r i f u g e d a t 7,000 x g f o r 15 minutes and t h e p e l l e t was r e d i s s o l v e d i n 0.2 ml I N NaOH f o l l o w e d by p r e c i p i t a t i o n - 52 -with 4 ml of,20% t r i e h l o r o a c e t i c acid-and centrifugation at 7,000 x. g for 15 minutes. This washing procedure was repeated twice and the residue was suspended i n water and dialysed against water (histone) or 10 mM Tris-HCl, (sarcolemma) pH 7.5, for 24 hours at room temperature. Assay of Phosphoprotein Phosphatase - Various.muscle f r a c t -ions (40-60 ug protein) were incubated at 30° i n a medium ( f i n a l volume 0.2 ml) containing 50 mM Tris-HCl, pH 7.5, 1 mM d i t h i o -t h r e i t o l and [ 3 2 P ] -labeled histone or sarcolemma. The reaction was started by the addition of substrate.and. stopped after 10 minutes by the addition of 4 ml of 20% cold t r i c h l o r o a c e t i c acid. To each tube, 0.2 ml of 1% albumin solution was added and the tubes were kept at 4° for 15 minutes before being centrifuged at 7,000 x g for 15 minutes. The p e l l e t was washed once by d i s -solving i n 0.1 ml of 1 N NaOH, followed by p r e c i p i t a t i o n with cold t r i c h l o r o a c e t i c acid and centrifugation. The f i n a l p e l l e t was dissolved i n 0.1 ml of 1 N NaOH, 0.2 ml water added and r a d i o a c t i v i t y of a 2 00 p i aliquot was determined i n 15 ml of Aquasol** s c i n t i l l a t i o n solution. Controls were carried with heat-denatured (100°C for 30 minutes) extracts or without added protein. Phosphoprotein phosphatase a c t i v i t y was calculated from the d i f -ference i n r a d i o a c t i v i t y between the experimentals and controls. RESULTS 1. C a + + binding and uptake i n cardiac sarcolemma In view of the s i g n i f i c a n t role of the plasma membrane in the regulation of ion transport, we studied the C a + + binding and uptake a c t i v i t i e s of cardiac sarcolemma. Calcium uptake i s defined as the amount of C a + + accumulated by membranes i n the presence of oxalate, a C a + + - p r e c i p i t a t i n g agent that diffuses i n and out of membrane v e s i c l e s , whereas calcium binding i s the amount of C a + + accumulated i n the absence of oxalate. As shown in Table VI, these membranes.bound a small amount of C a + + i n the absence of ATP, but binding was stimulated 5-fold when ATP (2 mM) was added. The binding process appeared to be a rapid one, since maximum binding was reached at 1 minute. The maximal amount of Ca bound .remained rather constant for up.to 5 minutes. In the presence of oxalate, C a + + uptake was almost l i n e a r up to 5 minutes of incubation. Since muscle mitochontria also possess C a + + up-take a c t i v i t i e s (129) which are sensitive to metabolic i n h i b i t o r s such as azide (130), we studied the e f f e c t of t h i s agent on C a + + uptake by sarcolemma. As shown i n Table VI, 5 mM sodium azide had no e f f e c t on C a + + uptake by sarcolemma. ++ 2. Ca -stimulated ATPase of cardiac sarcolemma Since both C a + + binding and uptake are dependent on the presence of ATP, it.was of in t e r e s t to know whether the membranes contained ATPase a c t i v i t i e s . As shown i n Table VII, i n the - 54 -TABLE VI Calcium binding and uptake a c t i v i t i e s of cardiac sarcolemma. Calcium binding and uptake were measured as described under "Methods" with the various additions i n d i c a t e d . When added, ATP ( t r i s s a l t ) was 2 mM; potassium oxalate, 5 mM; sodium azide, 5 mM. Values are the mean with S.E.M. of 4 separate membrane preparations. Calcium Binding and Uptake Additions 1 min 3 min 5 min nmoles/mg -ATP .67 + .28 .80 + .16 .75 + .13 +ATP 3.5 + .57 3.9 + .41 3.2 + .67 +ATP + oxalate 8.8 + 2.0 18 + 1.4 23 + 1.6 +ATP + oxalate azide 9.1 + 1.9 19 + 1.2 25 + 1.4 - 55 -TABLE VII Ca -stimulated ATPase a c t i v i t i e s of cardiac sarcolemma. ATPase a c t i v i t i e s were measured as described under "Methods" with 2 mM ATP ( t r i s s a l t ) . Values are the mean with S.E.M. of 4 separate membrane preparations. ATPase a c t i v i t i e s Additions nmoles/mg/min No addition 8 + 1.1 2+ 5 mM Mg 109+7.3 2+ 50 2 + 5 mM Mg + uM Ca 148 + 8.2 5 mM Ca 186 + 14 absence o f Mg"1"1", ATP h y d r o l y s i s was n e g l i g i b l e . W i t h added Mg' ' , Mg + +-dependent ATPase was found t o be p r e s e n t i n t h e sarcolemma, and t h i s a c t i v i t y w a s . f u r t h e r 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 o f C a + + (50 uM). I n a d d i t i o n , t h e sarcolemma p o s s e s s e d a CaATPase w h i c h was not dependent on M g + + . Our o b s e r v a t i o n s c o n f i r m e d p r e v i o u s r e p o r t s t h a t Mg -dependent and Mg -indep e n d e n t CaATPase a r e a c t i v e components o f plasma membranes from c a r d i a c and s k e l e t a l t i s s u e s (17, 32, 35, 47, 131). 3. P h o s p h o r y l a t i o n o f sarcolemma by exogenous p r o t e i n k i n a s e When c a r d i a c sarcolemma were, i n c u b a t e d w i t h [T - 3 2 P ] ATP and p u r i f i e d . b o v i n e p r o t e i n k i n a s e , t h e r e was a r a p i d i n c o r p o r a -t i o n o f t h e t e r m i n a l phosphate o f ATP i n t o t h e t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e r e s i d u e ( F i g u r e 3A).. I n t h e pr e s e n c e o f 1 uM c y c l i c AMP, t h e amount o f phosphate i n c o r p o r a t e d was more than d o u b l e d . The r e a c t i o n was not l i n e a r w i t h t i m e and began t o p l a t e a u a f t e r 3 m i n u t e s , p r o b a b l y due t o h y d r o l y s i s o f ATP by t h e ATPase and a d e n y l a t e c y c l a s e t h a t t h e s e membranes p o s s e s s e d . Under t h e s e i n c u b a t i o n c o n d i t i o n s i n whi c h r e a c t i o n was t e r m i n -a t e d a t 5 m i n u t e s , p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma was l i n e a r w i t h r e s p e c t t o p r o t e i n k i n a s e up t o 100 ug, as shown i n F i g u r e 3B. F i g u r e 3 (A) Time c o u r s e o f p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma by p u r i f i e d p r o t e i n k i n a s e i n t h e p r e s e n c e (•) and absence (A) o f 1 pM c y c l i c AMP. C a r d i a c sarcolemma (* 135 p g ) , p r o t e i n k i n a s e (10 p g ) . Membrane p h o s p h o r y l a t i o n by endogen-ous p r o t e i n k i n a s e has been c o r r e c t e d f o r . (B) E f f e c t o f v a r i o u s amounts o f p r o t e i n k i n a s e on p h o s p h o r y l a t i o n o f c a r d i a c sarcolemma (~100 pg) i n t h e p r e s e n c e (•) and absence (A) o f 1 pM c y c l i c AMP ( i n c u b a t i o n time was 5 m i n u t e s ) . nMOLES 32p I N C O R P O R A T E D / M G PROTEIN o o o o o —• rO CO K Ol to Ui VI — —' ° 01 b to - 85 -- 59 -4. Endogenous protein kinase andautophosphorylation of  cardiac sarcolemma Recent findings of protein kinase(s) associated with plasma membranes from various tissues (88-91, 114-117, 109, 119, 132-133) prompted us to determinate whether cardiac sarcolemma contained endogenous protein kinase. Freshly prepared membranes were incubated i n the presence of [Y - 3 2 P ] ATP and histone, and as shown i n Figure 4A, there was a low l e v e l of histone phosphoryl-ation which reached a plateau aft e r 2 minutes of incubation. With added c y c l i c AMP (1 pM), histone phosphorylation was stimulated 5 to 7-fold and did not reach a plateau at the end of 5 minutes. Since cardiac sarcolemma contained both protein kinase(s) as well as substrate(s) for phosphorylation,.it was of i n t e r e s t to see whether these membranes could e f f e c t autophosphorylation. When sarcolemma was incubated with [ Y - 3 2 P ~J ATP without added protein kinase or histone, phosphate incorporation was detected as early as 15 seconds and the reaction proceeded rapidly to a plateau. In the presence of added c y c l i c AMP, autophosphorylation was stimulated moderately (25% to 40%), but consistently i n 6 sep-arate membrane preparations studied. The r e s u l t s of a t y p i c a l experiment are shown in Figure 5A. Since membrane-associated protein kinase a c t i v i t y ob-served i n these preparations represented only about 1% of the t o t a l protein kinase a c t i v i t y of the s t a r t i n g homogenate, the p o s s i b i l i t y existed that the a c t i v i t y was due to adsorption of soluble enzyme F i g u r e 4 (A) Time course of h i s t o n e p h o s p h o r y l a t i o n (100 ug c a l f thymus IIA histone) by c a r d i a c sarcolemma ("130 pg) i n the presence (•) and absence (A) of 1 uM c y c l i c AMP. In a l l numbers g i v e n f o r . h i s t o n e p h o s p h o r y l a t i o n , v a l u e s due t o membrane autophos-p h o r y l a t i o n had been s u b t r a c t e d . (B) Assay c o n d i t i o n s were the same as i n (A), ex-cept t h a t c a r d i a c sarcolemma was washed wit h a b u f f e r c o n t a i n i n g 250 mM sucrose, 2 mM d i t h i o t h -r e i t o l , 10 mM Tris-HCL, pH 7.5 and c e n t r i f u g e d at 37,000 x g f o r 15 minutes. The washing procedure was repeated 5 times and the washed sarcolemma r e -suspended by a ground-glass homogenizer be f o r e use. (0) denotes presence and (A) absence of 1 uM cAMP i n the i n c u b a t i o n . MINUTES MINUTES Figure 5 (A) Time course of autophosphorylation of cardiac sarcolemma (~ 130 ug) by endogenous protein kinase i n the presence ( • ) and absence ( A ) of 1 uM cyc-l i c AMP. (B) Assay conditions were the same as i n (A), ex-cept that cardiac sarcolemma was washed 5 times be-fore use. Washing procedure i s as described i n legend to Fig. 4B. ( O ) denotes presence and ( A ) absence of 1 uM c y c l i c AMP i n the incubation medium. - 64 -t o t h e membranes. To r u l e o u t t h i s p o s s i b i l i t y , 2.0-3.0 mg o f sarcolemma was washed 5 t i m e s w i t h 25 ml o f i s o t o n i c b u f f e r c o n t a i n i n g 250 mM s u c r o s e , 2 mM d i t h i o t h r e i t o l and 10 mM T r i s -H C l , pH 7.5, each t i m e c e n t r i f u g e d a t 37,000 x g f o r 15 m i n u t e s . A p p r o x i m a t e l y 15% t o 25% membrane p r o t e i n was removed by t h i s p r o c e d u r e , b u t t h e a b i l i t y o f t h e s e "washed sarcolemma" t o e f f e c t a u t o p h o s p h o r y l a t i o n ( F i g u r e 5B) and h i s t o n e p h o s p h o r y l a -t i o n ( F i g u r e 4B) was u n a l t e r e d . 5. C a l c i u m a c c u m u l a t i o n by p h o s p h o r y l a t e d c a r d i a c sarcolemma I n v i e w o f the r e c e n t f i n d i n g s t h a t membranes d e r i v e d from c a r d i a c s a r c o p l a s m i c r e t i c u l u m (127, 140) and from s k e l e t a l sarcolemma (128) were c a p a b l e o f i n c r e a s e d C a + + uptake when p h o s p h o r y l a t e d by c y c l i c AMP-dependent p r o t e i n k i n a s e , i t was o f i m p o r t a n c e t o d e t e r m i n e whether C a + + a c c u m u l a t i o n by c a r d i a c sarcolemma c o u l d a l s o be a f f e c t e d as a r e s u l t o f p h o s p h o r y l a t i o n by e i t h e r exogenous o r endogenous p r o t e i n k i n a s e . R e s u l t s from 5 s e p a r a t e e x p e r i m e n t s are shown i n T a b l e V I I I . F r e s h l y p r e -p a r e d sarcolemma were i n c u b a t e d a t 30° f o r 5 minutes i n t h e C a + + uptake medium (see Methods) under a v a r i e t y o f p h o s p h o r y l a -t i o n c o n d i t i o n s p r i o r t o measurement o f C a + + u p t a k e . A t t h e 45 end o f t h e p r i o r i n c u b a t i o n p e r i o d , ATP (2 mM) and C a C l 2 were added t o g e t h e r and the amount o f C a + + uptake was measured a f t e r 5 m i n utes o f i n c u b a t i o n . When 25 uM ATP was p r e s e n t d u r i n g t h e p r i o r i n c u b a t i o n p e r i o d , ( a l l o w i n g endogenous p h o s p h o r y l a t i o n t o t a k e p l a c e ) , t h e r e was a s l i g h t , b u t c o n s i s t e n t l y o b s e r v e d i n c r e a s e i n C a + + uptake o v e r t h e c o n t r o l . A f u r t h e r i n c r e a s e - 65 -TABLE VIII E f f e c t of d i f f e r e n t preincubation conditions on Ca^ uptake-by cardiac sarcolemma. Freshly prepared sarcolemma (100-130 jig) was incubated f o r f i v e minutes under various addi^ t i o n s . When added, ATP ( t r i s s a l t ) was 25 uM; c y c l i c AMP, 1 uM; p r o t e i n kinase, 100 ug. At the end of the p r i o r i n -cubation period, 2 mM ATP and 100 pM 4 5 C a C l 2 was added, and calcium uptake was measured f o r 5 minutes as described under "Methods-. Results from 5 separate sarcolemmal pre-parations were shown. Values are averages of t r i p l i c a t e determinations. Additions i n p r i o r incuba-t i o n Calcium uptake Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 No addi t i o n ATP ATP + cAMP ATP + Protein Kinase ATP + cAMP + Protein Kinase ni 21.9 23.4 25.5 41.9 42.8 noles/mg 14.2 15.2 16.9 22.7 25.0 p r o t e i n / 16.2 17.3 18.2 35.2 37.0 5 minute 14.5 15.5 17.0 30.6 29.5 17.2 18.5 20.7 36.9 38.5 - 66 -was noted, when c y c l i c AMP (1 uM) was added. This indicated that as autophosphorylation of the membrane was.allowed to take place i n the p r i o r incubation period by the addition of ATP (25 pM), the sarcolemma was capable of accumulating more C a + + , and as autophosphorylation was stimulated by the presence of added c y c l i c AMP, there was a greater increase i n C a + + uptake. With protein kinase added to the pri o r incubation mixture, a 2-fold stimulation of C a + + uptake was observed. When present together, c y c l i c AMP and protein kinase produced the greatest increase above control. It should be pointed out that the en-hancement of C a + + uptake due to autophosphorylation was quite small (7% and 20% i n the absence and presence of c y c l i c AMP respectively,) and C a + + uptake had to proceed up to 5 minutes before any s i g n i f i c a n t difference could be observed. In contrast, increase i n C a + + uptake due to exogenous protein kinase was suf-f i c i e n t l y large and the time course could be e a s i l y followed. The r e s u l t s of a t y p i c a l experiment are shown i n Figure 6A. Protein kinase increased both the rate and the extent of C a + + up-take even i n the absence of added c y c l i c AMP. In the presence of 1 uM c y c l i c AMP, there was only a s l i g h t increment. This may be attributed to the fact that these membranes contained an active adenylate cyclase and produced c y c l i c AMP close to micromolar levels under these conditions. Since adenylate cyclase i s a l a b i l e enzyme and i t s a c t i v i t y can be s i g n i f i c a n t l y reduced by storage at 4° overnight, the e f f e c t of added c y c l i c AMP might be demonstrated more c l e a r l y by using such an "aged" preparation. F i g u r e 6 (A) Time c o u r s e o f c a l c i u m uptake by c a r d i a c sarcolemma. F r e s h l y p r e p a r e d membranes (<~135 pg) was i n c u b a t e d f o r 5 minutes i n the pr e s e n c e o f 25 pM ATP w i t h t h e f o l l o w i n g c o n d i t i o n s : (• ) c o n t r o l ; (A ) p r e s e n c e o f 1 pM cAMP; (• ) 100 ug p r o t e i n k i n a s e ; (<• ) p r e s e n c e o f 100 pg p r o t e i n k i n a s e and 1 pM cAMP. A t t h e end o f 5 m i n u t e s , 2 mM ATP and 100 pM 4 5 C a C l 2 were added and c a l c i u m uptake was measured f o r the n e x t 5 minut e s . (B) C o n d i t i o n s and le g e n d s were s i m i l a r t o ( A ) , e x c e p t t h a t c a r d i a c sarcolemma was s t o r e d a t 4° f o r 20 hours b e f o r e use. MINUTES MINUTES - 69 -Results of such an experiment are shown in Figure 6B. Although the values for C a + + uptake were lower than that of fr e s h l y -prepared sarcolemma (since C a + + uptake i t s e l f i s also a l a b i l e function), added c y c l i c AMP caused a larger increase i n C a + + up-take, both i n the absence and presence of protein kinase. In the previous experiments studying the e f f e c t of phos-phorylation i n C a + + uptake, p r i o r incubation of sarcolemma with protein kinase took place i n a mixture optimal for C a + + uptake, but not for phosphorylation, i t seemed necessary to establish that under the conditions of the p r i o r incubation, membranes were actually phosphorylated, and that during the C a + + uptake phase, an adequate l e v e l of membrane phosphorylation was maintained. Results i n Figure 7 show that when sarcolemma were incubated with protein kinase (10 to 200 pg), c y c l i c AMP (1 pM), and r 3 2 n ++ [V - PJ ATP (25 uM) i n a Ga uptake medium, they were rapidly phosphorylated, reaching a maximum l e v e l i n 3 to 4 minutes. When CaCl 2 ( f i n a l concentration 0.1 mM) was added at the end of 5 minutes and the l e v e l of phosphorylation determined at d i f f e r e n t time i n t e r v a l s up to 10 minutes, i t was found that, despite a small gradual decline, the maximal l e v e l of phosphorylation was maintained at the end of 10 minutes. Since phosphorylation of membranes would y i e l d polar phosphate groups, the increased C a + + uptake observed might simply r e f l e c t e l e c t r o s t a t i c i n t e r a c t i o n between p o s i t i v e l y charged calcium ions and the negatively charged s i t e s on the surface of the membrane. If t h i s were the case, there would be a simple Figure 7 E f f e c t of CaCl2 on phosphorylation of card-iac sarcolemma by various amounts of p u r i f i e d protein kinase. Cardiac Sarcolemma ("IOO ug) was phosphorylated i n a C a + + uptake medium i n the presence of 2 5 uM Tris-ATP and 1 uM c y c l i c AMP. At the end of 5 minutes, 100 uM CaCl 2 was added and the l e v e l of membrane phosphoryla-ti o n was followed for the next 5 minutes. Numb-ers on each curve r e f e r to the amount of protein kinase added i n ug. MINUTES - 72 -s t o i c h i o m e t r i c r e l a t i o n s h i p between t h e amount o f phosphate i n -c o r p o r a t e d and the n e t amount o f Ca bound due t o p h o s p h o r y l a t i o n . As shown i n F i g u r e 8, t h i s was not the case. I n c r e a s e d C a + + up-t a k e was l i n e a r w i t h t h e amount o f phosphate i n c o r p o r a t e d , and f o r e v e r y nmole o f phosphate i n c o r p o r a t e d , (up t o 1.5 n m o l e s ) , t h e r e was 15 nmoles o f n e t i n c r e a s e i n C a + + uptake. T h i s suggested t h a t t h e i n c r e a s e i n C a + + uptake was n o t due t o s i m p l e i o n i c i n t e r -a c t i o n on t h e membrane s u r f a c e . I n f a c t , membrane p h o s p h o r y l a t i o n o n l y a f f e c t e d the Ca uptake system and not Ca b i n d i n g . The e x a c t mechanism whereby membrane p h o s p h o r y l a t i o n l e a d s t o C a + + up-t a k e remains t o be e l u c i d a t e d . 6. R e v e r s i b i l i t y o f membrane p h o s p h o r y l a t i o n and d i s t r i b u t i o n  o f p h o s p h o p r o t e i n phosphatase F o r membrane p h o s p h o r y l a t i o n t o have importance i n p h y s i -o l o g i c a l r e g u l a t i o n o f C a + + t r a n s p o r t i n t o t h e c a r d i a c c e l l , t h e r e must a l s o be a mechanism f o r r a p i d d e p h o s p h o r y l a t i o n . I n an a t t e m p t t o demonstrate d e p h o s p h o r y l a t i o n , c a r d i a c sarcolemma was p h o s p h o r y l a t e d by exogenous p r o t e i n k i n a s e , and when p l a t e a u l e v e l o f p h o s p h o r y l a t i o n was r e a c h e d a f t e r 5 m i n u t e s , e x c e s s u n l a b e l e d 32 ATP (3 mM) was added t o s t o p f u r t h e r P i n c o r p o r a t i o n , and the l e v e l o f p h o s p h o r y l a t i o n was measured a t d i f f e r e n t time i n t e r v a l s d u r i n g t h e f o l l o w i n g 15 m i n u t e s . I n c o n t r o l e x p e r i m e n t s , an e q u i -v a l e n t volume o f w a t e r was added. As shown i n F i g u r e 9, i m m e d i a t e l y a f t e r the a d d i t i o n o f e x c e s s u n l a b e l e d ATP, the l e v e l o f membrane p h o s p h o r y l a t i o n began t o d e c l i n e w h i l e c o n t r o l l e v e l s remained Figure 8 Relationship between phosphorylation and stimulation of calcium uptake by cardiac sarcolemma. Sarcolemmal membranes (125 ug) were phosphorylated by various amounts of protein kinase (20 pg to 200 pg) i n the same medium used for calcium uptake. The amount of phosphorylation (corrected for endogenous a c t i v i t i e s ) i s plotted against the net amount of calcium uptake stimulated by phosphorylation. NET INCREASE IN PHOSPHORYLATION nMOLES 3 2 P INCORP. /MG PROTEIN/5 MIN. Figure 9 Phosphorylation and dephosphorylation of cardiac sarcolemma. Freshly prepared memb-ranes ( 1 mg) was phosphorylated i n the presence (c i r c l e s ) and absence (triangles) of 1 pM cAMP, 25 pM [ Y - 3 2 P ] ATP by exogenous protein kinase (175 pg) i n a f i n a l volume of 2 ml. Fluoride was excluded from the medium. Phosphorylation was measured at various time, i n t e r v a l s up to f i v e minutes by removing 200 p i aliquots (see Methods for d e t a i l s ) . At the end of 5 minutes, unlabeled ATP was added ( f i n a l concentration 2 mM) and dephosphoryla-t i o n was followed for the next 15 minutes (open symbols) again by removing 200 p i aliquots. In control tubes, an equal amount of water was added (closed symbols). MINUTES - 77 -unaltered. Although dephosphorylation was not complete i n 15 32 minutes, as much as 50% of P was removed from membranes phos-phorylated i n the presence or absence of c y c l i c AMP. This i s highly suggestive that phosphoprotein phosphatase may be associated, with cardiac sarcolemma as part of a regulatory complex c o n t r o l l i n g membrane phosphorylation and dephosphorylation. We, therefore, further examined the presence and d i s t r i b u t i o n of t h i s phospho-protein phosphatase a c t i v i t y i n sarcolemma and i n various fractions obtained during the i s o l a t i o n of the sarcolemma, using phosphoryl-ated sarcolemma and histones as substrates. As shown i n Table IX, at the stage of "washed p a r t i c l e s " (see Figure 1), 80% of the phosphoprotein phosphatase a c t i v i t y was removed. More than 75% of t h i s a c t i v i t y was recovered from the supernatant, which contained large l y soluble protein and the microsomal fraction.. Extraction of the washed p a r t i c l e s with KCI removed most of the remaining a c t i v i t y so that i n the f i n a l sarcolemmal membranes, only 1% of the t o t a l a c t i v i t y remained. Because of the small recovery of enzyme a c t i v i t y , i n order to e s t a b l i s h that the enzyme i s i n t r i n s i c to the memb-rane, we washed sarcolemmal membranes thoroughly (5 times) as we did previously i n the studies of membrane bound protein kinase. As shown i n Table IV, despite a reduction by the washing procedure, 0.5% of enzyme a c t i v i t y survived such treatment and remained assoc-iated with the sarcolemma. - 78 -TABLE IX D i s t r i b u t i o n of phosphoprotein phosphatase i n f r a c t i o n s obtained during i s o l a t i o n of sarcolemma. Measurement of phosphoprotein phosphatase a c t i v i t y , and i s o l a t i o n of various f r a c t i o n s from guinea p i g heart were described under "Methods". "Supernatant" was obtained by pooling a l l supernatant f r a c t i o n s from the 5 washings described i n the i s o l a t i o n procedure; "Washed sarcolemma" re f e r r e d to sarcolemma subjected to 5 more washings with bu f f e r containing 250 mM Sucrose, 2 mM d i t h i o t h r e i t o l , 10 mM Tri s - H C l pH 7.5 and centrifuged at 37,000 x g for 15 min-utes a f t e r each wash. Values given on the table were the means with S.E.M. of 4 d i f f e r e n t membrane preparations. Fractions Phosphoprotein Phosphatase S p e c i f i c A c t i v i t y , (pmoles "^P/mg/min^.: ute) '• Tbta'llactivity}; (Percent) '" 8:5 + .98 100 7.8 + 1.2 52.2 + 5.4 5.0 + .12 17.1 + 1.8 KCl-extracted P a r t i c l e s 3.1 + .70 3.2 + .57 2.8 + .47 .90 + .06 1.5 + .12 .51 + .03 - 79 -DISCUSSION Studies with i s o l a t e d cardiac sarcolemma provide evid-ence for the presence of an ATP-dependent C a + + binding system as well as C a + + dependent ATPase (Tables VI and VII). These a c t i v -i t i e s are not l i k e l y to be due to contamination by mitochondrial or microsomal fragments because of (1) t h e i r lack of s e n s i t i v i t y to azide (we have observed, as others (130) that 65% to 85% of mitochondrial C a + + accumulation i s abolished by 5. mM NaN^), (2) t h e i r r e l a t i v e l y low s p e c i f i c a c t i v i t y (freshly prepared card-iac microsomal preparations accumulate at least 6 to 10 times more calcium under si m i l a r conditions), and (3) t h e i r r e l a t i v e s t a b i l -i t y (cardiac microsomes lose 60%-80% of t h e i r a b i l i t y to accumu-lat e C a + + 3 hours after i s o l a t i o n whereas cardiac sarcolemma can be stored at 4° overnight with 90% of the Ca"1"1" accumulation a c t i v i t y i n t a c t ) . I t i s also u n l i k e l y that these a c t i v i t i e s are due to contamination by myofibrils, i n view of the high s a l t con-centration (1.25 M KCI) used to extract the membranes. Further-more, oxalate i s not known to exert any e f f e c t on m y o f i b r i l l a r Ca binding. Ca -stimulated ATPase a c t i v i t i e s have been reported i n plasma membrane fractions from dog (146) and rat (134) myo-cardium. Energy-dependent C a + + transport mechanisms have also been described i n plasma membranes from red c e l l s (135,136), f i b r o -b lasts (137), smooth muscle (138) and sk e l e t a l muscle (32). These reports have argued for an active C a + + transport at the plasma membrane analogous to the transport of Na + and K +. The present finding i n the cardiac sarcolemma provides further support to th i s - 80 -concept and suggests a role for the sarcolemma i n C a T T mobiliza-t i o n and excitation-contraction coupling i n the heart. Our studies show that cardiac sarcolemma possess memb-rane-bound, c y c l i c AMP-dependent protein kinase a c t i v i t i e s as well as endogenous substrates for auto-phosphorylation. The procedure to i s o l a t e sarcolemma involved vigorous homogenization, repeated washings with hypotonic and isotonic buffers and ex-t r a c t i o n with high' concentration of KCl. This makes i t un l i k e l y that the protein kinase a c t i v i t i e s observed were due to the ad-sorption of soluble enzymes to the membranes. Furthermore, the fact that auto-phosphorylation was unaltered after the sarcolemma were washed 5 times with large volumes of buffer makes i t rather convincing that both the protein kinase(s) and the membrane sub-strate (s) were indeed i n t r i n s i c components of the membrane. Phosphoprotein phosphatase a c t i v i t y was also found to be assoc-iated with the sarcolemma. Maeno and Greengard (139) have shown that i n rat cerebral cortex, more than 50% of the t o t a l phos-phoprotein phosphatase a c t i v i t y was found i n the pa r t i c u l a t e fractions and that the s p e c i f i c a c t i v i t y was highest i n subtract-ions r i c h i n synaptic membranes. The present data shows that cardiac sarcolemma was able to reverse membrane phosphorylation stimulated by r e l a t i v e l y high concentrations of p u r i f i e d protein kinase. This makes i t highly probable that at least part of the phosphoprotein phosphatase a c t i v i t y i s associated with the plasma membranes, conferring r e v e r s i b i l i t y to membrane phosphorylation. - 81 -A u t o - p h o s p h o r y l a t i o n o f sarcolemma ( F i g u r e 5A) was o n l y s l i g h t l y s t i m u l a t e d (25%-40%) by c y c l i c AMP, whereas p h o s p h o r y l a -t i o n o f h i s t o n e by the same membrane p r e p a r a t i o n s was g r e a t l y enhanced by t h i s c y c l i c n u c l e o t i d e ( F i g u r e 4A). S i m i l a r low l e v e l s o f c y c l i c A M P - s t i m u l a t i o n have been o b s e r v e d i n t h e auto-phos-p h o r y l a t i o n o f e r y t h r o c y t e membranes (133). I n t h e s e membranes, o f t h e t h r e e p r o t e i n components t h a t were p h o s p h o r y l a t e d , o n l y one minor component was u n e q u i v o c a l l y shown t o be s t i m u l a t e d by c y c l i c AMP. S i m i l a r l y , i n the p h o s p h o r y l a t i o n o f nerve s y n a p t i c membranes ( 9 ) , Johnson e t a l found t h a t o n l y 2 p r o t e i n components showed i n c r e a s e d p h o s p h o r y l a t i o n i n t h e p r e s e n c e o f c y c l i c AMP. A t l e a s t s i x o t h e r membrane components were p h o s p h o r y l a t e d , but t h e i r l e v e l o f p h o s p h o r y l a t i o n was u n a f f e c t e d by c y c l i c AMP. These o b s e r v a t i o n s suggested t h a t i n membrane a u t o - p h o s p h o r y l a t i o n , o n l y one o r a few p o s s i b l e s u b s t r a t e ( s ) i s s u b j e c t t o c o n t r o l by c y c l i c AMP whose e f f e c t can e a s i l y be o b s c u r e d by h i g h l e v e l s o f c y c l i c AMP-independent p h o s p h o r y l a t i o n o f o t h e r membrane compon-e n t s . A l t h o u g h we have not a t t e m p t e d t o d e t e r m i n e th e n a t u r e o f the s i t e p h o s p h o r y l a t e d , i t i s q u i t e l i k e l y t h a t our o b s e r v a t i o n i n c a r d i a c sarcolemma may be t h e r e s u l t o f a s i m i l a r phenomenon. The s i t u a t i o n was f u r t h e r c o m p l i c a t e d by the f a c t t h a t t h e p r e -s e n t sarcolemmal p r e p a r a t i o n p o s s e s s e d an e x t r e m e l y a c t i v e a d e n y l a t e c y c l a s e so t h a t s u f f i c i e n t l y h i g h l e v e l s o f c y c l i c AMP were a l r e a d y p r e s e n t t o p a r t l y s t i m u l a t e p r o t e i n k i n a s e a c t i v i t i e s . T h i s e x p l a i n s why, even i n the p r e s e n c e o f added s o l u b l e p r o t e i n k i n a s e ( F i g u r e 3A), membrane p h o s p h o r y l a t i o n . w a s s t i m u l a t e d no - 82 -more than 3 - f o l d by added c y c l i c AMP, whereas h i s t o n e phosphoryla-t i o n by the same p r o t e i n k i n a s e was s t i m u l a t e d more than 1 0 - f o l d by c y c l i c AMP. Our f i n d i n g o f a p h o s p h o r y l a t i o n and dephos p h o r y l a t i o n system i n the c a r d i a c sarcolemma d i f f e r s from the o b s e r v a t i o n s of Wray e t a l (117), La Raia e t a l (118) and Katz e t a l (127, 140, 147) mainly i n the l o c a l i z a t i o n of these enzyme systems. These authors used c a r d i a c microsomes which c o n s i s t l a r g e l y of f r a g -mented endoplasmic r e t i c u l u m . As mentioned above, t h e i r s t u d i e s i n d i c a t e d t h a t these membranes were phosphorylated by c y c l i c AMP-dependent p r o t e i n k i n a s e , and the formation of phosphoprotein c o r -r e l a t e d w i t h the s t i m u l a t i o n o f the r a t e o f C a + + uptake, suggesting a f u n c t i o n a l r e l a t i o n s h i p between p h o s p h o r y l a t i o n and C a + + t r a n s -p o r t i n s a r c o p l a s m i c r e t i c u l u m . Sulakhe and Drummond (12 8) have r e p o r t e d r e c e n t l y t h a t s i t e s o f p h o s p h o r y l a t i o n and dephosphoryla-t i o n e x i s t i n the s k e l e t a l sarcolemma; Andrew e t a l (110) a l s o r e p o r t e d the e x i s t e n c e o f a membrane-bound p r o t e i n k i n a s e i n s a r -colemma d e r i v e d from s k e l e t a l muscle. Recently, Krause e t a l (141) demonstrated a r e v e r s i b l e p h o s p h o r y l a t i o n system i n a plasma memb-ran e - e n r i c h e d p r e p a r a t i o n from p i g myocardium.. These o b s e r v a t i o n s , t o g e t h e r w i t h our s t u d i e s , suggest t h a t a s i m i l a r system of auto-p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n i n v o l v i n g c y c l i c AMP e x i s t s at. the plasma membrane l e v e l o f the myocardium. Although membrane p h o s p h o r y l a t i o n has been observed i n a number of t i s s u e s , the p h y s i o l o g i c a l importance of t h i s phenomenon - 83 -remains obscure. Membrane permeability to ions has been postul-ated to be regulated by t h i s process i n synaptic membranes (88, 90, 142), toad-bladder membranes (143), renal medullary memb-ranes (116), l i v e r plasma membranes (108), cardiac microsomes (118, 127, 140) and s k e l e t a l sarcolemma (128). This present study shows that cardiac sarcolemma, phosphorylated either by endogen-ous or exogenous protein kinase, was able to accumulate more Ca than control preparations. As shown i n Figure 8, the net stimula-t i o n of C a + + uptake p a r a l l e l e d the net increase i n kinase-catalyzed phosphorylation, suggesting that the enhanced C a + + transport i s indeed the r e s u l t of membrane phosphorylation. While r e l a t i v e l y l i t t l e i s known about the exact mechanism whereby phosphorylation controls C a + + transport, i t i s of i n t e r e s t to note that Wolff and Siegel (144) i s o l a t e d from pig brain a C a + + binding phospho-protein which when treated with phosphatase, l o s t i t s C a + + binding a c t i v i t y , suggesting a role for phosphorylation-dephosphorylation i n regulation of C a + + binding. In recent studies, Tada et a l (145, 147, 148) showed that c y c l i c AMP-dependent protein kinase catalyzed the phosphorylation of a 22,000-dalton protein i n cardiac microsomes which was d i s t i n c t from the phosphoprotein intermediate of the C a + + transport ATPase. It was further shown that b r i e f digestion with trypsin i n the presence of 1 M sucrose did not s i g n i f i c a n t l y a f f e c t microsomal C a + + transport a c t i v i t y , but prevented both phosphorylation of the 22,000-dalton protein and stimulation of C a + + uptake by c y c l i c AMP-dependent protein kinase, suggesting that t h i s protein i s a modulator of the C a + + pump. - 84 -From a p h y s i o l o g i c a l p o i n t of view, the most important f u n c t i o n of catecholamines i s the modulation o f myocardial, cont-r a c t i l i t y , which u l t i m a t e l y i n v o l v e s v a r i a t i o n s i n C a + + d e l i v e r y to the c o n t r a c t i l e p r o t e i n s . The r e l e a s e of C a + + f o r a c t i v a t i o n of the c o n t r a c t i l e process i s c o n t r o l l e d predominantly by two membrane systems: the sarcolemma and the s a r c o p l a s m i c r e t i c u l u m . C a + + i n f l u x a c r o s s the sarcolemma i s a s s o c i a t e d w i t h a slow inward c u r r e n t t h a t takes p l a c e d u r i n g s y s t o l e ; i t i s now g e n e r a l l y ac-cepted t h a t catecholamines augment i n t r a c e l l u l a r C a + + content by i n c r e a s i n g t h i s slow inward c u r r e n t , and t h i s e f f e c t may account f o r a t l e a s t p a r t of the p o s i t i v e i n o t r o p i c a c t i o n o f B-adrenergic a g o n i s t s . The data i n t h i s t h e s i s supports t h i s concept by p r o v i d -i n g a b i o c h e m i c a l b a s i s f o r the mediation o f these events. We have shown t h a t c a r d i a c sarcolemma c o n t a i n an a c t i v e adenylate c y c l a s e , a u t o - p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n systems as w e l l as ATP-dependent C a + + t r a n s p o r t a c t i v i t y . Thus the c a r d i a c s a r c o -lemma not o n l y c o n t a i n the apparatus f o r the p r o d u c t i o n of c y c l i c AMP i n response to hormones, but a l s o the machinery f o r the b i o -l o g i c a l a c t i o n o f the n u c l e o t i d e , i . e . , the a c t i v a t i o n of memb-rane-bound p r o t e i n k i n a s e and p h o s p h o r y l a t i o n of c e r t a i n r e g u l a t o r p r o t e i n ( s ) l e a d i n g to enhancement of C a + + t r a n s p o r t . The p h y s i o -l o g i c a l r e l e v a n c e of these data i s f u r t h e r strengthened by e l e c t r o n microscopy s t u d i e s which showed t h a t the i s o l a t e d sarcolemmal p r e p a r a t i o n c o n s i s t e d mainly of v e s i c l e s and open sacs. (EM s t u d i e s of the c a r d i a c sarcolemma were done by Dr. G. I. Drummond). We i n t e r p r e t the C a + + accumulation i n these v e s i c l e s and the enhancement - 85 -of t h i s a c t i v i t y by c y c l i c AMP-dependent p h o s p h o r y l a t i o n t o be analogous t o an i n v i v o s i t u a t i o n o f i n c r e a s e d transarcolemmal C a + + t r a n s p o r t i n response t o B-adrenergic s t i m u l a t i o n . P h y s i o -l o g i c a l data i n d i c a t e s t h a t the i n f l o w i n g C a + + f i r s t f i l l some i n t r a c e l l u l a r s t orage s i t e s from which C a + + can be r e l e a s e d dur-i n g subsequent d e p o l a r i z a t i o n to a c t e i t h e r d i r e c t l y on c o n t r a c t -i l e p r o t e i n s or a c t i n d i r e c t l y by r e l e a s i n g C a + + from other s i t e s w i t h i n the c e l l . The f i n d i n g s o f Katz e t a l on c a r d i a c micro-somes suggested the e x i s t e n c e o f a s i m i l a r system i n the sa r c o -p l a s m i c r e t i c u l u m . These authors p o s t u l a t e d t h a t the i n c r e a s e d C a + + uptake by the sarc o p l a s m i c r e t i c u l u m f o l l o w i n g phosphoryla-in t i o n by p r o t e i n k i n a s e has the e f f e c t of r e t a i n i n g w i t h the c e l l some of the C a + + which would otherwise be l o s t d u r i n g d i a s t o l e . T h i s i n c r e a s e d C a + + storage can then add to the amount of C a + + a v a i l a b l e f o r d e l i v e r y t o the c o n t r a c t i l e apparatus i n subsequent beats, thus p r o v i d i n g augmentation of myo c a r d i a l c o n t r a c t i l i t y . I t i s reasonable to.assume t h a t both the sarcolemma and the s a r c o -p l a s m i c r e t i c u l u m are a c t i v a t e d by c y c l i c AMP-dependent phosphor-y l a t i o n i n the m o b i l i z a t i o n o f C a + + i n response t o B-adrenergic s t i m u l a t i o n ; f u t u r e work w i l l be necessary not o n l y t o assess the r e l a t i v e importance of these two c o n t r o l s i t e s , but a l s o to ex-p l o r e other mechanisms, e.g., r e l e a s e o f C a + + from m i t o c h o n d r i a , whereby c y c l i c AMP mediates the i n o t r o p i c response. - 86 -REFERENCES' 1. SNELL, F., WOLKEN, J . , IVERSON, G., and LAM, J . 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