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Subcellular localization of adenylate cyclase in canine heart ventricle Dobovicnik, Bernhard Karl 1981

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SUBCELLULAR LOCALIZATION OF ADENYLATE CYCLASE IN CANINE HEART VENTRICLE by BERNHARD KARL DOBOVICNIK B.Sc, University of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmacology Faculty of Pharmaceutical Sciences University of B r i t i s h Columbia We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1981 (c) Bernhard Karl Dobovicnik I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e 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 t h a t t h e 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 a n d s t u d y . I f u r t h e r a g r e e 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 t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t 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 n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . 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 2075 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 V6T 1W5 D a t e 22, /. i<f$2~ DE-6 (2/79) - i i -ABSTRACT Crude microsomal f r a c t i o n s (resuspended i n 10% sucrose) of dog heart v e n t r i c l e s were subjected to discontinuous sucrose density gradient c e n t r i f u g a t i o n i n order to obtain p u r i f i e d f r a c t i o n s that were enriched i n sarcoplasmic reticulum (S.R.) and contained a r e l a t i v e l y lower concentration of plasma membrane than the crude f r a c t i o n . It was observed that the f r a c t i o n with the highest oxalate-2+ supported Ca -uptake a c t i v i t y contained an adenylate cyclase a c t i v i t y . The main objectives of t h i s study were to r e f i n e the technique of i s o l a t i o n of t h i s pure microsomal f r a c t i o n and to compare the properties of i t s indigenous adenylate cyclase a c t i v i t y to that contained i n a sarcolemma-enriched "washed p a r t i c l e " f r a c t i o n (Drummond, 1978). It was found that the enzyme found i n the S.R.—enriched f r a c t i o n d i f f e r e d from that i n the "washed p a r t i c l e " preparation i n that i t : a) was stimulated by 10~^M epinephrine i n a calcium-dependent manner b.) was s i g n i f i c a n t l y more stimulated by high Mg /ATP r a t i o s c) was more s e n s i t i v e to i n h i b i t i o n by free calcium ion i n the presence of epinephrine French-press treatment of the microsomes before sucrose density gradient f r a c t i o n a t i o n reduced plasma membrane contamination without a concomitant decrease i n c y c l i c AMP accumulating a b i l i t y . These findings are consistent with and complement the hypothesis that cardiac c e l l sarcoplasmic reticulum contains an adenylate cyclase - i i i -a c t i v i t y that i s not accounted for by sarcolemmal impurities i n the preparations studied and that exhibits f u n c t i o n a l c h a r a c t e r i s t i c s d i f f e r e n t from the sarcolemmal adenylate cyclase. - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 EXCITATION-CONTRACTION COUPLING IN CARDIAC MUSCLE-FUNCTIONAL AND ULTRA STRUCTURAL BASIS IN CARDIAC AND SKELETAL MUSCLE 1 CALCIUM AND EXCITATION-CONTRACTION COUPLING 2 THE SARCOPLASMIC RETICULUM AND EXCITATION-CONTRACTION COUPLING 3 CYCLIC NUCLEOTIDES AND CARDIAC CONTRACTION 6 SUBCELLULAR LOCALIZATION OF ADENYLATE CYCLASE 7 OBJECTIVES 13 METHODS AND MATERIALS 15 PREPARATION OF DOG MICROSOMAL FRACTIONS ENRICHED IN SARCOPLASMIC RETICULUM 15 PREPARATION OF A WASHED-PARTICLE FRACTION 16 PREPARATION OF SARCOLEMMAL?'MICROSOMES 16 OUABAIN-BINDING ASSAY 17 ASSAY OF ADENYLATE CYCLASE ACTIVITY 18 CALCIUM LOADING OF CRUDE MICROSOMES 18 ASSAY OF Ca 2 +-UPTAKE AND ATP-DEPENDENT Ca 2 +-UPTAKE 19 MATERIALS 20 - v -Page RESULTS 21 ISOLATION OF DOG HEART MICROSOMAL FRACTIONS 21 A. Standard sucrose gradient c e n t r i f u g a t i o n 21 B. E f f e c t of calcium loading 21 C. E f f e c t s of French-press treatment 23 CHARACTERIZATION OF DOG MICROSOMAL FRACTIONS OBTAINED BY SUCROSE DENSITY CENTRIFUGATION 25 A. Calcium uptake studies 25 B. Ouabain binding studies 25 C. C y c l i c AMP accumulation i n preparations from dog cardiac ventricle-hormonal stimulation 33 1. E f f e c t of Mg 2 +/ATP r a t i o and GTP on c y c l i c AMP accumulation 35 2+ 2. E f f e c t of free Ca ion on basal and hormone-stimulated c y c l i c AMP accumulation i n microsomal f r a c t i o n and washed p a r t i c l e s 37 DISCUSSION 40 REFERENCES - 47 - v i -LIST OF TABLES Page TABLE 1 Characterization of dog ccardiac microsomal f r a c t i o n s obtained by sucrose density c e n t r i f u g a t i o n 27 TABLE 2 S p e c i f i c a c t i v i t i e s of ouabain binding of ^ dog cardiac microsomal f r a c t i o n s 29 TABLE 3 C y c l i c AMP accumulation i n preparations from dog cardiac microsomes 34 2+ TABLE 4 E f f e c t of [Ca ] on c y c l i c AMP accumulation i n f r a c t i o n s from dog heart 38 - v i i -LIST OF FIGURES Page FIG. 1 E f f e c t of calcium loading on sucrose density gradient banding patterns 22 FIG. 2 Scheme for preparation of p u r i f i e d S.R. and French press-treated S.R 24 FIG. 3 Protein concentration-dependence of ouabain binding 26 FIG. 3a E f f e c t of 15000 P.S.I. French-press treatment on c y c l i c AMP accumulation i n p u r i f i e d S.R. enriched microsomes from dog heart 31 2+ FIG. 4 E f f e c t of Mg /ATP r a t i o on c y c l i c AMP accumulation i n cardiac membrane preparations 36 - v i i i -ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. Sidney Katz, whose guidance and encouragement have been invaluable during the course of t h i s project. I am also thankful for the constructive c r i t i c i s m s of Dr. John McNeill, Dr. B a s i l Roufogalis and Dr. Jack Diamond. The f i n a n c i a l assistance, provided i n part by the Medical Research Council of Canada, and the B r i t i s h Columbia Heart Foundation, i s thankfully acknowledged. I would also l i k e to thank Mrs. Betty Richter for her help and patience. - i x -LIST OF ABBREIVATIONS EC excitation-contraction AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate ATPase adenosine 5'-triphosphatase „ 2+ . . . Ca free calcium xon c y c l i c AMP c y c l i c 3',5'-adenosine monophosphate EDTA ethylene diamine tetraacetate, disodium s a l t EGTA ethyleneglycolbis- (3-aminoethyl), N-;-N'tetraacetate GTP guanosine triphosphate S.E.M. standard error of the mean T r i s t r i s (hydroxymethyl) aminomethane P.S.I. pounds per square inch Vs maximum v e l o c i t y 2+ Mg free magnesium ion M.W. molecular weight Adenylate cyclase ATP pyrophosphate lyase ( c y c l i z i n g ) NaF sodium f l u o r i d e A.C. adenylate cyclase g force of gravity w/v weight per volume DTT d i t h i o t h r e i t o l (Cleland's reagent) p[NH]ppG guanyl-5'-yl-imidophosphate MES (2 N-Morpholino-ethaner-sulfonic Acid) S.R. sarcoplasmic reticulum - 1 -INTRODUCTION EXCITATION-CONTRACTION COUPLING IN CARDIAC MUSCLE -FUNCTIONAL AND ULTRASTRUCTURAL BASIS IN CARDIAC AND SKELETAL MUSCLE It i s now w e l l established that the f i n a l event i n the e x c i t a t i o n -contraction sequence i n muscle tissue i s the binding of calcium ions to t r o -ponin C. This t r i g g e r s contraction which i s retained u n t i l the i n t r a -c e l l u l a r calcium concentration f a l l s below approximately 10 ^ M (Ebashi, 1976). I t i s thought that the mechanism at the l e v e l of the c o n t r a c t i l e proteins i s fundamentally s i m i l a r i n s k e l e t a l and cardiac muscle, yet i n i t i a t i n g events, regulation and hormonal control d i f f e r d r a s t i c a l l y between the two types of muscle (Chapman, 1979). Considering the physio-l o g i c a l functions of s k e l e t a l muscle as "voluntary" and cardiac muscle as "involuntary", many of these differences are to be expected. For instance, force generation i n s k e l e t a l muscle i s accomplished by the a c t i v a t i o n of s o - c a l l e d motor units (a group of myocytes innervated by branches of the same motor neuron), the t o t a l force obtained being proportional to the number of motor-units activated. Within each motor u n i t , every c e l l contracts with maximal force. Heart, i n contrast, does not have voluntary innervation and contracts rhythmically with i t s e n t i r e muscle mass being activated i n a t y p i c a l " a l l or none" manner. Gradation of force i s accomplished by a number of f a c t o r s , among the most important the extent of overlap of a c t i n and myosin filaments seen macroscopically i n the r e s t i n g length of the muscle f i b r e s . - 2 -CALCIUM AND EXCITATION-CONTRACTION COUPLING Co n t r a c t i l e force i s also regulated, i r r e s p e c t i v e of f i b r e length, by v a r i a t i o n i n the amount of calcium a v a i l a b l e to the myofilaments. In s k e l e t a l muscle, the major source of contractile^-dependent calcium i s most l i k e l y the sarcoplasmic reticulum, whereas i n heart muscle extra-c e l l u l a r calcium seems to play a c r u c i a l r o l e i n the e x c i t a t i o n - c o n t r a c t i o n sequence. Experiments i n which the action of lanthanum (La) on s k e l e t a l muscle and cardiac muscle was studied (Andersson, 1974; Armstrong et a l . , 1972; Rich et a l . , 1975;) showed that small amounts of lanthanum (uM concentrations) r a p i d l y uncoupled e x c i t a t i o n from contraction i n cardiac 2+ muscle and also prevented binding of Ca to i s o l a t e d sarcolemma (Pang, 1980) but had no e f f e c t on s k e l e t a l muscle. The lanthanum ions are apparently capable of d i s p l a c i n g the r a p i d l y exchangeable pool of calcium but don't enter the c e l l themselves. The exact l o c a t i o n of t h i s surface-bound calcium appears to be i n the glycocalyx, a layer on the outer surface of the sarcolemma composed of glycoprotein, g l y c o l i p i d and mucopolysaccaride. It has 2 components, one 20nm thick inner layer and an outer layer (the external lamina) of about 30 nm thickness (Frank et a l . , 1977; Langer, 1978). The major function of t h i s external coat of sarcolemma appears to be the regulation of the transmembrane f l u x of calcium, probably v i a a mechanism inv o l v i n g the s i a l i c acid component of the glycocalyx. - 3 -THE SARCOPLASMIC RETICULUM AND EXCITATION-CONTRACTION COUPLING Although e x t r a c e l l u l a r calcium i s indispensable.for contraction of cardiac muscle and calcium may be extruded through the sarcolemma to help + 2+ i n the i n i t i a t i o n of r e l a x a t i o n , (possibly v i a a Na -Ca exchange or a calcium dependent ATPase (Sulakhe et a l . , 1 9 8 0 ) , t h e r e l a t i v e importance of t h e s e mechanisms seems to be v a r i a b l e from species to species (Chapman, 1979), a fa c t that greatly complicates a c r i t i c a l evaluation of t h e i r true physio-l o g i c a l s i g n i f i c a n c e . Findings by Fabiato and Fabiato (1978) on skinned cardiac f i b r e s demonstrate conclusively that smaller quantities of calcium than necessary for f u l l force development can induce a release of calcium from the sarcotubular system, i n d i c a t i n g a f u n c t i o n a l l y s i g n i f i c a n t role of sarcoplasmic reticulum i n the EC -coupling sequence of cardiac muscle. The undisputed r o l e of S.R. i n calcium sequestration and release i n s k e l e t a l muscle stands i n contrast to the less well-established action of t h i s structure i n cardiac muscle (Chapman, 1979). As with the role + 2+ of the Na -Ca exchange mechanism, there e x i s t s considerable controversy about the importance of the sarcoplasmic reticulum with respect to regula-t i o n of myocardial c o n t r a c t i l i t y . Attempts to resolve the dispute have been complicated by a marked species d i v e r s i t y i n both u l t r a s t r u c t u r e (Liillmann et a l . , 1979) and response of cardiac muscle preparations to the extra-c e l l u l a r i o n i c environment (Chapman, 1979; Sulakhe, 1980). Opinions range from a minimal involvement of S.R. i n regulation of cardiac function (Liillmann et a l . , 1979) , supported i n part by the action of cardiac glycosides (Sulakhe et a l . , 1980), to a key r o l e of S.R. i n the .'EC-sequence. ( D h a l l a et a l . ' . , 1 9 T 7 - | 1 9 7 8 J F a b i a t o , a n d F a b i a t o , 1 9 7 7 ) . - 4 -The S.R. i n mammals i s l e s s abundant i n cardiac than i n s k e l e t a l muscle, occupying approximately 7% of the m y o f i b r i l l a r volume and may range from 9 to 30 times l e s s than the S.R. volume i n s k e l e t a l muscle (Chapman, 1979). Sarcoplasmic reticulum i s also i n close contact with invaginations of the sarcolemma (T-tubules). This s t r u c t u r a l feature r e f l e c t s the s p e c i a l i -zation of the plasma membrane i n the conduction of the d e p o l a r i z a t i o n wave which, i n s k e l e t a l muscle, causes Ca release from the terminal cisternae of the S.R. by an unknown mechanism, probably involving s o - c a l l e d "couplings" between T-tubules and S.R. (Ebashi, 1976). Af t e r a c t i v a t i o n of contraction, 2+ the Ca ions are transported back into the S.R., the plasma membrane repolarizes and the c o n t r a c t i l e apparatus relaxes. The action p o t e n t i a l duration i n cardiac muscle i s much longer and the s t r u c t u r a l associations between S.R. and T-tubules are present mainly as "Diads" i n contrast to the "Triads" found i n f a s t s k e l e t a l muscle. In the l a t t e r , the T-tubules are l i n e d along t h e i r e n t i r e length by numerous terminal cisternae. Cardiac cisternae are less organized i n shape, r e g u l a r i t y and frequency of couplings to the T-tubules (Lullmann, 1979). However, c a l c u l a t i o n s taking i n t o account the surface areas of S.R. i n the two types of muscle 2+ (Chapman, 1979) show that the actual Ca -sequestering capacities may be of a s i m i l a r magnitude. The a b i l i t y of c a f f e i n e , a compound known to release calcium from the sarcoplasmic reticulum, to produce contractures i n mammalian heart trabeculae, as shown by Chapman and Leoty (1976), i s further evidence for the c r i t i c a l r o l e of S.R. i n t h i s t i s s u e . The strength of these contractures was dependent on the dose of c a f f e i n e and thus suggests a v a r i a b i l i t y i n the amount of calcium released from the S.R. - 5 -Solaro and Briggs (1974) estimated that cardiac S.R. i n vivo i s -9 2+ capable of sequestering 300-400 x 10 M Ca /g heart wet weight which i s more than s u f f i c i e n t to relax the muscle. The general mechanism of t h i s Ca -ATPase system i n cardiac muscle has been elucidated i n d e t a i l (see MacLennan et a l . , 1975): k l ATP + E ATP E k - l (1) K Ca k + ATP E " ^ ATP E Ca _ out out ATP E Ca out ADP E - P Ca ADP E - P Ca out out ADP E - P Ca. i n (2) (3) (4) Over a l l s t o i -chiometry; 1 mole ATP Hydrolyzed per 2+ 2 moles of Ca  taken up ADP E - P Ca. ADP E - P + Ca. i n m (5) 6 + ADP E - P + H 2 0 E + P i + H + ADP m-6 (6) 2-In steps 1 and 2 calcium and substrate (Mg ATP ) bind to the enzyme protein (E) on the outer membrane surface. Step 3 shows the transfer of the terminal phosphate of ATP to an a s p a r t y l residue of the ATPase. E - P represents a phosphorylated intermediate i n both forward and reverse d i r e c t i o n s . Translocation and release of the calcium ion on the inner membrane surface follow i n steps 4 and 5. Hydrolytic phosphate cleavage occurs i n step 6, the rate l i m i t i n g step of the reaction. The possible r o l e of c y c l i c AMP and protein kinase i n t h i s scheme appears to l i e i n - 6 -f a c i l i t a t i o n of calcium binding i n step 2, brought about by a conformational change i n a 22000 Dalton regulatory protein, phospholamban (Tada et a l . , 1975). Phospholamban i s phosphorylated by the c y c l i c AMP/protein kinase system, and responds i n a fashion that a f f e c t s a negative cooperativity between the two Ca binding s i t e s on the 100000 M.W. ATPase, thus favouring an independent, non-sequential binding of the two calcium I | ions, which e f f e c t i v e l y increases Ca transport at low calcium concen-t r a t i o n s . (Katz, 1979). Important regulatory factors of the Ca -ATPase other than the c y c l i c AMP/protein kinase system are monovalent ions ( e s p e c i a l l y Na + and K +) and calmodulin, both of which function to increase the Ca -uptake capacity of i s o l a t e d S.R. membrane preparations (Shigekawa et a l . , 1976; Katz and Remtulla, 1979). CYCLIC NUCLEOTIDES AND CARDIAC CONTRACTION The preceding sections have attempted to show that S.R. i s e s s e n t i a l i n the regulation of cardiac c o n t r a c t i l i t y and that c y c l i c AMP stimulates calcium uptake into S.R. (Entman,1968; Tada et a l . , 1974). Hormones such as catecholamines stimulate production of c y c l i c AMP (Sutherland and R a i l , 1958).It i s no surprise that a l i n e of research attempting to l i n k the above fac t s i n a sequential cause and e f f e c t scheme, has evolved. Adenylate cyclase [ATP pyrophosphate lyase ( c y c l i z i n g ) ] i s a membrane-bound, multiunit enzyme subject to a wide array of regulatory f a c t o r s . The c a t a l y t i c u n i t , located on the i n s i d e of the cell-membrane, can be activated by the action of f l u o r i d e , hormones, guanine nucleotides, divalent ions and t r y p s i n v i a a number of regulatory u n i t s . Mobile hormone - 7 -receptors are located on the outer membrane surface and can i n t e r a c t with an apparently independent pool of c a t a l y t i c subunits (Schramm et a l . , 1977). R e l a t i v e l y l i t t l e i s known about the mechanism by which the enzyme a c t i v i t y i s a ffected by p o s i t i v e and negative e f f e c t o r s . Theories include dephos-phorylation of an active state, conformational changes of the enzyme following the e f f e c t o r binding and involvement of the membrane l i p i d s (Stole, 1979). There i s also evidence that the mechanism of regulation by GTP and i t s analogues involves a GTP-ase located on the guanine regula-tory subunit (Lefkowitz, 1980). Reconstitution experiments with soluble c e l l extracts from s k e l e t a l muscle suggest the existence of a factor or factors conferring f l u o r i d e and guanine nucleotide s e n s i t i v i t y to the c a t a l y t i c subunit (Drummond et a l . , 1980) . SUBCELLULAR LOCALIZATION OF ADENYLATE CYCLASE In the attempt to construct a consistent model of catecholamine regulation of cardiac c o n t r a c t i l i t y , i t was necessary to characterize the cardiac adenylate cyclase i n terms of i t s s u b c e l l u l a r l o c a l i z a t i o n , hormone s e n s i t i v i t y and other regulatory f a c t o r s . This i s s t i l l an area of much controversy and i s also the primary concern of t h i s t h e s i s . Sutherland and Robison (1966)established a number of c r i t e r i a e s s e n t i a l for i d e n t i f i c a t i o n of cAMP as a "second messenger" i n the action of catecholamines. Agents that a l t e r cardiac function v i a cAMP should: a) Produce corresponding changes i n the i n t r a c e l l u l a r l e v e l of cAMP. b) Have an e f f e c t on e i t h e r adenyl cyclase or phosphodiesterase or both. c) A l t e r cAMP l e v e l i n a time course consistent with the tiss u e response. d) Have t h e i r pharmacological actions potentiated by phosphodies-terase i n h i b i t o r s . e) Be mimicked by exogenous a p p l i c a t i o n of cAMP or one of i t s analogues to the tiss u e or c e l l preparation. Some of the most important findings i n support of the "second messenger" hypothesis include: i ) Perfused heart preparations show c l o s e l y r e l a t e d r i s e s i n t i s s u e c y c l i AMP content and c o n t r a c t i l e force a f t e r hormone stimulation by epinephrine and histamine (Cheung, 1965; McNeill et a l . , 1974) . i i ) Adenylate cyclase^'in microsomal preparations from hearts of several species i s stimulated by catecholamines (Murad et a l . , 1962) with an order of potency that corres-ponds to t h e i r i n o t r o p i c potency ; This stimulation can be blocked by 3-adrenergic receptor blocking agents. i i i ) P h o s p h o r y l a s e a c t i v a t i o n i s a l s o found to be associated with increased c o n t r a c t i l i t y and phosphorylase kinase a c t i v a t i o n i s f a c i l i t a t e d by catecholamines i n beating hearts (Drummond and Severson, 1979). i v ) T h e o p h y l l i n e produces an i n o t r o p i c e f f e c t on the heart but i t s actions are d i f f i c u l t to explain on the basis of cAMP alone. In perfused guinea p i g hearts, t h i s agent produces a modest increase i n c o n t r a c t i l i t y and augmentation of phosphorylase but f a i l s to increase cAMP l e v e l s (McNeill et a l . , 19 74). Exogenous e f f e c t s of cAMP on skinned (sarcolemma-free) cardiac muscle f i b r e s are p r i m a r i l y relaxing (Fabiato and Fabiato, 1975b). E x t r a c e l l u l a r cAMP f a i l s to produce an e f f e c t p r i m a r i l y because of the low permeability of sarcolemma to cAMP. This d i f f i c u l t y has been overcome - 9 -by using the d i b u t y r y l d e r i v a t i v e of cAMP which crosses membranes more e a s i l y and produces p o s i t i v e chronotropic and i n o t r o p i c responses (Drummond, 1979). The antagonism by propranolol of catecholamine e f f e c t s on heart i s a d i f f i c u l t subject to resolve since blockade of 3-receptors appears to be only one aspect of i t s action. Noack (1978) found that propranolol interferes s p e c i f i c a l l y with Ca translocation across S.R. i n s k e l e t a l muscle and that a negative i n o t r o p i c e f f e c t i s more c l o s e l y p a r a l l e l to an a b i l i t y to i n h i b i t Ca transport i n t h i s system than 0-adrenergic blocking potency. Katz (1979) also concludes that 3-blockade i s pharmaco-l o g i c a l l y l e s s important than a non-specific membrane s t a b i l i z i n g e f f e c t . The studies on microsomal f r a c t i o n s showing marked augmentation of 2+ S.R. Ca uptake by c y c l i c AMP and exogenous (Tada et a l . , 1974) and endogenous (Wray et a l . , 1973) protein kinases are complemented by the i d e n t i f i c a t i o n of a s p e c i f i c substrate for the phosphorylase activated by catecholamines. A 22000 Dalton S.R. protein, phospholamban (see above), has been i d e n t i f i e d by Tada et a l . (1975) and provides a further l i n k on the molecular l e v e l i n the mechanism of cAMP action. The model i s also strengthened by the observation by Kirchberger and Raffo (1977) that a phosphoprotein phosphatase which dephosphorylates phospholamban i s present 2+ i n the heart c e l l and can reduce the rate of Ca -transport i n sarcoplasmic reticulum. Entman (1969) reported that a s i g n i f i c a n t amount of adenylate cyclase was associated with the microsomal f r a c t i o n of canine heart. This a c t i v i t y was stimulated by epinephrine and glucagon and the epinephrine augmentation was blocked by propranolol. Epinephrine and glucagon were also found to increase calcium accumulation i n t h i s f r a c t i o n . Although further evidence - 10 -for the l o c a l i z a t i o n of a B-receptor and adenylate cyclase i n canine heart sarcoplasmic reticulum has accumulated (Katz e t ' a l . , 1974), adenylate cyclase i n t r i n s i c to these preparations in_ vivo has remained a subject of much controversy, due mainly to the inherent shortcomings of the sub-c e l l u l a r f r a c t i o n a t i o n techniques employed. These techniques employed have the p o t e n t i a l to produce a r t i f i c i a l structures and unpredictable cross-contaminations. Of p a r t i c u l a r i n t e r e s t i s the recent work of Lau et al.,(1977) and Caswell. et a l . , (1979) on rabbit s k e l e t a l muscle microsomal f r a c t i o n s demonstrating structures consisting of two terminal cisternae of sarcoplasmic reticulum attached to a ce n t r a l l o n g i t u d i n a l v e s i c l e , presumably formed by part of the T-tubule system. This structure .can be separated i n t o i t s components by french-press treatment with sub-sequent loss of beta-adrenergic receptor and adenylate cyclase a c t i v i t y (Caswell et a l . , 1978). Due to great differences between s k e l e t a l and cardiac muscle i n u l t r a s t r u c t u r e and response to catecholamines (Bowman et a l . , 1958), experimental r e s u l t s obtained i n one tissue cannot be generalized to the other and one objective of t h i s work was to attempt a separation of components i n cardiac muscle s i m i l a r to Caswell's work. Jones et a l . ( l 9 T 9 ) ' recently presented evidence that adenylate cyclase, (Na +, K +)-ATPase a c t i v i t i e s and beta-adrenergic receptors can be e n t i r e l y accounted for by the sarcolemmal content of microsomal preparations of the heart,although the p u r i f i e d SR s t i l l showed r e s i d u a l adenyl cyclase a c t i v i t y . Since there i s an obvious disagreement between the workers claiming to have found adenylate cyclase i n sarcoplasmic reticulum and those the a t t r i b u t e i t s presence there to sarcolemmal contamination, the controversy appears d i f f i c u l t to resolve. Techniques which avoid these drawbacks of f r a c t i o n a t i n g t i s s u e and subsequent density gradient separation - 11 -of s u b c e l l u l a r organelles include histochemical and cytochemical studies. Unfortunately, the bulk of the data on the s u b c e l l u l a r d i s t r i b u t i o n of adenylate cyclase a c t i v i t y i n a -wide v a r i e t y of t i s s u e s has been obtained by histochemical techniques. The c e l l s are incubated i n lead-containing media which some authors believe to produce a r t i f a c t u a l r e s u l t s due to non-enzymatic, lead-catalyzed formation of c y c l i c AMP i n combination with lead-induced i n h i b i t i o n of adenylate cyclase. Since the cytochemical methods depend on the use of lead ions to p r e c i p i t a t e the inorganic pyrophosphate formed i n the r e a c t i o n , A.C. ATP — • cAMP + PPi, the r e s u l t s are questionable (Kempen, 1978). A more promising technique recently employed by Ong and Steiner (1977) i s based on immuno-cytochemical demonstration of cAMP. This method reveals fluorescent antibody against cAMP i n the area of sarcolemma and sarcoplasmic reticulum i n both s k e l e t a l and cardiac muscle. These findings obtained by an e n t i r e l y independent methodology tend to lend c r e d i b i l i t y to the authors claiming to have found evidence for s u b c e l l u l a r l o c a l i z a t i o n of cAMP i n cardiac S.R. Recent work by Drummond et a l . , (1978) has shown that adenylate cyclase a c t i v i t y present i n rabbit heart v e n t r i c l e microsomal f r a c t i o n s had c h a r a c t e r i s t i c s s i g n i f i c a n t l y d i f f e r e n t from the a c t i v i t y found i n the sarcolemma-enriched washed p a r t i c l e f r a c t i o n i n a number of respects: i ) The' microsomal enzyme was dramatically stimulated by low concen-trations of non-ionic detergents Lubrol PX and Tritox-X-100 while basal a c t i v i t y i n a predominantly plasma membrane containing f r a c t i o n was only moderately stimulated and the f l u o r i d e - a c t i v a t e d enzyme was i n h i b i t e d by these treatments. - 12 -i i ) Microsomal adenylate cyclase a c t i v i t y also responded d i f f e r e n t l y to phospholipase A treatment,guanyl nucleotides and epinephrine stimulation compared to that a c t i v i t y present i n the sarcolemma-enriched f r a c t i o n . These findings were extended by Sulakhe et a l . (1978) who subjected microsomal and sarcolemmal f r a c t i o n s of guinea-pig v e n t r i c l e s to treatment with non-ionic detergents and observed that microsomal adenylate cyclase (basal as well as p [NH]ppG- and NaF-stimulated) was increased over 2-fold i n the presence of Lubrol-PX (0.01-0.1%). The sarcolemmal enzyme, however, showed concentration-dependent i n h i b i t i o n caused by the detergent except when p[NH]ppG was included i n the assay which resulted i n a modest (30-45%) increase i n enzyme a c t i v i t y . Triton-X-100 also stimulated the microsomal adenylate cyclase a c t i v i t y and i n h i b i t e d the a c t i v i t y of the sarcolemmal enzyme. Responses to isoproterenol also d i f f e r e d markedly i n the two f r a c t i o n s ; the sarcolemmal basal enzyme a c t i v i t y was stimulated 2-fold and the microsomal enzyme to a much les s e r degree (30%) . - 13 -OBJECTIVES The objectives of t h i s study were to develop a method to p u r i f y a crude canine cardiac microsomal f r a c t i o n and to study the e f f e c t s of t h i s p u r i f i c a t i o n on adenylate cyclase a c t i v i t y by comparison with a plasma-membrane enriched f r a c t i o n . The s p e c i f i c aims of t h i s project include: 1. To develop a technique of i s o l a t i o n of a homogenous microsomal f r a c t i o n enriched i n sarcoplasmic reticulum with a minimum of plasma membrane contamination. The methods include the discontinuous sucrose density gradient centrifugation of the crude microsomal f r a c t i o n and the determination of r e l a t i v e plasma membrane contamination by ouabain binding and the presence of active sarcoplasmic reticulum by active 2+ Ca -uptake studies. 2. To compare the c h a r a c t e r i s t i c s of basal, NaF-stimulated and epinephrine-stimulated adenylate cyclase a c t i v i t y i n p u r i f i e d sarcoplasmic reticulum, crude sarcoplasmic reticulum and washed p a r t i c l e (plasma membrane-enriched) preparations. The object of these studies was to determine i f any difference exists i n the adenylate cyclase enzyme(s) and t h e i r a b i l i t y to be stimulated dependent on t h e i r c e l l u l a r l o c a l i z a t i o n . These studies include a determination of the r e l a t i o n s h i p of divalent cations 2+ 2+ (Mg and Ca ) and GTP to adenylate cyclase stimulation i n these d i f f e r e n t membrane f r a c t i o n s . 2+ 3. To determine the use of Ca -loading as a f e a s i b l e technique of preparing sarcoplasmic reticulum free of sarcolemma. Recent reports i n the l i t e r a t u r e (see Methods) indi c a t e that loading - 14 -of microsomal v e s i c l e s with calcium w i l l allow the separation of i n t a c t r i g h t - s i d e out v e s i c l e s from leaky microsomes and membrane fragments. These studies constitute a continuation of techniques i n v o l v i n g density gradient centrifugation and determine i f adenylate cyclase a c t i v i t y and 2+ Ca -transport a c t i v i t y can be e f f e c t i v e l y measured i n t h i s preparation - 15 -METHODS AND MATERIALS PREPARATION OF DOG MICROSOMAL FRACTIONS ENRICHED IN SARCOPLASMIC RETICULUM Frozen pieces of dog v e n t r i c l e (approximately 4 g) were f i n e l y cut and homogenized i n 40 ml of 10 ml NaHCO^, 5 mM NaN3 and 0.2 mM ascorbic acid, pH 6.8, using a motorized homogenizer (Fisher DYNAMIX) and a t e f l o n pestle at 1500 rpm for 30 sec. The homogenate was then centrifuged at 12,000 x g for 15 min and the supernatant c o l l e c t e d and centrifuged at 45,000 x g for 50 min. The r e s u l t i n g p e l l e t was washed i n a medium containing 0.6 M KC1 and f i n a l l y i n 10 mM T r i s - C l , pH 7.4. The p e l l e t was suspended i n 10% sucrose with 10 mM T r i s - C l , pH 7.4 and t h i s f r a c t i o n designated as the microsomal f r a c t i o n . A portion of t h i s f r a c t i o n was then layered onto a discontinuous sucrose gradient of 25, 28, 32.7 and 35% (w/v) sucrose, pH 7.0, previously p u r i f i e d by passage through a Dowex 50-X8 column. P u r i f i c a t i o n of the sucrose was c a r r i e d out to remove trace impurities that have been found to r e s u l t i n loss of enzymatic a c t i v i t i e s of the f r a c t i o n s obtained (Carsten, 1964). In the studies using a French press before density gradient c e n t r i f u g a t i o n , the microsomal f r a c t i o n was separated into two portions, one of which was passed through an Aminco French pressure c e l l at 15,000 P.S.I, and 4°C. The sucrose gradient was centrifuged at 105,000 x g for 60 min i n an s.w.-TI 41 rotor using a Beckman L2-65 u l t r a c e n t r i f u g e . The r e s u l t i n g bands were c o l l e c t e d using a pasteur pipe t t e and d i l u t e d 1:4 i n 10 mM I | T r i s - C l , pH 7.4. Except for the Ca uptake experiments, f r a c t i o n s were quickfrozen and stored at -70°C . The sucrose present i n these f r a c t i o n s (representing 2-3% i n the f i n a l incubation volumes of the assay procedures that - 16 -followed) was not washed out as i t was found that these f r a c t i o n s maintained 2+ Ca -accumulating capacity for longer periods of time when stored i n t h i s manner. PREPARATION OF A WASHED-PARTICLE FRACTION This f r a c t i o n represents an e a s i l y sedimentable f r a c t i o n enriched i n plasma membranes and was prepared according to Drummond (1978). Frozen pieces of dog v e n t r i c l e (approximately 8 gm) were homogenized using a Polytron P20 homogenizer (5 strokes of 10 sec t o t a l duration at setting 5) in 20 ml of 10 mM NaHCOg, 5 mM NaNg and 0.2 mM ascorbic acid, pH 6.8 and centrifuged at 12,000 x g for 15 min. The p e l l e t was resus-pended i n a medium containing 0.6 M KC1 and homogenized using a Polytron P20 homogenizer (5 strokes at se t t i n g 5 f or a t o t a l duration of 30 sec and 2 sec at s e t t i n g 7) and centrifuged at 24,000 x g for 10 min. The r e s u l t i n g p e l l e t was resuspended i n 5 ml of 10 mM T r i s - C l , pH 6.8 and homogenized again with the Polytron (30 sec at setting 4) and centrifuged at 24,000 x g for 10 min. The p e l l e t was suspended i n 40% sucrose and d i l u t e d 1:4 in 10 mM T r i s - C l , pH 6.8 before use. In the following sections of t h i s t h e s i s , crude S.R. w i l l r e f e r to the microsomes obtained from the 45,000 x g p e l l e t ( F i g . 2) and " p u r i f i e d " or "Pure S.R." w i l l designate the f r a c t i o n on the 32.7/35% int e r f a c e on the " c o n t r o l " tube i n F i g ; 2. PREPARATION OF SARCOLEMMAL MICROSOMES The procedure used to i s o l a t e t h i s f r a c t i o n was s l i g h t l y modified from the one used by Van Alstyne (1979). Frozen pieces of dog v e n t r i c l e - 17 -were homogenized i n the NaHCO^/NaN^ buffer as described above and c e n t r i -fuged at 8700 x g for 20 min. The r e s u l t i n g p e l l e t was suspended i n the o r i g i n a l volume of the buffer and rehomogenized i n a Kontes glass homo-genizing vessel with a motor driven (Fisher DYNA-Mix) glass pestle with 10 passes at medium speed. This homogenate was then centrifuged at 8700 x g f o r 20 min. The two supernatants were combined and centrifuged at 35,000 x g for 20 min. The p e l l e t was suspended i n approx. 30 ml of 10 mM T r i s - C l with 0.6 M KC1 pH 7.4 using a glass homogenizing vessel and a hand-driven t e f l o n p e s t l e . The suspension was centrifuged at 75,000 x g for 20 min. The p e l l e t was resuspended i n 0.25 M sucrose, 10 mM T r i s C l , pH 8.2 containing 2 mM DTT and layered on top of a 22% (w/v) sucrose s o l u t i o n containing 20 mM T r i s - C l , pH 7.4. Centrifugation at 75,000 x g for 30 min yielded sarcolemmal membranes at the top of the 22% sucrose s o l u t i o n . These were aspirated, d i l u t e d with the 22% sucrose s o l u t i o n to 0.3 - 0.8 mg protein per ml, quickfrozen on methylbutane/dry i c e and stored at -70°C f o r further use. OUABAIN-BINDING ASSAY The method of Gelbart and Goldman (1977) was u t i l i z e d . Membranes (1.0 - 2.0 mg/ml) were incubated with 10 M [ H]-ouabain ( s p e c i f i c a c t i v i t y 12.7 Ci/mole) i n the presence of 3 mM MgC^, 100 mM NaCl, 1 mM EGTA, 25 mM T r i s - C l , pH 7.4, i n the presence and absence of 3 mM T r i s -ATP. Following 10 min of incubation at 37°C, an aliqu o t of the reaction mixture was f i l t e r e d through a M i l l i p o r e f i l t e r (0.45 uM) to separate 3 3 unbound [ H]-ouabain from tissue-bound [ H-]^ouabain. The f i l t e r s were washed and dried, dissolved i n Aquasol counting medium and assayed using 3 l i q u i d s c i n t i l l a t i o n counting techniques. Non-specific [ H]-ouabain - 18 -binding, determined i n the absence of ATP, i s subtracted from the t o t a l binding to determine the degree of ATP-dependent binding. ASSAY OF ADENYLATE CYCLASE ACTIVITY For the measurement of c y c l i c AMP accumulation, membranes (1.0 - 2.0 mg/ml) were incubated with 50 mM Tris-HCl, pH 7.4, 1 mM DTT, 1 mM EDTA, 5 mM theophylline, 10 mM KC1, 5 mM MgC^ (unless otherwise stated), 20 mM phosphoenolpyruvate and 0.35 mg/ml pyruvate kinase, 0.01 mM GTP, 2.5 mM Tris-ATP i n the presence and absence of NaF (10 mM) or Epinephrine (0.01 mM or 0.1 mM) i n a f i n a l volume of 0.25 ml. Following a 10 min preincubation period at 37°C, the reaction was started by the addition of ATP and terminated by placing the samples i n a b o i l i n g water bath. The samples are then centrifuged at 3,000 x g and aliquots of the clear supernatant assayed for the presence of c y c l i c AMP using the Gilman assay (1970). C y c l i c AMP accumulated i n the absence of added ATP was subtracted from the r e s u l t s obtained i n each case.. 2+ In the experiments where the concentration of free Ca was varied, free ion concentrations were obtained by so l v i n g simultaneous equations 2+ 2+ for Ca -EGTA and Mg -EGTA complexes. A l l s t a b i l i t y constants used were obtained from M a r t e l l (1971) and equations from Katz (1970) and Rodan et a l . , (1976). In these experiments, quickfrozen membranes were 2+ used i n the incubation and the Mg /ATP r a t i o was 10:1. CALCIUM LOADING OF CRUDE MICROSOMES The calcium loading was performed by a method adapted from Bonnet et a l . (1978), who have defined conditions where only the most active - 19 -calcium-accumulating v e s i c l e s were loaded and "inside out" v e s i c l e s or "leaky membranes" were eliminated by v i r t u e of lower density a f t e r c e n t r i f u g a t i o n . The "crude" f r a c t i o n described above was suspended i n 40% sucrose (w/v) to give a protein concentration of approximately 1-2 mg/ml. One ml of thi s preparation was d i l u t e d i n 50 ml of a loading medium containing 0.2 mM Ca C l 2 , 150 mM KC1, 30 mM HP0 4 buffer at pH 7.0, 7.5 mM MgCl 2, 5 mM DTT at 20°C. A c o n t r o l preparation was incubated i n an i d e n t i c a l manner except for the absence of C a C l 2 i n the medium. The suspension was immediately centrifuged at 12,000 x g for 30 min at 0°C. The p e l l e t s were suspended i n 1 ml of a buffer containing 10% sucrose, 30 mM phosphate buffer, 5 mM DTT at pH 7.0 and layered on a 28%/32.7%/35%/38% w/v sucrose density gradient. Fractions obtained at the in t e r f a c e s were withdrawn and d i l u t e d to approximately 50 yg protein/ml i n buffer deaerated by N 2 and containing 10 mM MES buffer, 100 mM MgCl 2 and 5 mM DTT at pH 6.0. Unloading was accomplished by incubation i n t h i s medium at 10°C for 120 minutes under anaerobic conditions to minimize oxidative i n a c t i v a t i o n of enzymatic components. Af t e r unloading the v e s i c l e s were concentrated by a 105,000 x g cent r i f u g a t i o n f o r 60 minutes and resuspended i n 40% sucrose containing 2 mM DTT. ASSAY OF Ca 2 +-UPTAKE AND ATP-DEPENDENT Ca 2 +-UPTAKE The assay was c a r r i e d out by the method of Tada et a l . (1974) with 2+ a few modifications. Ca -uptake was determined i n an incubation medium containing 40-50 yg protein of the microsomal preparation, 40 mM h i s t i d i n e , - 20 -pH 6.8, 5 mM MgCl2, 110 mM KC1, 5 mM Tris-ATP, 2.5 mM-Tris oxalate 45 and CaC^ containing [ C a j C ^ (10 Ci/mole). The desired free calcium concentration was maintained by the addition of ethylene-bis (3-aminoethyl ether) N,N'-tetra acetate (EGTA) and the free calcium concentrations present determined by the equations of Katz et a l . (1970). Following a preincubation of 7 min. at 30°C, the reaction was started by the 45 addition of [ C a j C ^ . A f t e r 5 min, the reaction was terminated by f i l t e r i n g an aliquot of the reaction mixture through a m i l l i p o r e f i l t e r (0.45 yM) . The f i l t e r was then washed twice with 15 ml of 40 mM T r i s - C l , pH 7.2 and dried and counted f o r r a d i o a c t i v i t y i n Aquasol using standard l i q u i d s c i n t i l l a t i o n counting techniques. When c y c l i c AMP-dependent protein kinase (Sigma type 1) and c y c l i c AMP were present i n the reaction mixture, the concentrations used were 0.25 mg/ml and 1 yM, res p e c t i v e l y . Protein concentrations were measured by the method of Lowry et a l . (1951). Student's " t " test f o r unpaired, common variance data was used as a measure of s i g n i f i c a n c e . Standard error of the mean (S.E.M.) was used as a measure of v a r i a t i o n . MATERIALS A l l chemicals used were reagent grade. Tris-ATP, C y c l i c AMP, GTP, EGTA, bovine cardiac protein kinase (type 1) and epinephrine were 45 purchased from Sigma Chemical Co., St. Louis, Mo. [ C a J C ^ and the c y c l i c AMP assay k i t s were obtained from Amersham-Searle Co. Aquasol was purchased from New England Nuclear Company and M i l l i p o r e f i l t e r s (HA 45) were obtained from the M i l l i p o r e Co. - 21 -RESULTS ISOLATION OF DOG HEART MICROSOMAL FRACTIONS A. Standard sucrose gradient c e n t r i f u g a t i o n Centrifugation of the crude microsomal f r a c t i o n on a 25, 28, 32.7 and 35% discontinuous sucrose gradient yielded f r a c t i o n s at each i n t e r -phase. The most abundant protein y i e l d was observed at the 32.7/35% interphase. The top layer was v i s i b l e as an opaque white band and a l l lower layers were found to be brownish i n colour. The reason for using the two sucrose concentration of such close d e n s i t i e s (32.7% and 35%) was that i t permitted i s o l a t i o n of a highly homogeneous microsomal f r a c t i o n . B. E f f e c t of calcium loading In t h e i r work on rabbit s k e l e t a l muscle, Bonnet et a l . (1978) chose conditions where the external amount of calcium i n the incubation medium was w e l l below the whole capacity of the v e s i c u l a r material to accumulate the cation; therefore, v e s i c l e s d i s p l a y i n g the highest rate of uptake were heavily loaded by sequestering a l l of the a v a i l a b l e cation before 2+ the l e s s a c t i v e accumulated a s i g n i f i c a n t amount of Ca that would increase t h e i r density. When s i m i l a r conditions were chosen for the 2+ Ca -loading of dog heart microsomes i n t h i s work, the microsomes at the 32.7% - 35% interphase exhibited a high degree of homogeneity i n 2+ t h e i r a b i l i t y to accumulate Ca F i g . 1 shows the bands obtained i n a control and calcium-loaded microsomal preparation. V i r t u a l l y the e n t i r e band of pure SR s h i f t e d from the top of the 35% sucrose layer to the top of the 40% layer a f t e r 2+ the Ca -loading, i n d i c a t i n g that the majority of the v e s i c l e s i n t h i s - 22 -C o n t r o l L o a d e d p e r c e n t s u c r o s e . 1. Effect of calcium loading on sucrose density gradient  banding patterns The above photograph depicts the effects of incubating the crude microsomal fraction in a medium containing 200 mM CaCl 2, 150 mM KC1, 30 mM phosphate buffer, pH 7.0, 7.5 mM MgCl2 and 5 mM DTT at 20°C. An identical control preparation was incubated in the absence of CaCl2. Fractions were layered on the gradients after repelleting at 12000 x g and resuspension in 10% sucrose, 30 mM phosphate, 5 mM DTT, pH 7.0. The figure shows one of three identical preparations. - 23 -gradient have a high calcium-accumulating capacity. It could be concluded that the l e s s dense band l e f t on top of the 35% layer probably constitutes impurities consisting of leaky and/or inside-out sarcoplasmic reticulum microsomes and possibly some sarcolemmal v e s i c l e s . Inside-out sarcolemmal 2+ v e s i c l e s can also accumulate Ca a c t i v e l y and one would expect them to behave, i n a s i m i l a r fashion as the SR microsomes. They could possibly 2+ contaminate the pure" preparation. However, the rates of Ca accumula-tio n that have been measured i n the inside-out sarcolemmal preparations are t y p i c a l l y one order of magnitude le s s than those i n S.R. (Morcos and Drummond, 1980; Tada et a l . , 1974) and therefore i t i s l i k e l y that they 2+ would also have been separated during Ca loading. Attempts to "unload" the f r a c t i o n s (see methods) i n v a r i a b l y resulted i n a loss of calcium-accumulating a b i l i t y compared to not previously loaded, fresh microsomes. On the basis of the r e l a t i v e l y low contamination evident i n the pure f r a c t i o n as demonstrated by the calcium loading study i t was decided not to include the loading as a p u r i f i c a t i o n step i n subsequent experiments. C. E f f e c t s of French press treatment Two passes of the crude microsomal suspension through an Aminco French pressure c e l l at 15,000 P.S.I, and 4°C produced a p a r t i a l c l e a r i n g of the f r a c t i o n and resulted i n a r e d i s t r i b u t i o n of protein bands i n the gradient. Whereas the control preparation showed a white band at the 10/25 i n t e r f a c e and brownish bands at the 25/32.7 and 32.7/35 i n t e r f a c e s , the French press-treated microsomes showed only brown bands throughout the gradient. (Fig. 2). E a r l i e r preparations at 5000 P.S.I, f a i l e d to produce any e f f e c t s on the protein d i s t r i b u t i o n of the gradient. - 24 -l O g V E N T R I C L E m l H O M O G E N A T E i s a a a x g P I 4 5 Q Q O x g K C I C.BM3 T R I S - C I C.Q1M) W A S H E S 8 m l C R U D E S .R . _ J 1 0 5 0 0 Q x g C O N T R O L 1 5 OOO P.S.I. no 3 S . 7 F1S- 2 - Scheme f o r p r e p a r a t i o n o f p u r i f i e d S.R. a n d F r e n c h p r e s s - t r e a t e d S.R. T h e b a n d s s h o w n i l l u s t r a t e t h e c h a n g e s i n b a n d i n g p a t t e r n a f t e r F r e n c h p r e s s t r e a t m e n t ( r i g h t ) t h i c k n e s s o f t h e b a n d s s h o w n i s a p p r o x i m a t e l y p r o p o r t i o n a l t o p r o t e i n c o n t e n t . - 25 -CHARACTERIZATION OF DOG MICROSOMAL FRACTIONS OBTAINED BY SUCROSE DENSITY  CENTRIFUGATION A. Calcium uptake studies Table 1 shows the r e l a t i v e rates of calcium accumulation i n the presence of oxalate by the microsomal f r a c t i o n s recovered from the sucrose density gradient. Calcium uptake capacity of the various f r a c t i o n s was stimulated to d i f f e r e n t degrees by optimal concentrations of c y c l i c AMP and c y c l i c AMP-dependent protein kinase. The f r a c t i o n at the 32.7/35% i n t e r f a c e showed the highest s p e c i f i c a c t i v i t y and was also stimulated to the greatest extent by the c y c l i c AMP/protein kinase system. Since calcium uptake a c t i v i t y was assumed to cor r e l a t e with the p u r i t y of the sarcoplasmic reticulum microsomes, the 32.7/35% i n t e r -face f r a c t i o n was chosen as p u r i f i e d sarcoplasmic reticulum. The amount of calcium accumulated by t h i s f r a c t i o n was l i n e a r with time and amount of microsomal protein used (G. Lopaschuk, unpublished observation). B. Ouabain binding studies The amount of ouabain bound was proportional to the amount of micro-somal protein i n the incubation procedure (Fig. 3) and the time of incuba-t i o n (not shown). Attempts to determine s p e c i f i c binding i n washed p a r t i c l e s was unsuccessful due to the excessive nonspecific binding a c t i v i t y present probably due to f i n e l y dispersed p a r t i c u l a t e matter generated during the vigorous polytron homogenization step. When 0.3 mg SDS (sodium dodecyl sulfate) was added to the reaction mixture i n an attempt to expose possible latent ouabain binding a c t i v i t y , no s p e c i f i c ouabain binding could be detected. I t appears that the concentration of I . O - 26 -.5 D Z D 0 03 Z 3 o < D 0 cn UJ J o / o • 1 1 1 1 X PROTEIN Fig. 3. Protein concentration-Dependence of ouabain binding. Values re-present specific binding (binding In presence of 3mM TRIS-ATP minus binding in absence of ATP.) Crude S.R:• Pure S.R:B The values represent duplicate determinations on a typical preparation. - 27 -TABLE 1 Characterization of dog cardiac microsomal f r a c t i o n s obtained by  sucrose density c e n t r i f u g a t i o n Fraction protein Ca -uptake average mg/ml nmoles/mg/min % stimulation - c y c l i c AMP + c y c l i c AMP (4 expmts.) - protein + protein kinase kinase crude microsomal 2.46 2.40 4.97 (107) a 80 10/25 sucrose i n t e r f a c e 0.77 2.58 3.21 (24) 33 25/28 sucrose i n t e r f a c e 0.89 6.25 8.81 (41) 67 28/32.7 sucrose i n t e r f a c e 0.89 7.27 10.05 (38) 52 32.7/35 sucrose i n t e r f a c e 2 . 2 8 6.81 12.69 (86) 93 Microsomal f r a c t i o n s prepared as described i n methods were fractionated on a discontinuous sucrose density gradient. The r e s u l t i n g protein bands recovered at the % sucrose interfaces indicated were compared to the crude microsomal preparation. The r e s u l t shown i s a t y p i c a l experiment of 4 s i m i l a r experiments. refe r s to the % stimulation of Ca -uptake i n the presence of c y c l i c AMP-dependent protein kinase. - 28 -detergent used by Jones et a l . (1979) may expose Na'-K' ATPase a c t i v i t y but i s unsuitable for the conditions of the ouabain binding assay used i n t h i s work. Since s o l u b i l i z a t i o n of the microsomes was probably responsible for the loss of a c t i v i t y (the membranes were washed through the 45 micron m i l l i p o r e f i l t e r ) , i t was decided to vary the concentration of SDS (from 0.001 to 0.5 mg/ml). The r e s u l t s (not shown) proved to be e r r a t i c and due to lack of consistency i n four preparations, i t was decided to discontinue the use of SDS. Table 2 shows the s p e c i f i c a c t i v i t i e s of selected microsomal f r a c t i o n s and percent recovery of the t o t a l amount of ouabain bound i n the 12,000 x g supernatant (SI). The microsomes prepared by the method of V. Alstyne (1979) ("22% sucrose membranes") showed the highest a c t i v i t y , i n d i c a t i n g a r e l a t i v e enrichment of t h i s f r a c t i o n with sarcolemmal membranes. The effectiveness of density gradient c e n t r i -fugation was demonstrated by an approximately threefold reduction of s p e c i f i c binding i n p u r i f i e d S.R. compared to crude S.R. (P < 0.001). French-press.treatment of the crude microsomal f r a c t i o n further reduced the a c t i v i t y of the plasma membrane marker by approximately 50% (P < 0.05), compared to the untreated "pure" S.R. The mechanism of t h i s p u r i f i c a t i o n may resemble that reported by Caswell et a l . (1978) who separated T-tubules bound to terminal cisternae ( i n s k e l e t a l muscle) with French press treatment. If cardiac S.R.-T-Tubule aggregates (consisting of diads) behave s i m i l a r l y to t h e i r counterparts (triads) i n s k e l e t a l muscle, t h i s would account for the a b i l i t y to reduce sarcolemmal markers by French press treatment. It i s , however, conceivable that French-press treatment merely fragmented the membranes to such a small s i z e that they were not retained by the 45u m i l l i p o r e TABLE 2 S p e c i f i c a c t i v i t i e s of ouabain binding of microsomal f r a c t i o n s Fraction Ouabain Binding p moles ouabain bound mg protein . min Percent Recovery N SI .059 ± .009 100 3 S2 <0.004 15 6 22°/Sucrose Membranes (V. Alstyne,1979) 1.69 ± 0.16 2 Crude S.R. 1.12 ± .09 36 6 Pure S.R. 0.41 ± .04 a 6 6 Pure S.R. (French Press-Treated) 0.18 ± .05 b 1 6 The values represent the average ± S.E.M. of N separate determinations c a r r i e d out on two i d e n t i c a l preparations a indicates s i g n i f i c a n t l y d i f f e r e n t from crude S.R. (p<.001) b indicates s i g n i f i c a n t l y d i f f e r e n t from pure S.R. (p<0.05) - 30 -f i l t e r which would represent an a r t i f a c t u a l reduction of H-ouabain retained on the f i l t e r . This p o s s i b i l i t y was not excluded i n t h i s work. However, Lau et a l . (1977) showed by the use of freeze-fracture electronmicroscopy that although the as s o c i a t i o n between t-tubules and terminal cisternae was disrupted, the microsomes retained t h e i r s t r u c t u r a l i n t e g r i t y a f t e r French-press treatment. This f i n d i n g reduces the likelyhood of an a r t i f a c t u a l reduction of ouabain binding i n t h i s study. The recovery figures were based on the t o t a l amount of ouabain bound by the low speed supernatant, SI, since crude homogenate and washed p a r t i c l e data proved to be inconsistent. About 85% of the binding was present i n the 45,000 x g p e l l e t P2. The KC1 and T r i s - C l washes eliminated an a d d i t i o n a l 49% and thus 36% was present i n the crude microsomes. Pure S.R. contains approximately 6% of the o r i g i n a l binding which was further reduced to 1% by French press treatment. Approximately 2% can be recovered at the 22% sucrose layer when sarcolemma was prepared as described i n the methods. Presumably, the 27% not accounted for by the f r a c t i o n s was either d i s t r i b u t e d i n the rest of the gradient above the p u r i f i e d f r a c t i o n or denatured. In addition to the measurements of ouabain binding, analysis of basal, epinephrine-stimulated and NaF-stimulated adenylate cyclase a c t i v i t y i n the 32.7/35% f r a c t i o n before and a f t e r French press treatment indicated a s t a t i s t i c a l l y s i g n i f i c a n t decrease i n ouabain binding i n a l l prepara-tions and an increase i n s p e c i f i c basal adenylate cyclase a c t i v i t y . (Fig. 3a). The decrease i n ouabain binding i s most l i k e l y a r e f l e c t i o n of an actual l o s s of sarcolemmal contamination but could be a flaw i n the methodology as mentioned above. Due to inconsistency i n epinephrine-- 31 -Fig. 3a. Effect of 15000 P.S.I. French press treatment on cyclic AMP  accumulation ln purified S.R. enriched microsomes from dog  heart. Microsomes were passed twice through an aminco French pressure c e l l at 15000 P.S.I, at 4°C. The fraction used was from the 32.7/35% Interface on the sucrose density gradient. Key. Basal activity I 1 A • Control 10 Si epinephrine B - French press treated lO'^NaF lllllllllll Ouabain binding -The values were obtained from a typical preparation of a total of 3 preparations and represent averages of 3 determinations ± S.E.M. denotes s t a t i s t i c a l l y d i f f e r e n t at p < 0.05. TO U) - 33 -and NaF-stimulation between preparations, no conclusions regarding the ef f e c t s of French-press treatment on these parameters could be drawn. The increase i n basal cAMP accumulation may be due to a p u r i f i c a t i o n of an adenylate cyclase i n S.R. or to loss of i n a c t i v e contaminating proteins during the French-press treatment. C. C y c l i c AMP accumulation i n preparations from dog cardiac v e n t r i c l e  - Hormonal stimulation Table 3 shows an i n i t i a l series of experiments performed to elucidate the c h a r a c t e r i s t i c s of cAMP accumulation of the crude microsomal f r a c t i o n , the 32.7/35 sucrose i n t e r f a c e subfraction and a washed-particle f r a c t i o n consisting mainly of plasma membranes (Drummond et a l . , 1978). C y c l i c AMP accumulation i n the washed-p a r t i c l e f r a c t i o n was s i g n i f i c a n t l y stimulated by epinephrine (10 ^ -4 and 10 M) and by NaF (10 mM) (p < 0.05 i n both cases). It was also shown that c y c l i c AMP accumulation i n the crude microsomal f r a c t i o n was s i g n i f i c a n t l y stimulated by 10 mM NaF (p < 0.05) and moderately though -5 -4 not s i g n i f i c a n t l y stimulated by epinephrine (10 - 10 M). The basal l e v e l of c y c l i c AMP accumulated i n the 32.7/35 sucrose i n t e r f a c e micro-somal subfraction was s i m i l a r to that observed i n the two other prepara-tions. It was observed, though, that epinephrine did not augment the degree of c y c l i c AMP accumulation i n t h i s f r a c t i o n ; NaF tended to stimulate c y c l i c AMP accumulation i n t h i s f r a c t i o n but the degree of stimulation observed was not s t a t i s t i c a l l y s i g n i f i c a n t . - 34 -TABLE 3 C y c l i c AMP accumulation i n preparations from dog cardiac v e n t r i c l e C y c l i c AMP accumulation (pmoles/mg/min) Preparation Basal Epinephrine NaF Washed-particle Crude microsomal 32.7/35 microsomal subfraction 43.7 ± 4.5 67.7 ± 5.8 51.8 ±14.0 0.01 mM 65.7 ± 3.7* 92.5 ± 9.3 55.2 ±18.6 0.10 mM 98.9 ± 15.3£ 101.8 ± 1 3 . 7 37.1 ± 14.5 10 mM 83.1 ± 10.1£ 197.5 ± 27.6l 101.3 ± 34.9 Fractions of dog cardiac v e n t r i c l e prepared as described i n methods were assayed f o r c y c l i c AMP accumulation i n the presence and absence of; no addition, 0.01 and 0.1 mM epinephrine and 10 mM NaF. Results shown represent the mean ± S.E.M. of at le a s t 3 preparations i n each case. ci S i g n i f i c a n t when compared to basal accumulation (p<0.05) k S i g n i f i c a n t when compared to basal accumulation (p<0.02) - 35 -2+ 1. E f f e c t of Mg /ATP r a t i o and GTP on c y c l i c AMP accumulation The a c t i v e adenylate cyclase complex contains separate binding u n i t s 2+ for agonist, p o s i t i v e regulators (guanine nucleotides and Mg ), substrate, negative regulator, associated proteins and adenosine (Stole, 1980). 2+ Figure 4 shows the e f f e c t of varying the Mg /ATP r a t i o on c y c l i c AMP 2+ accumulation. A trend of increasing responsiveness to high Mg /ATP r a t i o s i n the order of washed p a r t i c l e s < crude microsomes < pure S.R. was found. To optimize the expression of p u r i f i e d S.R. adenylate cyclase 2+ a c t i v i t y , a 10:1 Mg /ATP r a t i o was chosen for a l l subsequent studies. A 3.5:1 r a t i o produced saturation i n the adenylate cyclase a c t i v i t y of 2+ washed p a r t i c l e s and a 5:1 i n crude microsomes whereas at 10:1 Mg /ATP the p u r i f i e d S.R. enzyme was s t i l l not saturated. It was also observed -6 -4 that GTP (10 -10 M) was required for maximal c y c l i c AMP accumulation i n both crude and pure f r a c t i o n s (data not shown) and 10 ^ M GTP was included i n a l l assays shown. It i s possible that the combination of 2+ GTP and a 10:1 Mg /ATP i s not n e c e s s a r i l y a d d i t i v e since Alvarez et a l . , (1977) have shown a decrease of the magnesium ion requirement of cardiac 2+ adenylate cyclase by epinephrine and suggest an i n t e r a c t i o n of Mg with the nucleotide (GTP) s i t e . P ersistent stimulation of the enzyme by 2+ p[NH]ppG or f l u o r i d e ion also involves a decrease i n the Mg ion requirement and an increase i n maximum v e l o c i t y . Due to the complexity of these i n t e r a c t i o n s and the f a c t that our studies were not c a r r i e d out on a p u r i f i e d enzyme, the i n t e r a c t i o n s 2+ between GTP and Mg ion were not further investigated. 1 2 3 .5 5 DAQ/ATE> RATIO Fig.4 : E f f e c t of Mg /ATP r a t i o on c y c l i c AMP accumulation i n cardiac membrane preparations. C y c l i c AMP accumulation was determined as described i n F i g . 1 i n the presence of 0.1 mM GTP i n the absence of epinephrine at various Mg^+ .: ATP r a t i o s (ATP concentration maintained at 2.5 mM). So l i d l i n e s indicate the washed p a r t i c l e preparations and the hatched l i n e s Indicate the microsomal sub-fractions.Open c i r c l e s i n d i c a t e p u r i f i e d S.R., s o l i d t r i a n g l e s denote crude microsomes and s o l i d c i r c l e s indicate the washed p a r t i c l e s . The r e s u l t s shown are the mean ± S.E.M. of 3 experiments on d i f f e r e n t microsomal preparations. - 37 -2. E f f e c t of free Ca ion on basal and hormone-stimulated c y c l i c AMP  accumulation i n microsomal f r a c t i o n s and washed p a r t i c l e s Under standard assay conditions (see Methods), the p u r i f i e d S.R. -4 showed inconsistent responses when incubated with 10 M epinephrine. Values ranged from mild i n h i b i t i o n (-15%) to 50% stimulation. One possible explanation f o r t h i s v a r i a b i l i t y from preparation to preparation was thought to be a poorly controlled concentration of free calcium ion i n t h i s f r a c t i o n . In the microsomal f r a c t i o n , a high l e v e l of indigenous calcium retained in s i d e the v e s i c l e s might have contributed to the alt e r e d epinephrine response. It was thus decided to conduct a series of experiments over a large range of co n t r o l l e d free calcium concentrations (0-500 yM). EGTA, at 0.5 mM was assumed to reduce free calcium ion to v i r t u a l l y zero. The two preparations d i f f e r e d i n two important respects (see Table 4): 1) In the absence of epinephrine, the washed p a r t i c l e f r a c t i o n was s i g n i f i c a n t l y i n h i b i t e d by free calcium concentrations above 50 yM whereas the 32.7/35% S.R. microsome enzyme was not s i g n i f i c a n t l y i n h i b i t e d 2+ except at 50 yM [Ca ]. 2+ 2) At high [Ca ], epinephrine stimulation tended to be l o s t i n the S.R. whereas the washed p a r t i c u l a t e f r a c t i o n retained a s i g n i f i c a n t 2+ stimulation i n t h i s region of [Ca ]. 2+ Of p a r t i c u l a r i n t e r e s t are the values at 500 yM [Ca ]: The washed p a r t i c l e f r a c t i o n s t i l l shows a s i g n i f i c a n t increase i n a c t i v i t y by epinephrine (p < .05) (from 37% to 114% of basal value) but the S.R. i s not affected by the hormone (70% vs 79%). - 38 -TABLE 4 2+ Ef f e c t of [Ca ] on c y c l i c AMP accumulation i n f r a c t i o n s from dog heart uM WASHED PARTICLES PURE S.R. f r e e C a _ 4 _ 4 Basal 10 M epinephrine Basal 10 M epinephrine zero 100 (0) 169 ( 3 0 ) b 100 (0) 203 ( 4 3 ) b 1 95 (12) 149 ( 2 5 ) b 90 (2) 162 ( 2 9 ) b 5 90 (9) 152 ( 2 9 ) b 75 (11) 164 ( 2 7 ) b 10 84 (9) 149 (30) 82 (4) 156 ( 2 7 ) b 25 88 (12) 160 (21) 81 (7) 134 (33) 50 77 ( 3 ) a 161 (23) 81 ( 3 ) a 126 (34) 100 81 ( 3 ) a 162 (61) 90 (17) 112 (24) 250 69 (20) 137 (23) 82 (5) 114 ( 2 1 ) a 500 37 ( I D 3 114 ( 2 0 ) b 70 (16) 79 ( 1 8 ) a C y c l i c AMP accumulation was determined as described i n "methods" i n the presence of 0.1 mM GTP and i n the presence and absence of epinephrine. 2+ [Mg ] was 25 mM and [ATP] was 2.5 mM. The data was obtained from 3 i d e n t i c a l preparations with i n d i v i d u a l measurements ca r r i e d out i n duplicate. Values shown represent % of cAMP accumulation (± S.E.M.) i n the absence of epine-phrine and calcium (Zero C a 2 + = 0.5 mM EGTA). a 2+ indicates s i g n i f i c a n t l y d i f f e r e n t from value at zero [Ca ] (p<.05) b i n d i c a t e s s i g n i f i c a n t l y d i f f e r e n t from value i n absence of epinephrine (p<0.05) S t a t i s t i c a l analysis was performed with a two sample, paired observa-t i o n t - t e s t . - 39 -Although not s t a t i s t i c a l l y s i g n i f i c a n t due to the li m i t e d number 2+ of preparations, i t appears that at low [Ca ] (0-25 uM) and i n the presence of epinephrine, the S.R. adenylate cyclase i s more s e n s i t i v e „ 2+ . , . . to Ca i n h i b i t i o n . - 40 -DISCUSSION The microsomal f r a c t i o n i s o l a t e d by a combination of d i f f e r e n t i a l and sucrose density gradient c e n t r i f u g a t i o n was found to contain s i g n i -f i c a n t l y l e s s plasma membrane contamination than the f r a c t i o n i s o l a t e d by 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 techniques alone. Evidence i n support of t h i s claim i s derived from the s i g n i f i c a n t l y decreased ouabain binding (2.7-fold reduction of s p e c i f i c a c t i v i t y with respect to the crude micro-somes and 4.2-fold reduction with respect to a f r a c t i o n r e l a t i v e l y enriched i n sarcolemma) observed i n the p u r i f i e d S.R. f r a c t i o n . The microsomes at the 32.7/35% sucrose interphase correspond to s i m i l a r f r a c t i o n s i s o l a t e d from s k e l e t a l (Sarazala et a l . , 1978) and smooth muscle (Wuytack et a l . , 1978). The f a c t that ouabain binding could not be reduced to a greater extent by density gradient c e n t r i f u g a t i o n may i n d i c a t e a r e l a t i v e l y stable a s s o c i a t i o n between plasmalemmal and sarcoplasmic reticulum elements. U l t r a s t r u c t u r a l evidence for such j u n c t i o n a l structures i n s k e l e t a l muscle has been described (Franzini-Armstrong, 1975) and was confirmed, again i n s k e l e t a l muscle, by r e c o n s t i t u t i o n experiments on i s o l a t e d microsomes (Caswell, 1979). It appears l i k e l y that s i m i l a r structures also e x i s t i n cardiac muscle. In t h i s study we investigated whether French press treatment could further reduce the amount of ouabain binding and hence sarcolemmal contamination of the p u r i f i e d sarcoplasmic reticulum preparation. In s k e l e t a l muscle, French press treatment appears to be the method of choice for such a separation. Lau et a l . , 1977 have demonstrated that although 0.6 M KC1 did improve the separation of T-tubules from terminal cisternae - 41 -i n combination with French press treatment, the use of the potassium s a l t at high i o n i c strength can r e s u l t i n denaturation of microsomal proteins. This may be part of the reason why Jones et a l . (1979), who employed 0.6 M KCl-sucrose i n separation of cardiac sarcolemma from cardiac sarcoplasmic reticulum observed a very low indigneous adenylate cyclase a c t i v i t y i n t h e i r S.R. preparation. It was therefore decided to use a sucrose gradient without KC1 following French press treatment to minimize the time the microsomes are exposed to high i o n i c strength. The data shown i n F i g . 3a indic a t e that basal adenylate cyclase a c t i v i t y was increased whereas ouabain binding was decreased a f t e r French-press treatment. This r e s u l t i s i n d i r e c t agreement with the findings by Reiss et a l . (1979) that show a r e l a t i v e l y constant adenylate cyclase a c t i v i t y i n two S.R. preparations with widely varying (Na +-K +)-ATPase a c t i v i t i e s . Since SDS was not used to expose l a t e n t ouabain binding, the p o s s i -b i l i t y of an unknown amount of latent binding i n our preparation e x i s t s . This type of binding has been found i n inside-out plasma membrane v e s i c l e s (Peronne and Blostein,1 9 7 3 ).However,during Ca -loading, most of the 2+ v e s i c l e s exhibited very a c t i v e Ca -accumulation, which would not have been expected i f a s i g n i f i c a n t amount of inside-out plasma membranes had been present. Thus, la t e n t binding would not be expected to be present i n t h i s preparation. The data i n F i g . 3a suggest that there i s an indigenous adenylate cyclase present i n cardiac S.R.. The p o s s i -b i l i t y that the decreased ouabain binding i s due to a l o s s of membranes during f i l t r a t i o n , as already suggested i n the Results Section, becomes le s s p l a u s i b l e i n view of the fac t that electron microscopic studies on French-press treated s k e l e t a l muscle microsomes reveal retention of the - 42 -s t r u c t u r a l I n t e g r i t y of the v e s i c l e s (Lau et a l . , 1977). Assuming that the conclusion from the French press study i s v a l i d , a p l a u s i b l e explanation for the d i f f e r e n t f u n c t i o n a l c h a r a c t e r i s t i c s of the c y c l i c AMP production i n washed p a r t i c u l a t e (sarcolemmal) as opposed to S.Ri-enriched microsomal preparations i s that there e x i s t two d i f f e r e n t enzyme systems with d i f f e r e n t s u b c e l l u l a r l o c a l i z a t i o n s . A f t e r p u b l i c a t i o n of some of our i n i t i a l f i n d i n g s , (Katz and Dobovicnik, 1979) which appear i n tables 1 and 3, Reiss and Katz (1979) confirmed our data on the generally lower epinephrine and GTP s e n s i t i v i t y of the S .R .-associated enzyme. The authors also noted a s l i g h t l y lower pH optimum of the S.R. enzyme when compared to that of sarcolemmal adenylate cyclase (7.5 and 8.0, r e s p e c t i v e l y ) . The S.R. adenylate cyclase was also l e s s s e n s i t i v e to a l k a l i metal s a l t s than the sarcolemmal enzyme (the former was s l i g h t l y i n h i b i t e d whereas the l a t t e r was s i g n i -f i c a n t l y stimulated). The S.R.-enriched microsomes used i n t h e i r work were prepared i n the abscence of i s o t o n i c sucrose whereas t h e i r -sarcolemmal enzyme was exposed to t h i s medium. Reiss and Katz acknowledge the p o s s i b i l i t y that differences i n the c y c l i c AMP accumulation c h a r a c t e r i s t i c s may be due to t h i s d i f f e r e n c e . i n preparation. In addition,the above authors mention that sucrose has l i t t l e e f f e c t on • -e i t h e r f r a c t i o n . . -.Sin.ce. i n our work-sucrose was used i n preparation and storage of both f r a c t i o n s , ..the l i k e l i h o o d of a r t i f a c t u a l r e s u l t s has been reduced, Olson and Smith (1980), examining ATPase and adenylate cyclase d i s t r i b u t i o n i n s k e l e t a l muscle, provide evidence f o r an indigenous adenylate cyclase i n S.R. which shows a minimal i n h i b i t i o n of c y c l i c AMP accumulation by 0.1 mM epinephrine'. - 43 -The authors do not comment on (or seem to be unaware of) Caswell's study on s k e l e t a l muscle i n the same tissue (rabbit) which demonstrates a concomitant l o s s of 0-adrenergic receptors and adenylate cyclase a c t i v i t y from the microsomal f r a c t i o n containing t r i a d junctions a f t e r sarcolemmal T-tubules are separated from i t by French-press treatment. Caswell's work thus suggests that a further p u r i f i c a t i o n step would reveal a plasma membrane contamination i n the f r a c t i o n considered sarcoplasmic reticulum by Olson and Smith and others (Raible et a l . , 1978). Jones et a l . (1976, 1979) suggests that adenylate cyclase i s an enzyme l o c a l i z e d e x c l u s i v e l y i n the plasma membrane. But th e i r evidence r e s t s on a microsomal preparation obtained i n high [KC1] media, which may have contributed to a los s of enzymatic a c t i v i t y i n t h e i r " p u r i f i e d " S.R. N i j j a r et a l . (1980) have shown that s u b c e l l u l a r d i s t r i b u t i o n of adenylate cyclase i n r a t lung changes with age and i s not a r e l i a b l e marker for plasma membranes. This r a i s e s another point which i s also discussed by Reiss et a l . (1979): One of the reasons for adenylate cyclase a c t i v i t y i n an i n t e r n a l membrane component may be that i f the c e l l i s able to synthesize new adenylate cyclase c a t a l y t i c u n i t s , they would be synthesized i n the endoplasmic reticulum and pass through S.R.-associated membranes or S.R. during migration to the surface membrane. This would also imply some point of co n t i n u i t y between i n t e r n a l and external membrane systems. Entman et a l . , 1978, have gone as f a r as suggesting an i n t e r n a l 3-receptor associated with sarcoplasmic reticulum but t h i s proposition i s i n contrast to the findings by Reuter et a l . (1974) who demonstrated a lack of response of c a r d i a l purkinje f i b r e s to i o n t o p h o r e t i c a l l y administered isoproterenol - 44 -and noradrenaline. The r e s u l t s from t h i s work cannot c l e a r l y support or refute the concept of an i n t e r n a l 3,-receptor, since epinephrine stimulation of pure sarcoplasmic reticulum microsomes was inconsistent from preparation to preparation. If i t i s assumed that there i s no i n t e r n a l 3~receptor, the response of the 32.7/35% S.R. microsomal adenylate cyclase would be a consequence of i t s a s s o c i a t i o n with fragments of T-tubules which are continuous with the outer c e l l membrane. The e r r a t i c r e s u l t s obtained from B-agonist a c t i v a t i o n of the S.R.-associated adenylate cyclase (responses ranged from two-fold stimulation to s l i g h t i n h i b i t i o n ) contrast strongly with the highly reproducible data from washed p a r t i c l e s . This may well represent an u n i d e n t i f i e d factor i n the methodology employed i n prepara-t i o n of the membrane f r a c t i o n , such as highly v a r i a b l e sarcolemmal con-tamination (which i s made highly u n l i k e l y by the more consistent ouabain binding r e s u l t s ) , differences i n membrane-sidedness leading to i n a c c e s s i -2+ b i l i t y of the 3~receptor (also improbable i n view of the active Ca uptake) or a possible a r t i f a c t u a l migration of plasma membrane receptor to S.R. This i s a r e a l p o s s i b i l i t y , considering the recent demonstration of fusion of c e l l ghosts from mutant mammalian c e l l s (one lacking 3-receptor and another one lacking the c a t a l y t i c unit of adenylate cyclase) using sendai v i r u s (Schramm et a l . , 1 9 7 7 ) . A f t e r fusion the hybrid c e l l s exhibited hormone-sensitive adenylate cyclase a c t i v i t y . Therefore i t seemed appro-p r i a t e to search for modulating factors that could influence the S.R. adenylate cyclase i n a more consistent fashion. 2+ The studies employing varying Mg /ATP r a t i o s show a clear d i f f e r e n c e 2+ i n the response of the d i f f e r e n t f r a c t i o n s . Mg activates adenyl cyclase - 45 -4- 2-p a r t l y by combining with ATP to form the substrate MgATP (Alvarez, 1977) and p a r t l y by action on an independent i n t r a c e l l u l a r s i t e where 2+ 2+ Mg competes with Ca (Stole, 1979). Possible explanations for the 2+ resistance of the pure S.R. to saturation by a c t i v a t i n g Mg ions may l i e i n the microenvironment of the enzyme or o r i e n t a t i o n of the S.R. v e s i c l e s . The c h a r a c t e r i s t i c responses of the adenylate cyclases from d i f f e r e n t f r a c t i o n s may represent a true d i s t i n g u i s h i n g feature between two types of adenylate cyclase. Further support for such a f u n c t i o n a l heterogeneity i n the two forms of myocardial adenyl cyclases comes from 2+ the Ca - i n h i b i t i o n studies. S.R. adenylate cyclase appears more sen s i -2+ t i v e to i n h i b i t i o n by low [Ca ] (0-25 yM) than washed p a r t i c l e adenylate cyclase only i n the presence of epinephrine. The washed p a r t i c l e enzyme 2+ was i n h i b i t e d by Ca both i n the present and absence of epinephrine. It would be premature to assign any f u n c t i o n a l s i g n i f i c a n c e to these preliminary findings and more data over a greater concentration range of epinephrine i s necessary to f u l l y characterize the nature of , . „ 2+ . . the epinephrine- Ca i n t e r a c t i o n . Drummond and Duncan (1970) have also observed Ca - i n h i b i t i o n of cardiac adenylate cyclase and Raible et a l . (1978) have observed the phenomenon i n s k e l e t a l muscle S.R. adenylate cyclase. The l a t t e r authors also present r e s u l t s suggesting that the enzyme 2+ s i t e responsible for t h i s action of Ca faces the sarcoplasm and suggest that cAMP may stimulate release of calcium from terminal cisternae of S.R. This suggestion would agree with the controversial.observation of o s c i l l a t i o n s i n c y c l i c AMP l e v e l s i n frog v e n t r i c l e , showing a peak i n early s y s t o l e , (Brpoker, 1 9 7 . 3 ) p r e c i s e l y when Ca^ + release occurs. I f - 46 -the high C a ^ - s e n s i t i v i t y S.R. adenylate cyclase i s f u n c t i o n a l l y s i g n i f i -cant i n vivo, one would expect t h i s f a c t to integrate into the scheme proposed by Olson et a l . (1980). To propose a consistent hypothesis model of how cAMP i s supposed to do what the authors suggest i t does i s next to impossible and beyond the scope of t h i s discussion, considering 2+ the staggering amount of contradictory findings such as Ca -induced 2+ Ca -release from S.R. (see Introduction), compartmentalization of c y c l i c AMP-dependent protein kinases (Cor.bin et a l . , 1977), a c t i v a t i o n 2+ of sarcolemmal and S.R. Ca -fluxes by c y c l i c AMP and protein kinase induced phosphorylations of channels and c y c l i c AMP-independent increases i n cardiac c o n t r a c t i l e force. The main problem, even i f some doubtful findings that do not f i t into a scheme are cast out, i s to explain the involvement of the same regulator (cAMP) i n the con t r o l of both long-term (Inotropism) and short-term ( i n d i v i d u a l contraction cycles) events. This i s very d i f f i c u l t without invoking a highly e f f e c t i v e i n t r a c e l l u l a r compartmentalization. In conclusion, t h i s study presents further evidence that cardiac S.R. contains an indigneous adenylate cyclase a c t i v i t y . Evidence i s derived from an enzyme marker study (ouabain binding) and f u n c t i o n a l characterizations (responses to regulating factors) of washed p a r t i c l e and pure S.R. f r a c t i o n s . In addition, the use of French-press treatment has been shown to be us e f u l i n reducing contamination of S.R. preparations with plasma membranes. - 47 -REFERENCES Alvarez,R. and Brun, J . J . (1977): Proc. N a t l . Acad. S c i . USA 74(1), A c t i v a t i o n of cardiac adenylate cyclase: Hormonal modification of the magnesium ion requirement. Andersson, K.E. and Edman, K.A.P. (.1974): Acta Physiologica Scandinavica 90, 124-131. 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